Detection of Novel Strains Genetically Related to Anaplasma Platys in Tunisian One-humped Camels (camelus Dromedarius)

Introduction: Little information is currently available regarding the presence of Anaplasma species in North African dromedaries. To fill this gap in knowledge, the prevalence, risk factors, and genetic diversity of Anaplasma species were investigated in Tunisian dromedary camels. Methodology: A total of 226 camels from three different bioclimatic areas were sampled and tested for the presence of Anaplasma species by quantitative polymerase chain reaction (qPCR) and nested polymerase chain reaction (nPCR) assays. Detected Anaplasma strains were characterized by 16S rRNA sequence analysis. Results: Overall infection rate of Anaplasma spp. was 17.7%, and was significantly higher in females. Notably, A. marginale, A. centrale, A. bovis, and A. phagocytophilum were not detected. Animals were severely infested by three tick species belonging to the genus Hyalomma (H. dromedarii, H. impeltatum, and H. excavatum). Alignment, similarity comparison, and phylogenetic analysis of the 16S rRNA sequence variants obtained in this study suggest that Tunisian dromedaries are infected by more than one novel Anaplasma strain genetically related to A. platys. Conclusions: This study reports the presence of novel Anaplasma sp. strains genetically related to A. platys in dromedaries from various bioclimatic areas of Tunisia. Findings raise new concerns about the specificity of the direct and indirect diagnostic tests routinely used to detect different Anaplasma species in ruminants and provide useful molecular information to elucidate the evolutionary history of bacterial species related to A. platys.


Introduction
The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes Gram negative obligate intracellular bacteria of significant importance in veterinary and human medicine [1].Anaplasma marginale, the type species of Anaplasma genus, is highly pathogenic for ruminants and poses a considerable constraint to animal health in tropical and subtropical regions throughout the world [2].It causes a variety of clinical symptoms, including fever, weight loss, abortion, lethargy, icterus, and often death of animals older than two years of age [2].The closely related species A. centrale causes mild anaplasmosis in cattle [3,4]; for this reason, it has been used extensively as a live vaccine for anaplasmosis control in several countries [5].Indeed, infection with A. centrale induces long-lasting protective immunity in ruminants when challenged with highly virulent A. marginale strains [2].
A. phagocytophilum is zoonotic and infects neutrophil granulocytes of many host species [3], including domestic ruminants, in which it causes tickborne fever (TBF) [6,7].The most common symptoms of TBF are high fever, anorexia, dullness, and reduced milk production [8]. A. bovis, a monocytotropic species, has been detected in different ruminant species from many countries [9,10].It has been isolated from cattle and deer in Japan [11][12][13], cattle in Iran [14], water deer in South Korea [15], and goats in China [10].A. bovis infection can cause variable clinical conditions ranging from the absence of symptoms to fever, anemia, weight loss, abortion, and death [16].
In Sicily, Italy, strains closely related to A. platys have been detected in neutrophils of cattle, sheep and goats [17] and in platelets of cats [18].Based on genetic analyses using 16S rRNA and groEL genes, these strains revealed very high levels of nucleotide identity with canine A. platys strains (99% and 92%-93% identities with A. platys 16S rRNA and groEL genes, respectively) and were placed in a distinct monophyletic cluster closely related to A. platys sequences [17,18].
To date, few data on the presence of Anaplasma species in Tunisian domestic animals, especially in camels, are available.Molecular findings demonstrated the occurrence of A. phagocytophilum infections in dogs and horses [26,27], as well as A. ovis in sheep from the northern and central areas of the country [28].The presence of A. phagocytophilum in horses and dromedaries was investigated by serology [29,30].Indeed, surveys of anaplasmosis in camels have been focused mainly on A. marginale [31][32][33][34][35].
This study aimed to establish the presence and prevalence of Anaplasma species in Tunisian dromedaries by sampling three different bioclimatic areas.Molecular epidemiology of Anaplasma spp.strains infecting camels was also investigated by combining quantitative PCR (qPCR) with 16S rRNA sequence analyses.

Sampling and DNA extraction
Blood samples and ticks were collected in 2009 (May to October) from 226 apparently healthy dromedaries spread throughout three localities: Bouficha (governorate of Sousse, latitude 36°18'N, longitude 10°27'E), belonging to semi-arid bioclimatic area with a mean annual rainfall of 350 mm; Sidi Bouzid (governorate of Sidi Bouzid, latitude 35°0'N, longitude 9°29'E), belonging to arid bioclimatic area with a mean annual rainfall of 237 mm, and Douz (governorate of Kebili, latitude 33°27'N, longitude 9°01'E), belonging to the Saharan bioclimatic area with a mean annual rainfall of 89 mm (Figure 1).Blood was collected from jugular veins into EDTA tubes (Becton Dickinson, Franklin lakes, USA).For each animal, the studied region, approximate age, gender, and presence/absence of ticks were noted.Ticks collected from severely infected animals were preserved in 70% ethanol and identified at genus and species levels using diagnosis keys as described by Walker et al. [36].DNA was extracted from 300 µL volumes of EDTA-preserved whole blood using the Wizard Genomic DNA purification kit (Promega, Madison, USA) according to the manufacturer's instructions.DNA yields were determined with a spectrophotometer (Jenway, Genova, Italy).DNA samples were stored at -20°C until use.

Duplex real-time PCR
DNA samples were tested for the presence of A. marginale and A. centrale by using species-specific primers and TaqMan probes as described by Carelli et al. [37] and Decaro et al. [38], targeting, respectively, a fragment of the msp1b (77 bp) and groEL (95 bp) genes.PCR was performed using Premix Ex Taq (Perfect Real Time) (Takara Mirus Bio, Madison, USA) in a 7500/7500 Fast Real-Time PCR System quantitative thermal cycler (Applied Biosystems, Foster City, USA).PCR amplification for A. marginale and A. centrale detection was performed in a duplex format by optimal reaction conditions using primers AM-For and AM-Rev at 600 nM each, probe AM-Pb-FAM at 200 nM, primers AC-For and AC-Rev at 900 nM each, probe AC-Pb-VIC at 200 nM, and 2 μL of template DNA (Table 1).Thermal cycling conditions included an initial activation of the Taq DNA polymerase at 95°C for 15 minutes, followed by 50 cycles of denaturation for 1 minute at 95°C followed by a 1 minute annealing-extension step at 60°C.Negative and positive controls were included in all runs.

Single and nested PCR
Primers EE1 and EE2 were used in a simple PCR run for amplifying the 16S rRNA gene of all Anaplasma species in dromedaries (Table 1).Reactions were performed in a final volume containing 0.125 U/µL Taq DNA polymerase (Biobasic Inc., Markham, Canada), 1x PCR buffer, 1.5 mM MgCl 2 , 0.2 mM dNTPs, 2 µL genomic DNA, 0.5 µM of the primers, and autoclaved MilliQ water to 50 µL.Thermal cycling reactions were performed in an automated thermal cycler (Techne Flexigene, Cambridge, UK) as described previously by Liu et al. [10].Primers specific for A. phagocytophilum and A. bovis were used in two distinct nested PCRs (Table 1), in which 1 µL of the simple PCR run was used as DNA target.Thermal cycling profiles were as previously described by Kawahara et al. [11].Negative (distilled water) and positive (DNA extracted from A. phagocytophilum and A. bovis) were included in each experiment.PCR products were electrophoresed on 1% agarose gel to check the size of amplified fragments by comparison with a DNA molecular weight marker (1 Kb Plus DNA Ladder, Promega, Madison, USA).

DNA sequencing and data analysis
Nine selected positive Anaplasma spp.PCR products (three from each sampling region) obtained with primers EE1/EE2 were purified with the GF-1 Ambi Clean Kit (Vivantis Technologies, Subang Jaya, Malaysia) according to the manufacturer's instructions.Purified DNA fragments were sequenced in both directions, using the same primers as in the PCR amplifications (Table 1).Sequencing was  [39].Neighbor-joining (NJ) phylogenetic trees were constructed using the DNAMAN program based on Saitou and Nei distances [40] with bootstrap analysis of 1,000 reiterations.

Sequence accession number
The 16S rRNA partial sequences of Anaplasma spp.AspGDr1 to AspGDr4 variants were deposited in the GenBank under accession numbers KM401905 to KM401908, respectively.

Statistical analyses
Exact confidence intervals (CIs) for prevalence rates at the 95% level were calculated.To study the possible influence of location, gender, age and tick infestation on the molecular prevalence of Anaplasma spp., the Chi-square test or Fisher's exact test were performed using Epi Info version 6.01 with a cut-off value of 0.05.In order to consider any confusion factor, a Chi-square Mantel-Haenszel test was performed.

Tick identification and molecular survey of Anaplasma species
Ticks collected from the camels belonged to the genus Hyalomma (H.dromedarii, H. impeltatum, and H. excavatum).The overall tick infestation prevalence was 37.6% (85/226).Overall infection rate of Anaplasma spp., estimated by EE1/EE2 PCR (Table 1), was 17.7% (minimum 14.8% in Sidi Bouzid and maximum 31.3% in Bouficha) (Table 2).Moreover, the infection rate of Anaplasma spp. was significantly higher in female (24.5%) than in male camels (11.7%, p = 0.027) (Table 2).Using qPCR tests specific for A. marginale and A. centrale, and nPCRs for A. bovis and A. phagocytophilum (Table 1), none of the classified Anaplasma species analyzed in this study were detected in any of the tested camels.

Molecular characterization of Anaplasma sp. 16S rRNA genotypes
Nine PCR products obtained from nine randomly selected camels (three from each sampling site) with primers EE1/EE2 targeting 1,322 bp (88.5%) of the 16S rRNA gene of Anaplasma spp.were successfully sequenced on both DNA strands.Based on nucleotide alignments, the sequences were grouped in four different genotypes (AspGDr1 to AspGDr4; GenBank accession numbers KM401905 to KM401908).All 16S rRNA sequences obtained in this study shared 99.8% to 99.9% nucleotide similarity and differed from each other in three nucleotide positions (Tables 3  and 4).

A. sp (AspGDr1) 100
A. sp (AspGDr2) 99.9 100 Based on BLASTN analyses and nucleotide alignments, the four identified genotypes were 99.6%-99.8%similar to those of J3 and E10 Anaplasma sp.isolates (GenBank accession numbers JN558826 and JN558821, respectively) found on Chinese goats and considered as A. platys-like by Liu et al. [10] and differed in seven and six nucleotide positions, respectively (Tables 3 and 4).Obtained sequences also shared 99.7%-99.8%similarity with an A. platys Okinawa isolate recovered from a dog in Japan (GenBank accession number AY077619) and differed in five nucleotide positions (Tables 3 and 4).Lower nucleotide sequence identities were obtained on comparisons with other Anaplasma species (98.7%-98.9%with A. phagocytophilum; 97.0%-97.1% with A. marginale; 97.0% with A. bovis; 96.9%-97.0%with A. centrale, and 96.9%-97.0%with A. ovis; Table 4).Similarly, comparisons based on 763 bp of the 16S rRNA gene highlighted a similarity of 99.3% with strains BovineCaprine1 and Caprine2 found on Italian cattle and goats (GenBank accession numbers KC335220-KC335222) and classified as Anaplasma sp.strains closely related to A. platys [17].
Phylogenetic analysis placed all the sequences obtained in this study in monophyletic clusters including A. platys (Figure 2A, 2B).In particular, all Anaplasma sp.Tunisian strains were closely related to A. platys strains isolated from Chinese goats and to Italian strains isolated from goats and cattle [10,17].

Discussion
Dromedary camels can host different pathogens, including several Anaplasma species [35,41].In Tunisia, a molecular survey of Anaplasma species in dromedaries is still lacking [29].In this study, molecular epidemiology of selected Anaplasma species was investigated in dromedary camels from different bioclimatic areas of Tunisia.Results clearly indicate evidence of Anaplasma infection in camels from all studied localities with an average prevalence of 17.7% (minimum 14.8% in Sidi Bouzid and maximum 31.3% in Bouficha).This is the first estimate of the molecular prevalence of Anaplasma spp. in Tunisian camels.Despite the important difference in bioclimatic characteristics between the three investigated areas, the difference in prevalence rates is not statistically significant (p > 0.05) (Table 2).This is probably due to the frequent camel movement between these areas as well as the similarity of tick populations infesting camels in sampling locations [29].
Compared to other countries, the overall prevalence rate in Tunisia remains higher than that in Spain (0%) [35], and appreciably lower than that in Saudi Arabia (95.5%) [42].In Spain, a 3% Anaplasma spp.prevalence was established in camels by serology [35].This high discrepancy between prevalence rates may result from differences in tick control programs, farm management, husbandry practices, wildlife reservoir hosts, and/or abiotic factors.In fact, several studies have reported the variability of Anaplasma species prevalence in ruminants according to geographic location, associated with suitable tick habitats and animal management [10,28,43].Moreover, the infection rate of Anaplasma spp. was significantly higher in females compared to males (p = 0.027) (Table 2).This can be explained by the immunosuppression of females which may occur during pregnancy and lactation periods [41], which could last up to two years [44].
Notably, we failed to recover A. marginale, A. centrale, A. bovis, and A. phagocytophilum from investigated camels.It can be postulated that dromedaries are not relevant reservoirs for classified Anaplasma species in the studied regions, but alternative ruminants and other wild and domestic animal species could act as reservoir hosts in this area.The seroprevalence of A. phagocytophilum in the same animals was investigated in a previous study [29].Overall, 66 out of 226 camels (29.2%) were seropositive.The discrepancy between molecular and serological tests could be explained by cross-reactivity of the antigen used in serology with anti-cytoplasmic antibodies, as well as with other autoimmune antibodies and/or with antibodies related to other Anaplasma species closely related to those of A. phagocytophilum [17,18,45].Notably, previous studies reported a great degree of cross-reactivity in serological tests between Anaplasma species [46][47][48].
In the present study, H. dromedarii, H. impeltatum, and H. excavatum were collected from camels.These data are in agreement with what observed by Gharbi et al. [25], who reported the infestation of dromedaries by these tick species in Tunisia.All tick genera identified in investigated areas have never been reported as vectors of A. phagocytophilum, A. marginale, A. bovis, or A. centrale [36], suggesting that these tick species may be vectors of other Anaplasma species probably not yet classified.Further studies are needed to clarify the role of these tick species in transmission of Anaplasma species to camels in Tunisia.
The 16S rRNA gene is considered a sensitive molecular tool for the discrimination of Anaplasma species in phylogenic studies [3,49].Sequencing of 1,322 bp of the 16S rRNA gene isolated from randomly selected Anaplasma spp.-positive camels revealed four different and novel Anaplasma sp.variants.Alignment (Table 3) and percent sequence identity comparison (Table 4) of the 16S rRNA sequence variants obtained in this study suggests that Tunisian dromedaries are infected by Anaplasma strains genetically related to A. platys.Indeed, these sequence variants shared a similarity greater than 99% with the 16S rRNA sequences of the canine A. platys and related strains found in Chinese and Italian ruminants [10,17] (Tables 3 and 4).
Phylogenetic analysis of 16S rRNA partial sequences performed with Anaplasma sp.sequences isolated from camels and selected sequences of Anaplasma species obtained from GenBank confirmed what was observed by percent sequence identity comparison (Figure 2).In agreement with Ooshiro et al. [12], Liu et al. [10], Ybañez et al. [50], and Zobba et al. [17], the phylogenetic tree based on 1,322 bp of the 16S rRNA gene shows two main clusters, one containing A. phagocytophilum, A. platys, A. bovis sequences, and another containing A. marginale, A. centrale, and A. ovis sequences.Anaplasma sp.variants isolated from Tunisian dromedaries cluster with A. platys and related strains (Figure 2A).
A. platys, the etiologic agent of canine infectious cyclic thrombocytopenia, has been associated with thrombocytopenia and anemia [17,18].In this study, randomly selected dromedaries did not show any symptoms specifically referable to A. platys infection.Therefore the A. platys-like strains isolated in camels might not be pathogenic and not cause any symptoms, as previously observed in ruminants from China and Italy [10,17] and in cats from Italy [18].

Conclusions
This paper reports the presence of novel Anaplasma sp.strains genetically related to A. platys in dromedaries from various bioclimatic areas of Tunisia.Findings open new concerns about the specificity of the direct and indirect diagnostic tests routinely used to detect different Anaplasma species in ruminants and provide useful molecular information to elucidate the evolutionary history of bacterial species related to A. platys.Further studies are needed to investigate if these A. platys-like strains infect other animal species in Tunisia, to better characterize these different strains by more discriminative genes, and to identify vectors implicated in the transmission of the potentially novel Anaplasma to which these strains could be ascribed.Sequence variants from this study represented in bold and marked with asterisks.Numbers associated with nodes represent the percentage of 1,000 bootstrap reiterations supporting the nodes (only percentages greater than 50% were represented).The host or vector, the strain or isolate name, the country of origin and the GenBank accession number are indicated

Figure 1 .
Figure 1.Map of Tunisian studied regions This work was supported by the Laboratoire d'épidémiologie d'infections enzootiques des herbivores en Tunisie (LR02AGR03), funded by the Ministry of Higher Education, Scientific Research and Information and Communication Technologies of Tunisia, and the research project Epidémiologie de maladies bactériennes à transmission vectorielle des herbivores (06-680-0029), which was funded by the Ministry of Agriculture of Tunisia.The authors would like to thank Dr. Mounir Aloui, Dr. Amen Allah Djaïem, Dr. Mohamed Bayoudh, and Dr. Bacem Hadj Mohamed for their help in blood sampling and tick collection.

Figure 2 .
Figure 2. Phylogenetic trees of Anaplasma species inferred with partial sequences (1,322 and 763 bp for A and B, respectively) of the 16S rRNA gene using the neighbor-joining method showing the location of the four new sequences from Tunisian camels

Table 1 .
Primers and/or probes used for detection and/or characterization of Anaplasma spp., A. platys-like, A. phagocytophilum, A. marginale, A. centrale, and A. bovis in camels in the present study [38]ple PCR allowing the detection of all Anaplasma species; 2 Second PCR, performed after the Simple PCR, allowing the specific species detection of A. phagocytophilum and A. bovis;3Primers used in PCR reaction for the detection of A. phagocytophilum and A. bovis;4The quencher dye fluorophore for the A. marginale probe was modified on 6-carboxyl-tetramethyl-rhodamine (6TAMRA) instead of Black Hole Quencher 1 (BHQ1) used by Carreli et al.[37];5The reporter and quencher dye fluorophores for the A. centrale probe were modified on 4,7,2′-trichloro-7′-phenyl-6-carboxyfluorescein (VIC) and 6carboxyl-tetramethyl-rhodamine (6TAMRA) instead of Texas Red and Black Hole Quencher 2 (BHQ2), respectively used by Decaro et al.[38].

Table 2 .
Factors associated with molecular prevalence of Anaplasma spp. in camels from Tunisia 1CI: 95% confidence interval; * Significant test.

Table 3 .
Nucleotide diversity among 16S rRNA sequences (1,322 bp) from Anaplasma sp.related to A. platys isolated from camels and other Anaplasma species found in GenBank 3Numbers represent the nucleotide position with respect to the clone J3 from China for Anaplasma sp.related to A. platys (GenBank accession number JN558826); Conserved nucleotide positions are indicated with asterisks.Nucleotides: T: thymine; C: cytosine; G: guanine; A: adenine.

Table 4 .
Comparison of 16S rRNA sequences (1,322 bp) from Anaplasma sp.related to A. platys isolated from camels and other Anaplasma species found in GenBank.The numbers represent the nucleotide identity rates found between the sequences.