Diagnosis andmolecular characterization of Anaplasma platys in dog patients in Yogyakarta area , Indonesia

Anaplasma platys is a tick-borne, Gram-negative bacterium that causes anaplasmosis, a companion vector-borne disease impacting dogs. Information on this disease remains limited in Indonesia. Its symptoms are not specific, so molecular analysis is required for a rapid and accurate diagnosis. GroEL is an essential gene commonly used for classification and species identification of many groups of bacteria, including Anaplasma spp. In this study, a molecular diagnosis of anaplasmosis based on the groEL gene sequence was conducted using PCR. In addition, the genetic diversity of Anaplasma platys in infected dogs was determined. Blood samples were collected from 51 dogs suspected of anaplasmosis from Prof. Dr. Soeparwi Animal Hospital, animal clinics, andpet shops in theYogyakarta area, Indonesia, basedonanamnesis, histories of tick infestations, and clinical symptom examinations. DNA extraction and PCR targeting the groEL gene were performed, followed by sequencing. Phylogenetic tree analysis and construction were carried out using the BLAST and MEGA programs. Positive PCR sample results (amplicon length of 624 bp) were found in 6 of 51 dogs. Samples A1 (KHJ/C2), A2 (KHJ/A2), A3 (KSK/L), A4 (KHJ/L), and A5 (KNP/M2) had close ties to Anaplasma platys (AF478129.1) from GenBank. Phylogenetic analysis showed a very high homology value (100%) andbootstrap value of 100%. It can be concluded that therewas no genetic diversity in theAnaplasma platys found in infected dogs in the Yogyakarta area.


Introduction
Anaplasmosis in dogs is caused by the infection of Anaplasma phagocytophilum and Anaplasma platys (Sykes and Foley 2013).Anaplasma platys, which was previously known as Ehrlichia platys, is reported to cause canine cyclic thrombocytopenia in warm, tropical regions, such as the Mediterranean, Asia, Middle East, Africa, and Australia.The presence of Anaplasma in platelets is characterized by the presence of round, oval or peanut-shaped blue inclusion cells that have diameters ranging from 0.35 to 1.25 μm (Lillini et al. 2006;Carrade et al. 2009).Anaplasma platys is transmitted by the brown dog tick (Rhipicephalus sanguineus) and Dermacentor spp.tick.Co-infection can occur together with other pathogenic agents transmitted by the same or other tick species.Thus, it will affect the clinical manifestations of diseases (Sykes and Foley 2013).According to Arraga-Alvarado et al. (2014), like other Anaplasma species, A. platys is zoonotic and therefore can infect humans.
The clinical symptoms of A. platys infection in dogs have been described both experimentally in the laboratory and naturally in the field by researchers from Greece (Kontos et al. 1991), France (Beaufils et al. 2002), and Israel (Harrus et al. 1997).The symptoms are high fever, lethargy, anorexia, weight loss, pale mucous membranes, petechiae, nasal discharge, and lymphadenomegaly (Santos et al. 2009;Dyachenko et al. 2012).The incubation period of A. platys infection in dogs lasts for 1-2 weeks.The incubation period will continue to thrombocytopenia and fever, which appear and disappear cyclically every 1-2 weeks (Gaunt et al. 1990).
The diagnosis of anaplasmosis depends on detecting the presence or exposure of an infectious agent (de Farias Rotondano et al. 2012).Blood smear examination has a low sensitivity because the stage of morulae A. platys can only be detected in the initial phase of infection (Otranto et al. 2010).Serological tests such as indirect immunofluorescence are commonly used, but this examination sometimes interferes with cross-reaction of antibodies Faizal et al. Indonesian Journal of Biotechnology 24(1), 2019, 43-50 between Anaplasma species (Beaufils et al. 2002;Greene 2012).The polymerase chain reaction (PCR) method can make it possible to detect anaplasmosis active infections, because this method can directly amplify the presence of A. platys based on 16S rRNA, groEL, or msp2 gene targets (Fuente et al. 2006;Matei et al. 2016;Vargas-Hernandez et al. 2016;Lee et al. 2017;Ribeiro et al. 2017).GroEL protein is a part of the heat shock protein-group (HSP) (Yu et al. 2001).Heat shock proteins are regulated in physiological stressful situations, for example during an increase in temperature or toxicity, therefore this protein serves as a cell protection tool (Dasch et al. 1990).The sequence of genes is considered a useful tool for phylogenetic analysis of Anaplasma spp.(Dasch et al. 1990;Jahfari et al. 2014).The 'blind spot' in some genera makes the 16S gene not discriminatory enough to identify certain species (highly conservative), so that the groEL gene can support and expand phylogenetic results (Dasch et al. 1990;Jahfari et al. 2014).The PCR method can also be used to detect specific gene fragments after the amplification process.The sequencing process of PCR results can identify specific infecting species of Anaplasma spp.(Ybañez et al. 2012(Ybañez et al. , 2016;;Bonilla et al. 2017).
Anaplasmosis is an important zoonotic disease.Opportunistic infections can occur in humans and dogs, which will aggravate the patient's condition until death (Sykes and Foley 2013).Difficulties in diagnosing this disease also need to be considered, because it can cause significant economic losses due to the administration of drugs that are less precise and continuous.Studies on the diagnosis of anaplasmosis in dogs in Indonesia have not been widely carried out.Limited research by Hadi et al. (2016), on the prevalence of anaplasmosis in dogs, has been reported in several cities, namely Bogor, Jakarta, and Bandung.However, the specific species of Anaplasma spp.which infects the dogs was not specifically identified.The aim of this study was to conduct a molecular diagnosis of anaplasmosis based on the groEL gene sequence using the PCR method.In addition, this study was also intended to study the genetic diversity of A. platys species in infected dog patients from the Yogyakarta area, Java, Indonesia.This study is expected to be beneficial as a standard reference in diagnosing anaplasmosis in dogs more accurately in order to control the spread of anaplasmosis in Indonesia.

Sample
This study was conducted in March-November 2018.It was approved by the Ethical Committee of the Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yo-gyakarta, Indonesia, No: 034/EC-FKH/Int./2018(issued on November 27, 2018).Blood samples were collected from 51 dogs with clinical symptoms and were suspected to have been infected by A. platys.The dog patients showed symptoms such as fever, weakness, and haemorrhage.The supporting data were that the dogs were also infected by ticks, or previously reported to have a history of tick infection.The blood samples were collected from dog patients in Prof. Dr. Soeparwi Animal Hospital Faculty of Veterinary Medicine Universitas Gajah Mada, several animal clinics, and pet shops in the Yogyakarta area.

Anamnesis and physical examination
Anamnesis was done by interviewing the animal owners or nurses to obtain detailed information regarding the history of previous diseases suffered by the dog patients which were related to tick infection.Physical examination was carried out starting from an examination of body temperature, state of mucous or conjunctival membranes, the examination of hair condition (hair loss/presence of ticks), and observation of behavior (weakness/lethargy) (Kelly 1984;Beaufils et al. 2002).

Blood sample collection
Blood samples were taken through antebrachii cephalica veins as much as 0.5-2.5 mL using a 1 mL syringe (One Med, Indonesia) for small dog breeds and 3 mL syringe (One Med, Indonesia) for large dog breeds.Blood samples were collected into Vaculab tubes (One Med, Indonesia) with EDTA K3 as an anticoagulant (Beaufils et al. 2002), and then were stored at 4°C, tightly closed, and protected from light.

DNA isolation
Two hundred microliters of blood samples from dog patients were extracted using gSYNC DNA Extraction Kit (Geneaid Biotech Ltd., Taiwan).The procedure for DNA extraction from the blood was carried out according to the standard method recommended by the manufacturer.

DNA amplification
Amplification of isolated DNA from the blood was carried out using pairs of forward and reverse primers with the groEL target gene (Table 1).DNA amplification using KAPA Taq PCR Kits (Kapa Biosystems, South Africa) was done by mixing into the microtube as much as 6.5 µL H 2 O, 1 µL forward primer, 1 µL reverse primer, 4 µL DNA, and 12 µL KAPA Taq PCR Kits (Kapa Biosystems, South Africa) so that the final volume was 25 µL.All of these components were mixed until the mixture was ho- EphplgroEL-R TCTACTCTGTCTTTGCGTTC mogeneous.The microtube then was inserted into Thermal Cycler (GTC96S, Cleaver Scientific Ltd., UK) under the following conditions: initial denaturation at 94°C for 5 min, followed by denaturation at 94°C for 45 s, annealing at 56°C for 45 s, and extension at 72°C for 60 s.This process was carried out repeatedly for 40 cycles, followed by the final extension at 72°C for 10 min and ended with a final hold at 4°C.

DNA electrophoresis
The DNA electrophoresis started by the preparation of 1.5% agarose gel (0.75 g agarose in 50 mL Tri Borate EDTA / TBE buffer 1x).DNA samples of 5 μL and DNAloading dye (Geneaid Biotech Ltd., Taiwan) of 2 μL were loaded into the gel well.DNA ladder 100 bp markers (Geneaid Biotech Ltd., Taiwan) were included in the last well as much as 5 μL.Electrophoresis was carried out for 30-45 min with 100 volts.The gel was then visualized on a UV transilluminator.Analysis of amplification products was done based on the fragment size compared to the band position in the marker.

PCR product purification
Purification of PCR products was performed in PT Genetika Science, Jakarta, Indonesia, based on the standard protocol of the manufacturer.

Phylogenetic analysis
PCR products were sequenced at PT. Genetika Science, Jakarta.Then, the result of Anaplasma spp.groEL gene sequencing was analyzed using the Basic Local Alignment Search Tool (BLAST).The sequencing results of all samples were compared with groEL sequences of Anaplasma spp.obtained from GenBank using the Clustal W Algorithm multiple alignments methods.Phylogenetic trees were constructed based on the Neighbor-Joining method using Molecular Evolutionary Genetics Analysis (MEGA) software version 7.0.The analysis process was carried out by bootstrapping 1,000 times repetition; identification of genetic distance and the presence of nucleotide substitutions were analyzed according to the parameters of the Kimura-2 model.

DNA from dog blood samples were positive for groEL
The electrophoresis results from 6 dog samples showed the formation of DNA bands from PCR amplification which was parallel to the positive control at 624 bp (Figure 1).This shows that the primer attached to the groEL gene target which corresponded to the amplification length.The PCR results showed that the dog DNA samples studied were positive for Anaplasma spp.molecularly (Alberti et al. 2005;Bonilla et al. 2017).The PCR products of 51 dogs can be seen in Table 2. Five positive samples of unpurified PCR products (KHJ/C2, KHJ/A2, KSK/L, KHJ/L, and KNP/M2) were sequenced at PT. Genetika Science, Jakarta, Indonesia.Purification was carried out prior to the process of sequencing the PCR products.The electrophoresis result of the purified PCR products can be seen in Figure 2.

BLAST analysis
The sequencing results of the (purified) PCR products from five positive samples infected with Anaplasma spp.
were analyzed using the online Basic Local Alignment Search Tool (BLAST) on the National Center for Biotechnology Information (NCBI) website (https://blast.ncbi.nlm.nih.gov/Blast.cgi).The parameters observed included max score, total score, query coverage, identity, and E-value (Expectation value).Max score is the highest value between the query and the total segment in the database.The max score is generally the same as the total score.Query coverage is the percentage value of the total nucleotide length of the sample that is good enough to be aligned with the nucleotide sequence found in GenBank.Identity indicates the percentage similarity of the nucleotide sequences of samples that are aligned with the nucleotide sequence in GenBank.A higher value of the four parameters shows more similar sequence between query and database.E-value is the level of probability that the similarity between sequence pairs is the result of random events; lower E-value indicates more significant similarity with database sequence (Aprilyanto and Sembiring 2016).BLAST results from five samples on groEL gene forward primer Anaplasma spp.obtained the same maximum score and total score between 1059-1072 (high score).The value of the query cover is 97%, which means that as much as 97% of the length of the sequenced nucleotide sequence can be compared with the database.The E-value of all samples was 0.0.This indicates that the similarity between pairs was very convincing with 99% identity value.The homology level of all samples shows very high homology by producing flat and parallel lines.The details of the data from the BLAST results can be seen in Table 3.Nucleotide differences between sample sequences and GenBank could be identifies using Molecular Evolutionary Genetics Analysis (MEGA) software version 7.0.Nucleotide differences were analyzed between Anaplasma spp.groEL gene sequences from five samples; A1 (KHJ/C2), A2 (KHJ/A2), A3 (KSK/L), A4 (KHJ/L), and A5 (KNP/M2) and groEL gene sequence data from GenBank; Anaplasma platys (AF478129.1),Anaplasma phagocytophilum (KU519286.1),Ehrlichia canis (U96731.1),Wolbachia endosymbiont (EF468716.1),and Neorickettsia sp (Table 4).
The results of the analysis obtained the difference value between 0-198.A value of 0 states that there is no nucleotide difference, which means that there are genotypic similarities as shown between five samples (A1-A5) and Anaplasma platys (AF478129.1).Genetic distance is a genetic difference between species or between populations in one species.A small genetic distance or close to 0 value indicates a close ge-netic relationship, while a large genetic distance or close to a value of 1 indicates a distant genetic relationship.Genetic distance can be analyzed by using Molecular Evolutionary Genetics Analysis (MEGA) software version 7.0.Results of genetic distance analysis between sequences of groEL gene Anaplasma spp.from five samples; A1 (KHJ/C2), A2 (KHJ/A2), A3 (KSK/L), A4 (KHJ/L), and A5 (KNP/M2) with sequence data of the A. platys groEL gene from GenBank obtained a value of 0%.These results indicate that the five study samples had 100% homology with A. platys (AF478129.1)from GenBank, which suggests that there is no genetic diversity in A. platys infecting dogs in Yogyakarta, Indonesia.Data from the analysis are shown in Table 5.

Phylogenetic analysis
The phylogenetic tree was constructed using the Neighbor-Joining method using Molecular Evolutionary Genetics Analysis (MEGA) software version 7.0 (Kumar et al. 2016).Phylogenetic tree construction between groEL gene sequences of Anaplasma spp.from five samples;

Discussion
The GroEL protein is a part of the heat shock protein-group (HSP), and is also called eukaryotic HSP60 (based on its molecular weight of 60 kDa).Heat shock proteins are regulated in physiological stressful situations, for example during an increase in temperature or toxicity, therefore this protein serves as a cell protection tool.The groEL gene is one product of two genes (groEL ["L" means large] and groES ["S" means small]) that is united in the groESL gene.The sequence of genes is considered a useful tool for phylogenetic analysis of Anaplasma spp., especially in cases where analysis of 16S rRNA is limited due to high conservation, groEL genes can support and expand phylogenetic results (Dasch et al. 1990;Jahfari et al. 2014).Characterization based on groEL gene targets in diagnosing A. platys has been carried out in various countries such as the Philippines (Ybañez et al. 2012)), Taiwan (Yuasa et al. 2017), Italy (Fuente et al. 2006), and Venezuela (Huang et al. 2005).
According to Inokuma et al. (2002), the sequence determination of N Heat Shock Operon (groESL) genes and Citrate Synthase Gene (gltA) from A. platys for phylogenetic and diagnostic studies give results that groESL and gltA genes both have a greater variety of gene sequences compared to the 16S rRNA gene sequence.The specificity of these genes was examined using the DNA of three A. platys strains from different geographical locations-France, Japan, and Venezuela-and using DNA from nearby species, including A. phagocytophilum and A. marginale.The results showed that both PCR systems of groESL and gltA genes are specific to A. platys.The latest study in the Philippines by (Ybañez et al. 2016) regarding the phylogenetic analysis of A. platys using the groEL gene target reported thatA.platys co-infection of different variants has been reported.This was not found in phylogenetic analysis using the 16S gene target in the same study and had not even been explained in previous studies.
The phylogenetic analysis showed a close relationship between A. platys in Yogyakarta and A. platys from the Democratic Republic of Congo (DRC) in Africa.This indicated that there was a possibility that A. platys species from the African region can spread to Indonesia through the activity of importing animal trade or tourists entering to Indonesian territory if it was associated with the condition and clinical symptom of animals infected with A. platys tend to be asymptomatic (Sykes and Foley 2013).

Conclusions
The incidence of anaplasmosis in dog patients in Yogyakarta and its surrounding areas was confirmed based on molecular diagnosis using the PCR technique with the groEL gene target.Study samples A1 (KHJ/C2), A2 (KHJ/A2), A3 (KSK/L), A4 (KHJ/L), and A5 (KNP/M2) have a close kinship with Anaplasma platys (AF478129.1)from the Democratic Republic of Congo (DRC) in Africa.The results of a phylogenetic analysis show very high homology values (100%) with a bootstrap value of 100%.This shows that there is no genetic diversity in A. platys that infected dogs in the Yogyakarta area.

TABLE 2
Data of PCR products from 51 dogs.

TABLE 3
The results of data analysis using BLAST.

TABLE 4
Matrix of differences between sequence of groEL nucleotide of Anaplasma spp.from research samples and sequences from GenBank in several species.

TABLE 5
Genetic distance of groEL sequences of Anaplasma spp.from research samples with sequences from GenBank in several species.