Genetic diversity of groEL and msp4 sequences of Anaplasma ovis infecting camels from Tunisia

Journal Pre-proof Genetic diversity of groEL and msp4 sequences of Anaplasma ovis infecting camels from Tunisia

Rachid Selmi, Mourad Ben Said, Mokhtar Dhibi, Houcine Ben Yahia, Hedi Abdelaali, Lilia Messadi PII:

S1383-5769(19)30331-9

DOI:

https://doi.org/10.1016/j.parint.2019.101980

Reference:

PARINT 101980

To appear in:

Parasitology International

Received date:

16 July 2019

Revised date:

10 August 2019

Accepted date:

30 August 2019

Please cite this article as: R. Selmi, M.B. Said, M. Dhibi, et al., Genetic diversity of groEL and msp4 sequences of Anaplasma ovis infecting camels from Tunisia, Parasitology International(2019), https://doi.org/10.1016/j.parint.2019.101980

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© 2019 Published by Elsevier.

Journal Pre-proof Genetic diversity of groEL and msp4 sequences of Anaplasma ovis infecting camels from Tunisia

Rachid Selmi a,c,d, Mourad Ben Said a, Mokhtar Dhibi b, Houcine Ben Yahia c, Hedi Abdelaali c, Lilia Messadi

a

a,*

Service de Microbiologie et Immunologie, Ecole Nationale de Médecine Vétérinaire, Université

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b

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de Manouba, 2020 Sidi Thabet, Tunisie.

Service de Parasitologie, Ecole Nationale de Médecine Vétérinaire, Université de Manouba,

Ministère de la Défense Nationale, Direction Générale de la Santé Militaire, Service

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c

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2020 Sidi Thabet, Tunisie.

Institut National Agronomique de Tunis, Université de Carthage, Tunisie.

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d

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Vétérinaire, Tunis, Tunisie.

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* Corresponding author:

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Pr. Lilia Messadi

Service de Microbiologie, Ecole Nationale de Médecine Vétérinaire, 2020 Sidi Thabet, Tunisie Tel: +216 71 552 200

Fax: +216 71 552 441

E-mail: [email protected]

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Journal Pre-proof Abstract To date, no information is available regarding the infection of camels (Camelus dromedarius) by Anaplasma ovis in North African region. Several animal species can be infected by A. ovis which further complicates its natural infection cycle. In this paper, we investigated the occurrence and the genetic diversity of A. ovis in camels and ticks collected from them in Tunisia and the risk factor analysis. Camel blood samples (n = 412) and tick (n = 300) samples, identified as Hyalomma dromedarii (n = 149, 49.6%), H. impeltatum (n = 142, 47.3%) and H. excavatum (n =

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9, 3%), were analyzed by conventional PCR followed by the sequencing of msp4 and groEL genes. A. ovis DNA was identified in five camels (1.2%), but not in infesting ticks (0%). The

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microscopic examination revealed the specific infection of camel erythrocytes by Anaplasma

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inclusions. The msp4 and groEL typing confirmed the natural infection of camels by A. ovis and

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revealed two different msp4 genotypes earlier detected in Tunisian small ruminants and their infested ticks, and five different and novel groEL genetic variants forming a separately sub-

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cluster within A. ovis cluster. The occurrence of different A. ovis strains specific to camels

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associated with a low prevalence of this Anaplasma species in camels may enrich knowledge

of Tunisia.

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regarding the distribution and the transmission cycle of this bacterium in arid and Saharan areas

Keywords : Anaplasma ovis; Camels (Camelus dromedarius); Molecular detection; msp4 and groEL typing; Phylogenetic analysis; Tunisia

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Journal Pre-proof 1- Introduction Several Anaplasma species were identified in animals from Tunisia. Particularly, Anaplasma ovis was identified with high infection rates in sheep and goats [1]. However, it has been demonstrated that the one humped camel harbored strains genetically related to A. platys (A. platys- like) [2] and antibodies anti-A. phagocytophilum (29.2%) [3]. The natural cycle of Anaplasma spp. involves domestic and wild animal species, and ticks that serve as vectors [4]. The infection by this bacterium did not respect the preferential animal species rule [4]. This

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could be strictly dependent to the extremely important genetic diversity which may be associated with the development of adaptation and resistance mechanisms [5].

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Anaplasma ovis was detected and genetically characterized based on the phylogenetic

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analysis of several partial sequences including ARNr 16S, ARNr 23S, rpoB, msp4, groEL and

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glta genes [6, 7]. The msp4 gene, routinely used, seems to be more informative regarding the genetic diversity and the evolutionary phylogeography of strains [8]. Msp4 protein modulates the

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interaction between A. ovis and host cells and regulates the immune response [9]. Besides, the

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combined analysis of msp4 and groEL partial sequences allows not only to discriminate between

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Anaplasma species but also to explain heterogeneity that could be observed at the animal, herd and region level [10-12]

In this study, we investigated the occurrence of A. ovis in camels and infesting ticks from Tunisia in order to better understand the molecular epidemiology of this Anaplasma species in arid and Saharan bioclimatic areas [13]. Therefore, this study was carried out to investigate the occurrence of A. ovis in camels and infesting ticks from Tunisia in order to better understand the molecular epidemiology of this Anaplasma species in arid and Saharan bioclimatic areas.

2- Materials and Methods 2.1 Areas of study and sample collection

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Journal Pre-proof This study was performed between 2015 and 2017 in seven camel herds from five Tunisian localities (Figure 1). From Southern Tunisia, visited governorates are Kebili (state farm, n= 250, 151 ticks), Tataouine (private farms, n= 54, 34 ticks) (Saharan bioclimatic areas; temperature average: 35°C; annual rainfall average: 134 mm), and Gabes (private herd; n= 26, 46 ticks) (arid area; oasis type; temperature average: 33°C; annual rainfall averages: 177 mm). From the center, sampling was also performed in the governorate of Kairouan (state herd, n= 40, 22 ticks) (arid area; temperature average in the summer: 35°C; annual rainfall average: 293 mm) and the

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governorate of Sousse (private farm, n= 42, 47 ticks) (semi-arid bioclimatic area; temperature average: 25°C; annual rainfall average: 361 mm) [14]. A total of 412 camel blood samples were

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withdrawn from the jugular and tail vein of apparently healthy camels. Besides, 118 camels were

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found to be infested by ticks. A total of 300 partially engorged ticks were removed (2.5

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specimens per animal) from different parts of animal body (ears, tail, scrotum and udder) and

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2.2 Sample size determination

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identified morphologically using the published taxonomic key [15].

The minimal required number of samples may be estimated and determined using the

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following formula: N= 1.96 ² * Pexp (1-Pexp) / d² [16]. The required sample size (N) depends on the expected prevalence (Pexp=50%) with a confidential interval of 95% and the accepted absolute error (d) of 5%. According to this formula, a total of 384 samples were required in this study. Since we have visited 7 camel herds, 54 samples were required from each herd. However, this number was not already available. Apparently healthy camels were selected and sampled randomly.

2.3 Nucleic acid extraction DNA was extracted from the whole-blood samples using the Wizard® Genomic DNA purification kit (Promega, Madison, USA). Tick specimens were crushed individually and DNA 4

Journal Pre-proof was extracted by using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. DNA yields were determined by the Qubit 3.0 Fluorimeter (Thermo Fisher).

2.4 Specific detection and genetic characterization of Anaplasma ovis genotypes In order to specifically detect A. ovis in camel and tick DNA samples, single PCR reaction, targeting the major surface protein (msp4) gene, was performed using Aovismsp4fw/

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Aovismsp4rev primers [17]. For further characterization and genotyping, selected camel samples positive to A. ovis by single PCR based on AovisMSP4Fw and AovisMSP4Rev primers were

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used in two single PCRs with MSP45/MSP43 and groelAoF/groelAoR primers for the

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respectively (Supplementary file 1) [18, 19].

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amplification of partial sequences of the msp4 and the heat-shock operon groEL genes,

PCR reaction was carried out in a final volume of 50 μl containing 0,125U/μl Taq DNA

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polymerase (Biobasic Inc, Canada), 1x PCR buffer, 1.5mM MgCl2 , 0.2mM dNTPs, 2 μl genomic

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DNA (50 to 150 ng) and 0.5μM of primers. Positive DNA of A. ovis isolated from sheep blood

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[19] and distilled water were included as positive and negative controls, respectively. Thermal cycling profile was as described by de la Fuente et al. [18, 19] and Belkahia et al. [18, 19] for msp4 and groEL genes, respectively. Amplified DNA products were electrophoresed in 1.5% agarose gel stained by ethidium bromide for 1 h at 120 V and then visualized by UV light transilluminator.

2.5 Microscopic examination To investigate the tropism of A. ovis in camel blood, thin smears prepared from three A. ovis positive camel blood were deposited on clean slide, fixed using methanol for 3 min and stained with Giemsa stain during 20 minutes. Slides were examined by scanning 30 microscopic

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Journal Pre-proof fields for each sample. All abnormalities and morphological features reminding intracellular bacterial inclusions were recorded (Supplementary file 2).

2.6 Sequencing and phylogenetic analysis Ten revealed positive PCR products by primers msp45/msp43 (n = 5) and groelAoF/groelAoR (n = 5) were purified (GF-1 Ambi Clean kit, Vivantis, USA) and sequenced in both directions using Big Dye Terminator cycle sequencing ready reaction kit (Applied

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Biosystems, Foster City, USA) and an ABI3730XL automated DNA sequencer). The chromatograms were evaluated with Chromas Lite v 2.01. The DNAMAN software (Version

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5.2.2; Lynnon Biosoft, Que., Canada) was used to perform multiple sequence alignment of msp4

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and groEL sequences. BLAST analysis of GenBank was used to assess the level of similarity

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with previously reported sequences [20]. By using the DNAMAN software, genetic distances among the sequences were computed by the maximum composite likelihood method [21] and

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were used to construct neighbor-joining trees [22]. Statistical support for internal branches of the

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trees was evaluated by bootstrapping with 1,000 iterations [23].

2.7 Statistical analysis

Exact confidence intervals (CI) for prevalence rates at the 95% level were calculated. Comparison of the prevalence of A. ovis in camel blood according to bioclimatic areas, governorates, season, tick infestation, contact with ruminants (sheep and goats) , age and gender of dromedaries were performed with Epi Info 6.01 (CDC, Atlanta), using the χ2 test and Fisher’s exact test with a threshold value of 0.05.

2.8 GenBank accession numbers

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Journal Pre-proof The identified msp4 and groEL partial sequences of A. ovis isolates infecting dromedaries have been deposited under GenBank accession numbers MN094834 to MN094838 and MN094839 to MN094843, respectively.

3- Results Regarding tick identification, all specimens were identified as Hyalomma genus and three

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predominant species followed by H. excavatum (n = 9, 3%).

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species namely, H. dromedarii (n = 155, 52%) and H. impeltatum (n = 136, 45%) were the most

Estimated by Aovismsp4fw/Aovismsp4rev primers, only five samples (1.2%) of camel

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blood harbored DNA of A. ovis. However, DNA of this bacterium was not amplified from any of

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tested ticks. Positive camels derived from both semi-arid (governorate of Sousse) and Saharan

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(governorate of Kebili) bioclimatic areas. Differences in prevalence rates were not statistically significant according to age, gender, breed, contact with other ruminants and tick infestation

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(Table 1).

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Based on the microscopic examination, reddish-violet, thin, homogenous and punctiform

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inclusions, reminding A. ovis organisms found in small ruminants blood, were observed in erythrocytes of three revealed positive A. ovis camels (out of 5 PCR positive samples) (Supplementary file 2). These inclusions measured 0.1-0.3 µm of diameter and appeared to be relatively attached to the inner side of the membrane cells. However, these microscopic inclusions were absent in neutrophile granulocytes, monocytes and platelets of these tested animals. Anaplasma ovis infection was validated by sequencing of 808 bp msp4 fragments from all positive A. ovis camels. Alignment of obtained sequences revealed two genotypes (Aomsp4Cd1 and Aomsp4Cd2; GenBank accession numbers MN094834 to MN094838) (Table 2). These two genotypes were 99.8% identical to each other. Aomsp4Cd1 genotype was 100% identical to

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Journal Pre-proof various genotypes isolated from Tunisian sheep and to the genotype “Italy147” isolated from Italian sheep. However, Aomsp4Cd2 genotype were 100% identical to the Panagcy strain infecting Human in Cyprus [24], to the genotype Italy20 isolated from Italian sheep and to several other genotypes isolated from small ruminants and their infesting ticks located in Tunisia. Phylogenetic analysis shows that A. ovis cluster is formed by four sub-clusters. In particular, the first three sub-clusters included several isolates from the Old World countries such

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as Tunisia, Italy, Spain, Cyprus, Serbia, Hungary, Iran and China. The last sub-cluster contained two isolates from the New World countries exclusively represented by the USA (Figure 2).

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Tunisian strains Aomsp4Cd1 and Aomsp4Cd1 were, respectively, assigned to the first and the

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second sub-cluster with strains mainly isolated from small ruminants and their associated ticks

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located in the Mediterranean countries including Tunisia, Italy, Cyprus and Spain (Figure 2). Infection by A. ovis was also confirmed by sequencing of 620 bp groEL fragments from all

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positive A. ovis camels. Five novel and distinct genotypes were obtained (AogroELCd1-5;

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GenBank accession numbers MN094839 to MN094843) (Table 2, Figure 3). Nucleotide identity

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among genotypes ranged from 98.7 to 99.8%. These genotypes were 99% identical to genotypes AogroElRt1-6 and AogroElRs1/2 (GenBank accession numbers MH292907 to MH292916) isolated from Rhipicephalus turanicus and R. sanguineus sensu lato removed from Tunisian small ruminants and to two A. ovis genotypes isolated from South African and Chinese sheep (OVI and S43 strains, GenBank accession numbers AF441131 and KX579069, respectively). Phylogenetic analysis was performed on the basis of the alignment of these five genetic variants with all available genotypes of A. ovis strains, as well as other Anaplasma species found in the GenBank (Figure 3). Identified variants were classified among A. ovis cluster and formed, separately, the second sub-cluster closely related to the first sub-cluster composed by all

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Journal Pre-proof genotypes isolated from Chinese and South African sheep and from ticks infesting small ruminants in Tunisia (Figure 3).

4- Discussion This study demonstrated the infection of Tunisian camels by A. ovis with a low infection rate (1.2%). This finding confirmed previous report of Noaman et al. [18, 19] in Iran who reported similar results (2%). This data suggests that camels may be accidentally exposed to this

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bacterium during close and prolonged contact (gathering in the same pasture, water sources, traditional fiestas and livestock market) with small ruminants that have been found to be heavily

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infected by this species in our country (93.8% in sheep and 65.3% in goats) [25, 26]. However,

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A. ovis was not detected neither in cattle nor in horses from Tunisia although the contact of these

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species with small ruminants is more important than camels [4]. In fact, competent arthropods feeding on camels and small ruminants like sheep and goats may transfer this pathogen between

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these ruminant species. Based on this result, camels could probably be one of the possible

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accidental hosts of A. ovis in arid and Saharan areas of Tunisia.

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In the current study, infection was absent in all analyzed specimens of Hyalomma spp. infesting camels. This may be explained by the specific vectors’ activities involved in the transmission of A. ovis. This bacterium was adapted to several arthropod vectors. However, ticks of Rhipicephalus genus, including R. turanicus, R. bursa and R. sanguineus s.l. are considered as the main vectors of A. ovis in European, African and Asian countries [19, 27-30]. These tick species are commonly infesting watchdogs and/or small ruminants present in close contact with camels [19, 31]. Furthermore, Hyalomma sp. can also harbor, passively, this pathogen through the co-feeding process [32]. Besides, infection by A. ovis could be accidentally transmitted by hippoboscid flies of small ruminants given that it has been identified in the sheep blood sucking flies Melophagus ovinus from China [33].

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Journal Pre-proof The microscopic examination of Giemsa stained blood films revealed a round, purple inclusions with a polar or sub-polar position within the outer margins of the erythrocytes. These inclusions found in camel erythrocytes were considered as Anaplasma organisms in previous microscopic investigations on camels from Egypt [34], Iran [35, 36], Saudi Arabia [37], Nigeria [38] and Somalia [39]. A. ovis and A. marginale were the two main Anaplasma species suggested to be found in camel erythrocytes [34]. Based on the msp4 and groEL phylogenetic analysis, genetic diversity was recorded in A.

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ovis strains infecting Tunisian camels. The obtained A. ovis msp4 genotypes were closely similar to those isolated from small ruminants and their infesting ticks located in the Mediterranean

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regions [19, 26]. This result is similar to those reported in Turkey [40] and China [41] indicating

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low genetic diversity with only two A. ovis msp4 genetic variants. Contrariwise, more than five

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A. ovis msp4 genotypes were isolated from goats in France [27] and from sheep in Hungary [11]. Regarding the groEL gene analysis, five different genotypes were identified among

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camels. This interesting finding demonstrated that this gene is more informative and provides a

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higher diversity sequences according to host and geographic region compared to other genes

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routinely used like msp4, gltA and 16S rRNA [42]. This gene is particularly useful to study the genetic diversity of A. ovis strains infecting animals other than habitual hosts like sheep and goats [27, 43].

The clinical expression of A. ovis infection is strictly dependent on the level of strain virulence and to the animal host susceptibility. In some cases, pathogenic strains may lead to severe clinical form expressed by anemia, emaciation, mortality among young animals particularly in small ruminants [44] and abortion of pregnant females [45]. In contrast, in the current study, all camels revealed positive to this bacterium were apparently healthy. This may be explained by the fact that identified strains were probably non-pathogenic and/or camels are sufficiently resistant.

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Journal Pre-proof In conclusion, this study identified a low prevalence of A. ovis in blood of apparently healthy camels in Tunisia. High genetic diversity of A. ovis especially in groEL gene was revealed. In order to improve knowledge and to manage the control of this bacterium, further studies should investigate vectors incriminated in the transmission of A. ovis to camels and mechanisms of cellular and humoral immune responses of this animal species to A. ovis infection.

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Ethical approval

Animal were restrained, examined and sampled in accordance with the national legislation

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regarding ethics and animal welfare. Blood and tick samples were, professionally, collected by

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veterinarians from restrained camels. Verbal acceptation was obtained from the camel handlers

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participating in this study.

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Conflict of interest

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The authors declare that they have no competing interests.

Acknowledgements

We would like to express our sincere gratitude and deep thanks to Tunisian Ministry of Higher Education and Scientific Research for financial supporting this research project (Project No. LR16AGR01). Great thank to Prof. Ali Bouattour for his valuable help in tick identification.

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[37] A.B. Ismael, A.A.A. Swelum, A.F. Khalaf, A.N. Alowaimer, First evidence of natural anaplasmosis in Camelus dromedarius in Saudi Arabia, J Camel Pract Res. 23 (2016) 95-

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mortality in a neonatal elk calf, J. Vet. Diagn Invest. 31 (2019) 267-270.

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[45] M. Obaidat, A. Salman, Anaplasma spp. in dairy ruminants in Jordan: high individual and

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herd-level seroprevalence and association with abortions, J Vet Diagn Invest. 31 (2019) 1-9.

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Journal Pre-proof Figure legend Figure 1 (A) Geographic position of Tunisia in the African continent. (B) Map of Tunisia showing studied regions.

Figure 2 Neighbor-joining tree based on the alignment of partial msp4 sequence (805 bp) of Anaplasma

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ovis obtained in this study with selected sequences representative of the Anaplasma genus. Numbers over the branches indicate the percentage of replicated trees in which the associated

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taxa clustered together in the bootstrap test (1,000 replicates, only perc entages greater than 50%

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were represented). The two different sequences obtained in this study are indicated with camel

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picture. The host or vector, the genotype, strain or isolate name, the country of origin and the GenBank accession number are indicated. One A. phagocytophilum msp4 partial sequences was

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Figure 3

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added as out-group.

Phylogenetic tree inferred with partial sequences (620 bp) of the groEL gene of Anaplasma ovis isolated from camels and other Anaplasma species found in GenBank using the neighbor-joining method.

Bootstrap values (1,000 replicates) are indicated in each node (only percentages greater than 50% are shown). The five novel genotypes of A. ovis obtained in the present study are indicated with camel picture. The host or vector, the genotype, strain or isolate name, the country of origin and the GenBank accession number are represented. One Ehrlichia ruminantium groEL partial sequence was added as out-group.

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Journal Pre-proof Supplementary file legend Supplementary file 1 Primers used in the present study for the detection and characterization of Anaplasma ovis strains in examined dromedaries.

Supplementary file 2 Blood smears from camels naturally infected with Anaplasma ovis at the periphery of infected

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red blood cells (Figure 2A) compared to a non infected sample (Figure 2B) and the infected

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sheep of used positive control (2C) Giemsa x100.

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Journal Pre-proof Table 1 Molecular prevalence of Anaplasma ovis in camels according to different risk factors

Season2

Gender Age Breed3

Total

0.750 0.687

0.750

0.302 0.281

0.493 0.804 0.119

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Tick infestation

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Contact with ruminants

P-value

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Geographic region

Positive camels (%±C.I.1 ) 4 (1±0.03) 1 (1±0.04) 0 (0) 4 (2±0.03) 0 (0) 1 (2±0.09) 0 (0) 2 (3±0.07) 0 (0) 2 (2±0.06) 1(3±0.09) 1 (1±0.03) 4 (2±0.03) 0 (0) 3 (2±0.04) 2 (2±0.05) 5 (1±0.02) 0 (0) 2 (1±0.02) 3 (1±0.02) 3 (3±0.05) 2 (1±0.01) 5 (1±0.01)

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Saharian Arid and semi-arid Gabes Kebili Kairouan Sousse Tataouine Autumn Winter Spring Summer Male Female < 5 years 5 to 10 years > 10 years Local Other breeds Yes No Yes No

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Bioclimatic area

Examined camels 304 108 26 250 40 42 54 73 46 91 40 176 236 132 148 132 377 35 243 291 118 294 412

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Categories

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Risk factors

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Abbreviations: 1 : C.I.: 95% confidence interval. 2 : The effect of the season on A. ovis infection has been only investigated on 250 dromedaries located in the governorate of Kebili. 3 : Local breed is Arab breed and the other breeds are Arnouti and Tergui.

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Journal Pre-proof Table 2 Designation and information about sequencing of Anaplasma ovis genotypes isolated from camels in this study Gene

Genotype

Isolate

Infected host species (Ref. num.1 )

msp4

Aomsp4Cd1

Cd30 Cd31 Cd32 Cd34 Cd33 Cd35 Cd36 Cd37 Cd38 Cd39

Camelus dromedarius Camelus dromedarius Camelus dromedarius Camelus dromedarius Camelus dromedarius Camelus dromedarius Camelus dromedarius Camelus dromedarius Camelus dromedarius Camelus dromedarius

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Aomsp4Cd2 AogroELCd1 AogroELCd2 AogroELCd3 AogroELCd4 AogroELCd5

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groEL

(Do71) (Do85) (So234) (Do397) (So252) (Do71) (Do85) (So234) (Do397) (So252)

Geographical location Douz Douz Sousse Douz Sousse Douz Douz Sousse Douz Sousse

GenBank a number MN094834 MN094835 MN094836 MN094837 MN094838 MN094839 MN094840 MN094841 MN094842 MN094843

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Abbreviations: 1 : Reference number. FJ460443 is the GenBank accession number of “Panagcy” strain found in human from Cyprus [24]. AY702923 is the GenBank accession number of “Italy20” strain isolated from Italian sheep [18]. MH292916 is the GenBank accession number of “clone BzRs195” clone found in Rhipicephalus sanguineus sensu lato tick from Tunisia [19].

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Figure 1

Figure 2

Figure 3

Figure 4