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First clinical case of canine granulocytic anaplasmosis in Korea and genotypic analyses of Anaplasma phagocytophilum
G Model TTBDIS-790; No. of Pages 4
ARTICLE IN PRESS Ticks and Tick-borne Diseases xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Short communication
First clinical case of canine granulocytic anaplasmosis in Korea and genotypic analyses of Anaplasma phagocytophilum Seung-Hun Lee a,b , Nara Kim a , Dongmi Kwak a,c,∗ a b c
College of Veterinary Medicine, Kyungpook National University, Daegu, Republic of Korea Foreign Animal Disease Division, Animal and Plant Quarantine Agency, Gimcheon, Republic of Korea Cardiovascular Research Institute, Kyungpook National University, Daegu, Republic of Korea
a r t i c l e
i n f o
Article history: Received 2 November 2016 Received in revised form 12 January 2017 Accepted 1 February 2017 Available online xxx Keywords: Anaplasma phagocytophilum Canine granulocytic anaplasmosis 16S rRNA msp2 groEL
a b s t r a c t A 2-year-old male Jindo dog was presented to a local veterinary clinic for anorexia, lethargy, edema around neck, and vomiting. Based on the clinical history, physical examination, hematology, blood chemistry, serology, and PCR, the dog was diagnosed with canine granulocytic anaplasmosis (CGA). PCR and phylogenetic analyses targeting the 16S rRNA, groEL, and msp2 genes of Anaplasma phagocytophilum revealed that the A. phagocytophilum identified in this study subgrouped into alanine and USA groups according to the groEL and msp2 gene sequences, respectively. To the best of our knowledge, this is the first clinical case of CGA in Korea. In addition, detection of clinical CGA belonging to the alanine group is meaningful because only A. phagocytophilum belonging to the serine group has been reported to develop clinical signs. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction Anaplasma phagocytophilum (syn. Ehrlichia phagocytophila, E. equi, and human granulocytic ehrlichiosis agent) is a tick-borne zoonotic pathogen and causes canine granulocytic anaplasmosis (CGA) in dogs (Dumler et al., 2001). The clinical signs of CGA vary from mild to severe. Among the clinical manifestations, lethargy and fever are most common, and reluctance to move, lameness, polydipsia, anemia, pale mucous membranes, vomiting, diarrhea, hemorrhage, petechia, splenomegaly, and enlarged lymph nodes may also occur (Carrade et al., 2009; Greig et al., 1996; Silaghi et al., 2011). Although various studies on CGA have been reported worldwide such as North America including USA (Greig et al., 1996) and Canada (Lester et al., 2005) and Europe including Austria (Kirtz et al., 2005), France (Canelas Domingos et al., 2011), Germany (Kohn et al., 2008; Kohn et al., 2011), Italy (Dondi et al., 2014), and Slovenia (Tozon et al., 2003), there has been no previous report of CGA in Asia. Recently, several studies reported A. phagocytophilum in dogs in Asia detected by serological (Jung et al., 2012; Levi et al., 2006; Lim et al., 2010) and molecular techniques (Lee et al., 2016b). However,
∗ Corresponding author at: College of Veterinary Medicine, Kyungpook National University, 80 Daehakro, Bukgu, Daegu 41566, Republic of Korea. E-mail address: [email protected] (D. Kwak).
molecular or serological detection of A. phagocytophilum in dogs differs from clinical cases of CGA because not all A. phagocytophilum infections result in clinical signs (Carrade et al., 2009). Considering the increasing distribution of vector ticks due to global warming, tick-borne disease is also expected to be increasingly prevalent (Lee et al., 2016b). In addition, a recent report of human granulocytic anaplasmosis (HGA) in Korea raises awareness of the disease (Kim et al., 2014a). Therefore, we report the first clinical case of CGA in Asia as well as the molecular characterization of A. phagocytophilum.
2. Case presentation A 2-year-old male Jindo dog was presented to a local veterinary clinic in Korea in November 2015 with anorexia, lethargy, local edema around neck, and vomiting. The dog was reared outdoors without any vaccination. The dog ran away to rural area for a few days prior to developing clinical signs. The physical examination revealed a body weight of 14 kg and the rectal temperature of 38.2 ◦ C. The dog was emaciated, dehydrated, and anemic with no biochemical profile abnormalities in urine analysis. Based on the clinical history and physical examination, differential diagnosis suggested physical impairment of the throat, rickettsial infection, piroplasmosis, pancreatitis, gastroenteritis, or heartworm infection.
http://dx.doi.org/10.1016/j.ttbdis.2017.02.003 1877-959X/© 2017 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Lee, S.-H., et al., First clinical case of canine granulocytic anaplasmosis in Korea and genotypic analyses of Anaplasma phagocytophilum. Ticks Tick-borne Dis. (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.02.003
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Table 1 Hematology and blood chemistry data in a dog with granulocytic anaplasmosis. Parametera
Unit
Value
Reference range
Hematology Erythrocytes Hematocrit Hemoglobin MCV MCH MCHC RDW Reticulocytes Leukocytes Neutrophils Lymphocytes Monocytes Eosinophils Basophils Platelets
(M/l) (%) (g/dl) (fl) (pg) (g/dl) (%) (K/l) (K/l) (K/l) (K/l) (K/l) (K/l) (K/l) (K/l)
5.22 29.7 10.8 56.9 20.6 36.2 15.8 35.4 18.0 14.1 1.54 2.17 0.16 0.02 214
5.5–8.5 37.0–55.0 12.0–18.0 60.0–77.0 18.5–30.0 30.0–37.5 14.7–17.9 10.0–110.0 5.5–16.9 2.0–12.0 0.5–4.9 0.3–2.0 0.10–1.49 0–0.1 175–500
Blood chemistry Glucose BUN Creatinine Calcium Total Protein Albumin Globulin ALT ALKP GGT Total bilirubin Amylase Lipase
(mg/dl) (mg/dl) (mg/dl) (mg/dl) (g/dl) (g/dl) (g/dl) (U/l) (U/l) (U/l) (mg/dl) (U/l) (U/l)
139 >130 8.3 9.9 7.5 4.1 3.5 87 44 8 0.6 1494 >6000
74–143 7–27 0.5–1.8 7.9–12.0 5.2–8.2 2.3–4.0 2.5–4.5 10–100 23–212 0–7 0–0.9 500–1500 200–1800
a MCV, mean corpuscular volume; MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration; RDW, red blood cell distribution width; BUN, blood urea nitrogen; ALT, alanine aminotransferase; ALKP, alkaline phosphatase; GGT, ␥-glutamyl transferase.
Since the owner declined further diagnosis using radiography, hematology, blood chemistry, and serology, only treatment administered was against clinical signs and the dog was released. On the following day the patient came back with the same clinical signs. On the second day post-treatment, the dog was presented again without improvement of its symptoms. According to the owner’s testimony, the symptoms were relieved for several hours after administration of the drugs, but relapsed. Physical examination revealed a body weight of 13.6 kg and rectal body temperature of 37.8 ◦ C. The dog was still emaciated, dehydrated, and anemic, as observed in the first physical examination. For this trial, the owner agreed to a further analysis of hematology and chemistry, and blood chemistry evaluated using IDEXX ® VetLab Station (IDEXX Laboratories Inc., Westbrook, ME, USA). ® Furthermore, a commercial ELISA test kit (SNAP 4Dx Plus; IDEXX Laboratories Inc.) was used to detect Dirofilaria immitis antigens as well as antibodies to Anaplasma spp., Borrelia burgdorferi, and Ehrlichia spp. Hematology and blood chemistry revealed decreased erythrocyte, hematocrit, hemoglobin, and MCV values and increased leukocytes, neutrophils, monocytes, BUN, creatinine, albumin, GGT, and lipase levels. The blood chemistry results suggested symptoms of renal failure. The detail values and reference ranges are described in Table 1. ® Using a commercial SNAP ELISA test kit, the dog tested positive for D. immitis antigens and Anaplasma spp. antibodies. For molecular diagnosis, DNA was extracted from the whole blood ® using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). PCR was performed to amplify 16S rRNA gene fragments of A. phagocytophilum and A. platys as described by Barlough et al. (1996) and Inokuma et al. (2000), respectively. For molecular characterization, A. phagocytophilum groEL and msp2 gene fragments were
amplified using primer sets as described by Lee et al. (2016b). Piro® plasms were screened using a commercial PCR kit (AccuPower Babesia & Theileria PCR Kit, Bioneer, Daejeon, Korea). The DNA of A. phagocytophilum isolated from a Korean water deer was used as positive control. To verify the absence of PCR inhibitors, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was amplified as Birkenheuer et al. (2003). The PCR amplification of A. phagocytophilum 16S rRNA, groEL, and msp2 gene fragments confirmed the dog was infected by A. phagocytophilum (Biggs et al., 2016). The GAPDH gene fragments were amplified, thus confirming the absence of PCR inhibitors. In addition, the PCR results were negative for A. platys or piroplasms. Sequences of 16S rRNA (927 bp), groEL (1097 bp), and msp2 (457 bp) gene fragments were submitted to GenBank (accession nos. KU513794, KU519287, and KU519283). Although we could not distinguish the primary and secondary reasons for the abnormal hematology and blood chemistry values from the diagnosis, treatments were applied for CGA, renal failure, and heartworm disease. However, the case was lost to follow-up because the owner refused to revisit the clinic.
3. Phylogenetic analysis After confirmation of the CGA diagnosis based on A. phagocytophilum 16S rRNA amplification (Biggs et al., 2016), the groEL and msp2 genes were amplified and analyzed for further molecular characterization using phylogenetic analysis. Phylogenetic trees were constructed with maximum likelihood method with 100 bootstrap replicates using MEGA 6.0 (Tamura et al., 2013). Based on phylogenetic analysis, the A. phagocytophilum 16S rRNA sequences did not show characteristic differences (Fig. 1A). Phylogenetic analysis of the groEL gene revealed that the A. phagocytophilum identified in the current study belonged to the alanine group (Fig. 1B), which has alanine at position 242 of the amino acid sequence (Lee et al., 2016a; Lee et al., 2016b; Petrovec et al., 2002; Rymaszewska, 2008). In addition, msp2 gene analysis showed regional association (USA and Europe), and the A. phagocytophilum in this study showed relatedness to sequences obtained in Korea that belonged to the USA group (Fig. 1C) (Lee et al., 2016a).
4. Discussion A. phagocytophilum is transmitted by ticks such as Ixodes spp., Dermacentor spp., and Haemaphysalis spp. (Lee et al., 2016b). In Korea, H. longicornis is known as a dominant species (Kim et al., 2014b) and previous studies reported molecular detection of A. phagocytophilum in H. longicornis, I. nipponensis, and I. persulcatus (Chae et al., 2008; Kim et al., 2003). Although ticks were not detected from the animal, we think that the dog was bitten by ticks and infected by A. phagocytophilum when the dog ran away for a few days. Laboratory abnormalities in CGA commonly include thrombocytopenia, neutropenia, lymphopenia, and anemia (Carrade et al., 2009; Greig et al., 1996). However, some studies have reported contradictory results, such as neutrophilia and monocytosis (Carrade et al., 2009; Kohn et al., 2011; Silaghi et al., 2011), and in experimental infection, the A. phagocytophilum-infected dogs did not show fever, anemia, and low hemoglobin (Nair et al., 2016). In this study, anemia was observed, consistent with the common symptoms of CGA. In addition, the number of platelets was decreased but within the normal range. However, the leukocytosis, neutrophilia, and monocytosis were also observed that are contradicted symptoms of CGA. Therefore, diagnosis based only on hematology and blood chemistry is insufficient (Nair et al., 2016), and additional studies
Please cite this article in press as: Lee, S.-H., et al., First clinical case of canine granulocytic anaplasmosis in Korea and genotypic analyses of Anaplasma phagocytophilum. Ticks Tick-borne Dis. (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.02.003
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Fig. 1. Phylogenetic trees of the Anaplasma phagocytophilum (a) 16S rRNA, (b) groEL, and (c) msp2 gene sequences. A. phagocytophilum obtained in this study are indicated by arrows. The tree was constructed based on the maximum likelihood method and the reliability was estimated with 100 bootstrap replicates. The scale bar indicates the number of substitutions per nucleotide.
are needed to specify the laboratory abnormalities associated with CGA. In this study, abnormal hemoglobin, MCV, BUN, creatinine, albumin, GGT, and lipase values were observed, and these abnormalities have not been previously reported in other cases of CGA. Due to the association between heartworm disease and increased liver enzyme activity, azotemia, and proteinuria (Ware, 2014), we suspected that heartworm infection might have contributed to these abnormal values. Moreover, the dog was reared outdoors in a poor environment, without vaccination and frequent feeding of poorly processed food such as raw chicken. We believe that this poor living environment contributed to the heartworm disease and renal failure in the current case, which resulted in abnormal hemoglobin, MCV, BUN, creatinine, albumin, GGT, and lipase values. It is noteworthy that the A. phagocytophilum in this study belonged to the alanine group. Previous studies suggested that the pathogenicity of A. phagocytophilum belonged to the serine group because this group is commonly detected in patients and animals with clinical symptoms (Alberti et al., 2005; Lotric-Furlan et al., 1998; Petrovec et al., 1999; Rymaszewska, 2008; Rymaszewska, 2014). However, the phylogenetic analysis in this study revealed that the clinical CGA belonged to the alanine group, and we found another clinical HGA belonging to alanine group (Fig. 1B, accession no. KJ677107) (Kim et al., 2014a). Therefore, this study supports the potential pathogenicity of A. phagocytophilum belonging to the alanine group. This study reported the first clinical case of CGA in Asia. Although different studies have reported CGA (Canelas Domingos et al., 2011; Dondi et al., 2014; Greig et al., 1996; Kirtz et al., 2005; Kohn et al., 2008; Kohn et al., 2011; Lester et al., 2005; Tozon et al., 2003), CGA has been reported mainly in Europe and USA, but not in Asia. Because not all A. phagocytophilum-infected dogs develop clinical symptoms and most infected dogs remain healthy (Carrade et al., 2009), clinical CGA and its molecular characterization are meaningful for future studies. In addition, phylogenetic analysis revealed that the A. phagocytophilum in this study belonged to the alanine group by groEL analysis and the Korean sub-group by msp2 analysis. This study suggests the pathogenicity of a novel A. phagocytophilum isolate belonging to the alanine group in dogs.
Acknowledgement This research was supported by a grant from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (Grant No. NRF2016R1D1A1B02015366).
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Please cite this article in press as: Lee, S.-H., et al., First clinical case of canine granulocytic anaplasmosis in Korea and genotypic analyses of Anaplasma phagocytophilum. Ticks Tick-borne Dis. (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.02.003
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Please cite this article in press as: Lee, S.-H., et al., First clinical case of canine granulocytic anaplasmosis in Korea and genotypic analyses of Anaplasma phagocytophilum. Ticks Tick-borne Dis. (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.02.003
ARTICLE IN PRESS Ticks and Tick-borne Diseases xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Short communication
First clinical case of canine granulocytic anaplasmosis in Korea and genotypic analyses of Anaplasma phagocytophilum Seung-Hun Lee a,b , Nara Kim a , Dongmi Kwak a,c,∗ a b c
College of Veterinary Medicine, Kyungpook National University, Daegu, Republic of Korea Foreign Animal Disease Division, Animal and Plant Quarantine Agency, Gimcheon, Republic of Korea Cardiovascular Research Institute, Kyungpook National University, Daegu, Republic of Korea
a r t i c l e
i n f o
Article history: Received 2 November 2016 Received in revised form 12 January 2017 Accepted 1 February 2017 Available online xxx Keywords: Anaplasma phagocytophilum Canine granulocytic anaplasmosis 16S rRNA msp2 groEL
a b s t r a c t A 2-year-old male Jindo dog was presented to a local veterinary clinic for anorexia, lethargy, edema around neck, and vomiting. Based on the clinical history, physical examination, hematology, blood chemistry, serology, and PCR, the dog was diagnosed with canine granulocytic anaplasmosis (CGA). PCR and phylogenetic analyses targeting the 16S rRNA, groEL, and msp2 genes of Anaplasma phagocytophilum revealed that the A. phagocytophilum identified in this study subgrouped into alanine and USA groups according to the groEL and msp2 gene sequences, respectively. To the best of our knowledge, this is the first clinical case of CGA in Korea. In addition, detection of clinical CGA belonging to the alanine group is meaningful because only A. phagocytophilum belonging to the serine group has been reported to develop clinical signs. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction Anaplasma phagocytophilum (syn. Ehrlichia phagocytophila, E. equi, and human granulocytic ehrlichiosis agent) is a tick-borne zoonotic pathogen and causes canine granulocytic anaplasmosis (CGA) in dogs (Dumler et al., 2001). The clinical signs of CGA vary from mild to severe. Among the clinical manifestations, lethargy and fever are most common, and reluctance to move, lameness, polydipsia, anemia, pale mucous membranes, vomiting, diarrhea, hemorrhage, petechia, splenomegaly, and enlarged lymph nodes may also occur (Carrade et al., 2009; Greig et al., 1996; Silaghi et al., 2011). Although various studies on CGA have been reported worldwide such as North America including USA (Greig et al., 1996) and Canada (Lester et al., 2005) and Europe including Austria (Kirtz et al., 2005), France (Canelas Domingos et al., 2011), Germany (Kohn et al., 2008; Kohn et al., 2011), Italy (Dondi et al., 2014), and Slovenia (Tozon et al., 2003), there has been no previous report of CGA in Asia. Recently, several studies reported A. phagocytophilum in dogs in Asia detected by serological (Jung et al., 2012; Levi et al., 2006; Lim et al., 2010) and molecular techniques (Lee et al., 2016b). However,
∗ Corresponding author at: College of Veterinary Medicine, Kyungpook National University, 80 Daehakro, Bukgu, Daegu 41566, Republic of Korea. E-mail address: [email protected] (D. Kwak).
molecular or serological detection of A. phagocytophilum in dogs differs from clinical cases of CGA because not all A. phagocytophilum infections result in clinical signs (Carrade et al., 2009). Considering the increasing distribution of vector ticks due to global warming, tick-borne disease is also expected to be increasingly prevalent (Lee et al., 2016b). In addition, a recent report of human granulocytic anaplasmosis (HGA) in Korea raises awareness of the disease (Kim et al., 2014a). Therefore, we report the first clinical case of CGA in Asia as well as the molecular characterization of A. phagocytophilum.
2. Case presentation A 2-year-old male Jindo dog was presented to a local veterinary clinic in Korea in November 2015 with anorexia, lethargy, local edema around neck, and vomiting. The dog was reared outdoors without any vaccination. The dog ran away to rural area for a few days prior to developing clinical signs. The physical examination revealed a body weight of 14 kg and the rectal temperature of 38.2 ◦ C. The dog was emaciated, dehydrated, and anemic with no biochemical profile abnormalities in urine analysis. Based on the clinical history and physical examination, differential diagnosis suggested physical impairment of the throat, rickettsial infection, piroplasmosis, pancreatitis, gastroenteritis, or heartworm infection.
http://dx.doi.org/10.1016/j.ttbdis.2017.02.003 1877-959X/© 2017 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Lee, S.-H., et al., First clinical case of canine granulocytic anaplasmosis in Korea and genotypic analyses of Anaplasma phagocytophilum. Ticks Tick-borne Dis. (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.02.003
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S.-H. Lee et al. / Ticks and Tick-borne Diseases xxx (2017) xxx–xxx
Table 1 Hematology and blood chemistry data in a dog with granulocytic anaplasmosis. Parametera
Unit
Value
Reference range
Hematology Erythrocytes Hematocrit Hemoglobin MCV MCH MCHC RDW Reticulocytes Leukocytes Neutrophils Lymphocytes Monocytes Eosinophils Basophils Platelets
(M/l) (%) (g/dl) (fl) (pg) (g/dl) (%) (K/l) (K/l) (K/l) (K/l) (K/l) (K/l) (K/l) (K/l)
5.22 29.7 10.8 56.9 20.6 36.2 15.8 35.4 18.0 14.1 1.54 2.17 0.16 0.02 214
5.5–8.5 37.0–55.0 12.0–18.0 60.0–77.0 18.5–30.0 30.0–37.5 14.7–17.9 10.0–110.0 5.5–16.9 2.0–12.0 0.5–4.9 0.3–2.0 0.10–1.49 0–0.1 175–500
Blood chemistry Glucose BUN Creatinine Calcium Total Protein Albumin Globulin ALT ALKP GGT Total bilirubin Amylase Lipase
(mg/dl) (mg/dl) (mg/dl) (mg/dl) (g/dl) (g/dl) (g/dl) (U/l) (U/l) (U/l) (mg/dl) (U/l) (U/l)
139 >130 8.3 9.9 7.5 4.1 3.5 87 44 8 0.6 1494 >6000
74–143 7–27 0.5–1.8 7.9–12.0 5.2–8.2 2.3–4.0 2.5–4.5 10–100 23–212 0–7 0–0.9 500–1500 200–1800
a MCV, mean corpuscular volume; MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration; RDW, red blood cell distribution width; BUN, blood urea nitrogen; ALT, alanine aminotransferase; ALKP, alkaline phosphatase; GGT, ␥-glutamyl transferase.
Since the owner declined further diagnosis using radiography, hematology, blood chemistry, and serology, only treatment administered was against clinical signs and the dog was released. On the following day the patient came back with the same clinical signs. On the second day post-treatment, the dog was presented again without improvement of its symptoms. According to the owner’s testimony, the symptoms were relieved for several hours after administration of the drugs, but relapsed. Physical examination revealed a body weight of 13.6 kg and rectal body temperature of 37.8 ◦ C. The dog was still emaciated, dehydrated, and anemic, as observed in the first physical examination. For this trial, the owner agreed to a further analysis of hematology and chemistry, and blood chemistry evaluated using IDEXX ® VetLab Station (IDEXX Laboratories Inc., Westbrook, ME, USA). ® Furthermore, a commercial ELISA test kit (SNAP 4Dx Plus; IDEXX Laboratories Inc.) was used to detect Dirofilaria immitis antigens as well as antibodies to Anaplasma spp., Borrelia burgdorferi, and Ehrlichia spp. Hematology and blood chemistry revealed decreased erythrocyte, hematocrit, hemoglobin, and MCV values and increased leukocytes, neutrophils, monocytes, BUN, creatinine, albumin, GGT, and lipase levels. The blood chemistry results suggested symptoms of renal failure. The detail values and reference ranges are described in Table 1. ® Using a commercial SNAP ELISA test kit, the dog tested positive for D. immitis antigens and Anaplasma spp. antibodies. For molecular diagnosis, DNA was extracted from the whole blood ® using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). PCR was performed to amplify 16S rRNA gene fragments of A. phagocytophilum and A. platys as described by Barlough et al. (1996) and Inokuma et al. (2000), respectively. For molecular characterization, A. phagocytophilum groEL and msp2 gene fragments were
amplified using primer sets as described by Lee et al. (2016b). Piro® plasms were screened using a commercial PCR kit (AccuPower Babesia & Theileria PCR Kit, Bioneer, Daejeon, Korea). The DNA of A. phagocytophilum isolated from a Korean water deer was used as positive control. To verify the absence of PCR inhibitors, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was amplified as Birkenheuer et al. (2003). The PCR amplification of A. phagocytophilum 16S rRNA, groEL, and msp2 gene fragments confirmed the dog was infected by A. phagocytophilum (Biggs et al., 2016). The GAPDH gene fragments were amplified, thus confirming the absence of PCR inhibitors. In addition, the PCR results were negative for A. platys or piroplasms. Sequences of 16S rRNA (927 bp), groEL (1097 bp), and msp2 (457 bp) gene fragments were submitted to GenBank (accession nos. KU513794, KU519287, and KU519283). Although we could not distinguish the primary and secondary reasons for the abnormal hematology and blood chemistry values from the diagnosis, treatments were applied for CGA, renal failure, and heartworm disease. However, the case was lost to follow-up because the owner refused to revisit the clinic.
3. Phylogenetic analysis After confirmation of the CGA diagnosis based on A. phagocytophilum 16S rRNA amplification (Biggs et al., 2016), the groEL and msp2 genes were amplified and analyzed for further molecular characterization using phylogenetic analysis. Phylogenetic trees were constructed with maximum likelihood method with 100 bootstrap replicates using MEGA 6.0 (Tamura et al., 2013). Based on phylogenetic analysis, the A. phagocytophilum 16S rRNA sequences did not show characteristic differences (Fig. 1A). Phylogenetic analysis of the groEL gene revealed that the A. phagocytophilum identified in the current study belonged to the alanine group (Fig. 1B), which has alanine at position 242 of the amino acid sequence (Lee et al., 2016a; Lee et al., 2016b; Petrovec et al., 2002; Rymaszewska, 2008). In addition, msp2 gene analysis showed regional association (USA and Europe), and the A. phagocytophilum in this study showed relatedness to sequences obtained in Korea that belonged to the USA group (Fig. 1C) (Lee et al., 2016a).
4. Discussion A. phagocytophilum is transmitted by ticks such as Ixodes spp., Dermacentor spp., and Haemaphysalis spp. (Lee et al., 2016b). In Korea, H. longicornis is known as a dominant species (Kim et al., 2014b) and previous studies reported molecular detection of A. phagocytophilum in H. longicornis, I. nipponensis, and I. persulcatus (Chae et al., 2008; Kim et al., 2003). Although ticks were not detected from the animal, we think that the dog was bitten by ticks and infected by A. phagocytophilum when the dog ran away for a few days. Laboratory abnormalities in CGA commonly include thrombocytopenia, neutropenia, lymphopenia, and anemia (Carrade et al., 2009; Greig et al., 1996). However, some studies have reported contradictory results, such as neutrophilia and monocytosis (Carrade et al., 2009; Kohn et al., 2011; Silaghi et al., 2011), and in experimental infection, the A. phagocytophilum-infected dogs did not show fever, anemia, and low hemoglobin (Nair et al., 2016). In this study, anemia was observed, consistent with the common symptoms of CGA. In addition, the number of platelets was decreased but within the normal range. However, the leukocytosis, neutrophilia, and monocytosis were also observed that are contradicted symptoms of CGA. Therefore, diagnosis based only on hematology and blood chemistry is insufficient (Nair et al., 2016), and additional studies
Please cite this article in press as: Lee, S.-H., et al., First clinical case of canine granulocytic anaplasmosis in Korea and genotypic analyses of Anaplasma phagocytophilum. Ticks Tick-borne Dis. (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.02.003
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Fig. 1. Phylogenetic trees of the Anaplasma phagocytophilum (a) 16S rRNA, (b) groEL, and (c) msp2 gene sequences. A. phagocytophilum obtained in this study are indicated by arrows. The tree was constructed based on the maximum likelihood method and the reliability was estimated with 100 bootstrap replicates. The scale bar indicates the number of substitutions per nucleotide.
are needed to specify the laboratory abnormalities associated with CGA. In this study, abnormal hemoglobin, MCV, BUN, creatinine, albumin, GGT, and lipase values were observed, and these abnormalities have not been previously reported in other cases of CGA. Due to the association between heartworm disease and increased liver enzyme activity, azotemia, and proteinuria (Ware, 2014), we suspected that heartworm infection might have contributed to these abnormal values. Moreover, the dog was reared outdoors in a poor environment, without vaccination and frequent feeding of poorly processed food such as raw chicken. We believe that this poor living environment contributed to the heartworm disease and renal failure in the current case, which resulted in abnormal hemoglobin, MCV, BUN, creatinine, albumin, GGT, and lipase values. It is noteworthy that the A. phagocytophilum in this study belonged to the alanine group. Previous studies suggested that the pathogenicity of A. phagocytophilum belonged to the serine group because this group is commonly detected in patients and animals with clinical symptoms (Alberti et al., 2005; Lotric-Furlan et al., 1998; Petrovec et al., 1999; Rymaszewska, 2008; Rymaszewska, 2014). However, the phylogenetic analysis in this study revealed that the clinical CGA belonged to the alanine group, and we found another clinical HGA belonging to alanine group (Fig. 1B, accession no. KJ677107) (Kim et al., 2014a). Therefore, this study supports the potential pathogenicity of A. phagocytophilum belonging to the alanine group. This study reported the first clinical case of CGA in Asia. Although different studies have reported CGA (Canelas Domingos et al., 2011; Dondi et al., 2014; Greig et al., 1996; Kirtz et al., 2005; Kohn et al., 2008; Kohn et al., 2011; Lester et al., 2005; Tozon et al., 2003), CGA has been reported mainly in Europe and USA, but not in Asia. Because not all A. phagocytophilum-infected dogs develop clinical symptoms and most infected dogs remain healthy (Carrade et al., 2009), clinical CGA and its molecular characterization are meaningful for future studies. In addition, phylogenetic analysis revealed that the A. phagocytophilum in this study belonged to the alanine group by groEL analysis and the Korean sub-group by msp2 analysis. This study suggests the pathogenicity of a novel A. phagocytophilum isolate belonging to the alanine group in dogs.
Acknowledgement This research was supported by a grant from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (Grant No. NRF2016R1D1A1B02015366).
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Please cite this article in press as: Lee, S.-H., et al., First clinical case of canine granulocytic anaplasmosis in Korea and genotypic analyses of Anaplasma phagocytophilum. Ticks Tick-borne Dis. (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.02.003