Molecular detection and phylogeny of Ehrlichia canis and Anaplasma platys in naturally infected dogs in Central and Northeast Thailand

Background and Aim: Ehrlichia canis and Anaplasma platys are tick-borne, Gram-negative bacteria that cause canine monocytic ehrlichiosis and canine cyclic thrombocytopenia, respectively. These diseases are of great importance and are distributed globally. This study aimed to create new primers for the identification of E. canis and A. platys in naturally infected dogs using polymerase chain reaction (PCR), DNA sequencing, and phylogenetic analysis using the 16S rDNA and gltA genes. Materials and Methods: In total, 120 blood samples were collected from dogs in three different locations (Saraburi, Buriram, and Nakhon Ratchasima provinces) in Central and Northeast Thailand. The molecular prevalence of E. canis and A. platys was assessed using PCR targeting the 16S rDNA and gltA genes. All positive PCR amplicons were sequenced, and phylogenetic trees were constructed based on the maximum likelihood method. Results: Ehrlichia canis had an overall molecular prevalence of 15.8% based on the 16S rDNA gene, compared to 8.3% based on the gltA gene. In addition, the overall molecular prevalence of A. platys using the 16S rDNA gene was 10.8%, while the prevalence rate was 5.8% using the gltA gene. Coinfection was 0.8% in Saraburi province. The partial sequences of the 16S rDNA and gltA genes of E. canis and A. platys in dogs in Central and Northeast Thailand showed 96.75%–100% identity to reference sequences in GenBank. Phylogenetic analysis of the 16S rDNA and gltA genes revealed that E. canis and A. platys sequences were clearly grouped into their own clades. Conclusion: This study demonstrated the molecular prevalence of E. canis and A. platys in Central and Northeast Thailand. The 16S rDNA and gltA genes were useful for the diagnosis of E. canis and A. platys. Based on the phylogenetic analysis, the partial sequences of the 16S rDNA and gltA genes in E. canis and A. platys were related to prior Thai strains and those from other countries.


Introduction
Ehrlichia canis and Anaplasma platys are Gram-negative, obligate, intracellular, tick-borne bacteria in the order Rickettsiales and family Anaplasmataceae [1,2]. Ehrlichia canis and A. platys are important blood pathogens of dogs worldwide, especially in tropical and subtropical areas, and have recently been considered as zoonosis [3,4]. Ehrlichia canis and A. platys are the causative agent of canine monocytic ehrlichiosis (CME) and canine infectious cyclic thrombocytopenia (CCT), respectively [5,6].
The prevalence of E. canis in Thailand is in the range of 1.3%-38.3% [7][8][9][10][11][12], while the prevalence of A. platys in Thailand is in the range of 4.4%-30.6% [7-9, 11, 12]. Similar distributions of E. canis and A. platys have been reported in dogs in East and Southeast Asia [13,14]. The CME can be characterized into three phases: Acute, subclinical (usually without clinical signs), and chronic. The clinical signs of a dog infected with E. canis may vary from asymptomatic to severe life-threatening disease [3,14]. Symptoms of CME include depression, lethargy, high fever, anorexia, weight loss, pale mucous membranes, enlarged lymph nodes, petechiae caused by low platelets, anemia, bleeding, splenomegaly, hepatomegaly, lymphadenomegaly, and blindness [1,15]. Cyclic thrombocytopenia caused by A. platys infection usually has mild or asymptomatic clinical signs [15,16]. However, coinfections with E. canis can lead to severe thrombocytopenia [17]. Available at www.veterinaryworld.org/Vol.15/December-2022/ 16.pdf The diagnosis of E. canis and A. platys infection can be performed based on blood smear examination under a 1000× light microscope. The morula stage of E. canis can be found in the monocytes and macrophages of infected dogs, whereas the morula of A. platys can be detected in the platelets of dogs [18,19]. Although this method is easy to perform, it has low sensitivity, time-consuming, and requires experienced personnel to correctly identify the pathogens [9,11,17]. In addition, serological methods, such as indirect immunofluorescence assay and the enzyme-linked immunosorbent assay, have been widely used to diagnose E. canis and A. platys infection [6,18]. These methods require specific equipment and may have specificity problems due to cross-reactions with other pathogens [17]. Polymerase chain reaction (PCR) is a highly sensitive and specific molecular method used for E. canis and A. platys detection [4,20] and can be further used for phylogenetic analysis. Phylogenies are crucial tools for analyzing the evolutionary connections among different species or genes [21]. There have been recent reports on the use of 23S rDNA [22], 16S rDNA [22,23], heat-shock operon (groESL) [22], and the gltA [22,23] genes for phylogenetic analyses and characterization of E. canis and A. platys strains. The 16S rRNA gene has been most commonly used for identifying Ehrlichia spp. [21]. Phylogenetic analysis of the gltA gene, the gene that encodes enzymes of the tricarboxylic acid cycle [24], exhibited higher variation among Ehrlichia and Anaplasma spp. [25,26]. Therefore, gltA is one of the best genes for phylogenetic analysis of Ehrlichia species [26]. Previously, phylogenetic tree construction of E. canis and A. platys has been carried out in dogs based on the 16S rDNA and gltA genes in many countries, such as the Philippines [27], Cuba [25], and China [28]. The 16S rDNA and gltA nucleotide percentage identities vary from 99% to 100%. E. canis and A. platys are closely related genera that are commonly found coinfected [22]. However, there have been few reports on phylogenetic and epidemiological studies of E. canis and A. platys in Thailand, and no studies have been conducted in Saraburi, Buriram, and Nakhon Ratchasima provinces of Central and Northeast Thailand.
This study aimed to develop new primers for the detection of E. canis and A. platys in naturally infected dogs in Central and Northeast Thailand using PCR and to conduct phylogenetic analysis of E. canis and A. platys using the 16S rDNA and gltA genes.

Ethical approval
This study was approved by the Animal Ethics Committee of the Faculty of Veterinary Technology, Kasetsart University, Bangkok, Thailand (ACKU62-VTN-0011).

Study period and location
The samples were obtained from free-roaming, owned dogs from January 2021 to June 2022.
The samples were collected from three different locations in Central and Northeast Thailand. The sample were processed at Kasetsart University, Bangkok, Thailand.

Dog blood samples collection and genomic DNA extraction
The sample size was estimated using EpiTools (https://epitools.ausvet.com.au/) based on the previous prevalence [9]. To study both E. canis and A. platys, an estimated proportion of 0.044 has been used. The calculated sample size was 112 samples. In this study, 120 dog blood samples were obtained for better representation and to prevent data loss. Blood samples were collected from three different locations in Central and Northeast Thailand from Saraburi (n = 50), Buriram (n = 36), and Nakhon Ratchasima (n = 34) provinces ( Figure-1). Blood samples (2 mL) were collected from the cephalic vein or saphenous vein and kept in ethylenediaminetetraacetic acid (EDTA) tubes, stored at −20°C until used. Blood smears were prepared on the day of collection. Positive control was made up of a sample that was positive for E. canis in a blood smear and confirmed using PCR as described by Wichianchot et al. [29]. Sterile distilled water was used as a negative control. Genomic DNA was extracted from 300 μL of each EDTA blood sample using a Genomic DNA Mini Kit (Geneaid ® , New Taipei, Taiwan), according to the manufacturer's instructions. The DNA was stored at −20°C until use and the DNA concentration was assessed using a Nanodrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Primer design
The Primer3 software (https://bioinfo.ut.ee/ primer3-0.4.0/) was used to create new PCR primers. The 16S rDNA sequences of E. canis (GenBank accession no. GU810149.1) and A. platys (GenBank accession no. MK121782.1) and the gltA gene sequences of E. canis (GenBank accession no. AF304143.1) and A. platys (GenBank accession no. EU516387.1) were retrieved from the NCBI nucleotide database and used as DNA templates. The sequences for oligonucleotide primers and their predicted annealing temperatures as well as PCR product sizes are presented in Table-1.

Amplification of E. canis and A. platys of 16S rDNA and gltA genes
Polymerase chain reaction (PCR) was performed to detect the 16S rDNA and the gltA genes of E. canis and A. platys. All PCR reactions were prepared in 50 μL total volume in a 0.2 mL PCR tube, with each reaction containing 1X DreamTaq Green buffer (Thermo Scientific), 0.2 mM dNTP, 1 μM PCR primer, 1.25 units of DreamTaq DNA polymerase (Thermo Scientific), and 2 μL of DNA sample. The PCR conditions were as follows: All amplifications involved initial denaturation at 95°C for 2 min, 40 cycles of denaturation at 95°C for 30 s, annealing at 57°C for 30 s (for E. canis 16S rDNA), 54°C for 30 s (for E. canis gltA and A. platys 16S rDNA), or 58°C for 30 s (for A. platys gltA) followed by the extension step at 72°C for 30 s. Then, all amplifications were completed with a final extension at 72°C for 5 min. The PCR products were resolved on a 2% agarose gel with DNA gel stain to identify predicted amplicons using the gel documentation system. All positive PCR amplicons were purified using a commercial gel extraction kit (Geneaid ® ) according to the manufacturer's instructions and quantified using a Nanodrop spectrophotometry. Polymerase chain reaction products were sequenced from both ends using the Sanger method with a 3730XL automatic sequencer (Applied Biosystems, Foster City, CA, USA).

Sequence analysis and phylogenetic tree construction
Nucleotide sequences were analyzed on the basis of the BlastN suite, available from the National Center for Biotechnology Information website (https://blast. ncbi.nlm.nih.gov/Blast.cgi). The similarity of nucleotide sequences was compared to the highest score hit.

Statistical analysis
Univariable analysis was performed using McNemar's Chi-squared test for prevalence comparison between the 16S rDNA and gltA genes. The statistical analysis was performed using the STATA software package version 15.1 (Stata Corporation, College Station, TX, USA). Results were considered significantly different for p < 0.05.

Phylogenetic analysis
A total of 19, 10, 13, and 7 positive sequences were used for phylogenetic tree construction based on the ML method for E. canis 16S rDNA, E. canis gltA, A. platys 16S rDNA, and A. platys gltA, respectively.
The data indicated that sequences of E. canis 16S rDNA in this study were closely related to other E. canis sequences obtained from the USA, Turkey, Japan, India, Nigeria, Italy, Cuba, Spain, Brazil, Greece, Thailand, and China (GenBank accession numbers U26740, AY621071, AF536827, JX861392, JN982339, EU439944, MK507008, KC479022, EF195135, EF011110, EU263991, and MW412717, respectively) ( Figure-2) with the percentage of identity ranged from 99.59% to 100% (Table-3). The tree also showed that all E. canis sequences in this study were clustered in one clade, separated from E. muris, E. chaffeensis, E. ewingii, E. ruminantium, and R. rickettsii. The phylogenetic tree derived from the gltA gene showed that E. canis sequences in this study were clustered into one clade with other E. canis sequences from Italy, the USA, the Philippines, Thailand, Spain, and China (GenBank accession numbers AY647155, AF304143, JN391409, KU765198, AY615901, and MW428302, respectively) ( Figure-3) with the percentage of identity ranged from 98.41% to 100% (Table-4). All E. canis sequences in this study were clustered in one clade, separated from E. muris, E. chaffeensis, and E. ewingii, E. ruminantium, and R. rickettsii.

Discussion
In this study, we developed new primers for E. canis and A. platys detection using the 16S rDNA and gltA genes and used both genes for phylogenetic analysis. The overall prevalence rates using the 16S rDNA gene (15.8% for E. canis and 10.8% for A. platys) were greater than those determined solely using the gltA gene (8.3% for E. canis and 5.8% for A. platys). Although only the overall prevalence between the 16S rDNA and gltA genes of E. canis differed significantly (p = 0.0027), we suggest that 16S rDNA is the better option for identifying species for both pathogens due to the higher detection rate. Molecular identification of E. canis and A. platys was performed in dog samples from three different locations (Saraburi, Buriram, and Nakhon Ratchasima provinces) in Central and Northeast Thailand. In this study, the overall prevalence of E. canis using the 16S rDNA (15.8%) was higher than previously reported in Khon Kaen (1.3%) province, Thailand [8], but lower than previously reported from several parts of Thailand, including Bangkok (38.3%) [7], Maha Sarakham (21.5%) [10], Kalasin (25%) [11], and Buriram (36.7%) [12] provinces. The overall molecular prevalence of A. platys based on the 16S rDNA gene was 10.8%; however, this prevalence was lower than previously reported in Bangkok (13.9%) [7], Buriram (30.6%) [12], and    [11] provinces, Thailand. The fluctuation in the prevalence percentage was possibly due to the living environment of the dogs [30]. The coinfection rate of E. canis and A. platys (0.8%) in this study was lower than our previous study in Buriram (14.2%) [12] and Kalasin (11.8%) [11] provinces, Thailand. In addition, coinfection rates of E. canis and A. platys in this study were lower than reported in studies in Saint Kitts (19%) [6], Grenada (4.5%) [22], and Nicaragua (4.7%) [31]. All dogs in this study showed no clinical symptoms of blood pathogen infection but several of them tested positive for either E. canis (15.8%) or A. platys (10.8%) using 16S rDNA detection. These dogs may act as potential sources of zoonotic infection because it appears that most infections are asymptomatic [16]. Thailand is in a tropical area where infections with rickettsial pathogens are common. The results showed that the prevalence of E. canis and A. platys in this study was comparable to other tropical countries such as Indonesia, Malaysia, and Philippines [30,32], India [33], Argentina [34], and Brazil [35].
The partial sequences of the 16S rDNA and gltA genes of E. canis and A. platys in dogs in Central and Northeast Thailand were above 99.59% (16S rDNA) and 98.41% (gltA) identity to genotypes in GenBank. The current phylogenetic analysis for the 16S rDNA and gltA genes of E. canis and A. platys agreed with the taxonomic separation of members of the family Anaplasmataceae into the Ehrlichia and Anaplasma genera [16]. The phylogenetic analysis of 16S rDNA and gltA genes demonstrated that E. canis sequences are clustered tightly in E. canis subclade, whereas E. muris, E. chaffeensis, E. ewingii, E. ruminantium, and R. rickettsii clustering into their own subclades ( Figures-2 and 3). However, it should be noted that even though E. canis were grouped in one clade; we found a small variation among E. canis 16S (MW412717) and gltA (MW428302) from China. For this specific sample from China, 12 widespread base substitutions were found in the DNA alignment to other E. canis gltA sequences. Other studies also reported no heterogeneity among E. canis groups using the 16S rDNA gene [36]. A previous study in Thailand also showed that E. canis strains were linked with multiple connected branches and found little genetic diversity, suggesting slow and homogeneous evolution [17].
Anaplasma platys 16S rDNA phylogenetic trees revealed that all of A. platys sequences from this study were clustered in the same clade with sequences from other countries (Figures-4 and 5). However, we found a small variation among A. platys 16S rDNA sequences from Saraburi and Buriram (AG026, AG048, AG050, and B11) as subclades in the tree. Although another sequence sample from Cuba (MK506833) contained  a base substitution to other A. platys 16S rDNA sequences, a high similarity of 96.75%-100% was observed in A. platys 16S rDNA sequences in concordance with a recent study in Khon Kaen province, Thailand [20]. The same pattern was also observed in A. platys gltA sequences (98.94%-100% similarity) which were clearly grouped into one clade with other A. platys gltA sequences.

Conclusion
Using new PCR primers targeting the 16S rDNA and gltA genes, this study provided the first molecular prevalence and phylogeny of E. canis and A. platys in asymptomatic dogs from Saraburi, Buriram, and Nakhon Ratchasima provinces, Central and Northeast Thailand. Phylogenetic analysis of the 16S rDNA and gltA genes showed that E. canis and A. platys in Thailand were highly related to sequences from other countries. Future investigations on the genetic diversity of E. canis and A. platys should be conducted in different regions of Thailand.