Molecular Detection of Some Anaplasma Species in Blood of Dogs in Baghdad Province , Iraq

A total of 150 blood samples were collected from dogs and examined by the polymerase chain reaction (PCR) technique, which was used to detect the 16S RNA gene of Anaplasma platys and Anaplasma phagocytophilum. Subsequent analysis of the PCR amplicons was achieved by nucleotides sequencing of some positive samples. Totally, the findings show the presence of PCR products (i.e., Anaplasma spp. infection) in 12/150 (8.0%) of the dogs under study. While 5/150 (3.33%) of the cases were A. platys, 7/150 (4.66%) were A. phagocytophilum. Nucleotide sequencing confirmed the identity of the amplified genes whose sequences were compared with other references belong to 15 of 16S rRNA gene of A. platys and 14 references of 16S rRNA gene of A. phagocytophilum, and the isolate sequences of this study were deposited on the Gene Bank. The identity and similarity scores between the isolates of this study and reference strains ranged from 98 to 99%. In conclusion, canine anaplasmosis prevalence in dogs could be underestimated in Iraq, and the phylogenetic tree of the local A. platys and A. phagocytophilum isolates were found to resemble other worldwide strains of Anaplasma spp. with a high degree of similarity.


Introduction
Dogs are among the most common pets which can be influenced by many tick-borne diseases of parasitic, bacterial, and viral origins resulting in considerable impacts on the health (1)(2)(3). Anaplasma phagocytophilum and A. platys, two bacterial pathogenic agents belonging to Anaplasma taceae family of Rickettsia in less order, are known to infect livestock and domestic animals including dogs (4). Canine anaplasmosis caused by A. phagocytophilum is characterized primarily by granulocytic vacuoles (canine granulocytic anaplasmosis); whereas, A. platys specifically infects the platelets resulting in cyclic thrombocytopenia (canine thrombocytopenic anaplasmosis) (5,6).
Recently, many molecular assays are available commercially for diagnosis of canine anaplasmosis such as polymerase chain reaction (PCR) that demonstrated a high efficacy in diagnosis of acute as well as chronic infection (7,8). Nucleic acid (DNA or RNA) based sequencing methods are greatly effective tools, which were broadly used for both diagnosis and characterization of these bacteria (9,10). This study aimed to detect the incidence of A. platys and A. phagocytophilum in blood of dogs in Baghdad province by PCR technique in Iraq where neither molecular nor sequencing data are available on these bacteria in dogs.

Blood Sample Collection
Whole blood samples were collected from the cephalic vein of 150 dogs into EDTA anticoagulant tubes. The samples were transferred in a cooling box to the research laboratory at the Department of Internal and Preventive Veterinary Medicine, College of Veterinary Medicine, University of Baghdad. The blood samples were used for the molecular diagnosis of Anaplasma spp. The study was carried out from October 2018 till April 2019and approved by the Animal Care and Use Committee.

DNA Extraction
Genomic DNA from the whole 150 blood samples was extracted using a G-spin DNA extraction kit (Cat. No. 17045, Life Science/Korea). This kit was used for DNA extraction from the bacteria in a short time, and company instructions were followed. Concentration of genomic DNA was checked by using a Nanodrop spectrophotometer. The DNA purity (absorbance at 260/280 nm) was also checked.

Primers Used
The primers used for amplification of the 16S rRNA gene of A. platys and A. phagocytophilum from blood samples of dog are shown in Table 1. They were produced by IDT (Integrated DNA Technologies company, Canada).

PCR Reaction
The PCR reaction mixture involved the use of iNtRONs Maxime PCR PerMix kit. The components of the kit included: i-Taq DNA Polymerase5U/µl, dNTPmixture 2.5 mM, reaction buffer 1X, gel loading buffer1X. The PCR working solution in a final volume of 25µlincluded: 5µl of Taq PCR PreMix,10 picomoles/µl (1 µl) of each of forward and reverse primers,1.5µl of the DNA templates well as 16.5 µl of distilled water.

PCR Conditions
The conventional PCR thermocycler was used in this study, and the conditions of the reactionincludedinitialdenaturationat94 °C for 8 min and 1 cycle, followed by the second denaturation at 94 °C for 45 sec, annealing at 58 °C for 45 sec and extension at 72 °C for 45 sec, and these three steps were repeated for 35 cycles to end with the final extension at 72 °C for 5 min.

Agarose Gel Electrophoresis of DNA
The PCR products were analyzed by agarose gel electrophoresis. The agarose gel was made in 1.5%concentration by melting 1.5 g of agarose in 100 ml of previously made TBE buffer. The gel was heated to boil, then left to cool down to 45-50ᵒC. Afterwards, the gel was poured in its tray, a comb was placed into its suitable place to make holes that would hold the samples, and finally the gel was left to solidify at room temperature for approximately 30 min. Later, the comb was removed gently from the solid gel, and the gel tray was fixed inside electrophoresis tank filled with TBE buffer that covered the gel surface.

Preparation of Samples
Before loading the DNA samples into the agarose gel, 3 µl of the processor loading buffer (Intron/Korea) were mixed with 5 µl of each DNA sample to be electrophoresed. Following mixing, the samples were loaded into the holes of the gel. Then, the electrophoresis was run at an electric current of 7 V/c2 for 1-2 h. The gel was tested by a source of UV light after placing the gel in pool containing 3µl Red safe nucleic acid staining solution to make DNA bands visible.

Results
The DNA concentration of the samples ranged between 1.6-1.9 ng/L. All isolates from which the DNA was extracted showed bands of approximately 1500 bp of the proposed 16S rRNA gene amplicon on the agarose gel ( Figure  1). Figure 2 shows the results of the PCR amplification to the 16S rRNA gene of A. platys.

Phylogenetic of A. phagocytophilum
The phylogenetic tree analysis based on the partial sequence of the 16SrRNAgene in the local A.phagocytophilum of dogs isolates analyzed the genetic relationship of this Anaplasma spp.. When comparing between A. phagocytophilum isolated from dogs of this study with other strains from different sources, including: (ID:  HM366584.1, GQ450276.1 ), the degree of identify and similarity among our isolates with reference strain ranged from 99% to 100% (Figure 3).

Phylogenetic Tree of A.platys
The phylogenetic tree based on the partial sequence of the16SrRNAgene in the local A.  AY530806.1), the degree of identify ranged from 99% to 100% as seen in Figure 4.

Discussion
The results of the present study reported low anaplasmosis infection rate (12/150; 8.0%) in dogs of Baghdad city using PCR. While the cases infected with A. platys were 5/150 (3.33%), 7/150 (4.66%) of the cases were infected with A. phagocytophilum. When comparing the results with previous studies carried out in other countries, the overall prevalence of canine anaplasmosis obtained from the present study was compatible with many other studies, such as that of Turkey where the infection rates obtained using the PCR were 4.0% for A. phagocytophilum and 6.0% for A. platys (11). In Colombia, A.platys registered 2.2%, and A. phagocytophilum accounted for 3.3% out of 91 sampled dogs (12). The infection rates of anaplasmosis in dog population in different areas of the world have been investigated. These studies have reported higher prevalence of A. phagocytophilum in the world than that observed in Iraq, in which the infection rate was 2.62% (12,13). In Italy, 1.25% of the samples were positive for A. phagocytophilum using PCR (13,14). In China, the prevalence of this bacterium in stray dogs was 0.4% (15). Concerning A. platys, the rate of infection in Qatar was 1.6% (16). It was found that 4.0% of dogs in Sicily, Italy were infected with A. platys (17). The variations in worldwide prevalence of anaplasmosis can be associated with the infection status, differences in climate and ecology, distribution of vectors, socioeconomic factors, and the tests used to evaluate infection, such as nested PCR or Real time PCR (18). Therefore, it is expected that the source of anaplasmosis in dogs might come from importing animals from neighboring and non-neighboring countries of Iraq as a high degree of homology was observed between the sequences described in the literature and those determined in the present study. In addition, the phylogenetic analysis of Anaplasma spp. by using the 16S rRNA gene indicated that all of these strains might have similar genealogical species relationships, and these findings are in agreement with (19,20). In conclusion, canine anaplasmosis prevalence in dogs could be underestimated in Iraq and the phylogenetic tree of the local. A. platys and A. phagocytophilum isolates resemble other worldwide strains of Anaplasma spp. with a high degree of similarity.