Babesiosis is an emerging tick-borne hemolytic disease caused by intraerythrocytic protozoan parasites of the genus Babesia (10). To date, there are over 100 Babesia species that can infect various vertebrate hosts, occasionally including humans (13). Canine babesiosis is mainly caused by Babesia canis, B. gibsoni, and B. canis vogeli (32). B. gibsoni was first identified in 1910 in India (8), and it has since been reported in the Middle East, Africa, Asia, Europe, the United States, and in some Latin American and Caribbean countries (5,20). Dogs become infected when ticks feed and release sporozoites, which then enter the red blood cells usually within two to three days (29). The clinical manifestation of babesiosis typically occurs after an incubation period of 10 to 28 days (12), and it varies widely from hyper-acute or acute to chronic, and it can even present as a subclinical infection (23). Babesia species are mainly transmitted by tick vectors; however, they can alternatively be transmitted via dog bites, blood transfusions, and transplacental transmission (10). The four genera of ticks that have been reported as vectors of Babesia species are: Rhipicephalus, Ixodes, Haemaphysalis, and Hyalomma, all of which belong to the family Ixodidae (9).

In the Republic of Korea (ROK), the tick species most commonly infected with Babesia species are Haemaphysalis longicornis and H. flava, which can be collected from grass, vegetation, and human patients (11). According to a survey, H. longicornis, H. flava, Ixodes nipponensis, and Rhipicephalus sanguineus sensu late were found to be the tick species that most frequently infest domestic dogs in Korea (6); however, the main tick vector of B. gibsoni in the ROK has not yet been elucidated, especially considering that there have been few surveys of B. gibsoni infection in canines in the ROK. The first molecular detection was performed in German Shepherd dogs in 2004, in which an infection rate of 1.8% (9/501) was identified (27). Subsequently, in 2009, an infection rate of 24.8% (29/117) was reported (14). In 2017, B. gibsoni DNA was identified in 23/440 (5.2%) strictly outdoor dogs using a real-time polymerase chain reaction (PCR) assay (28). Moreover, one of the most recent studies carried out in the country demonstrated the effectiveness of using the ultra fast real-time PCR, giving an accurate diagnosis of babesiosis. In this study it was identified an infection rate of 38.3% (62/162) (33). Currently, there has only been one instance of B. gibsoni in serological evidence from Korean domestic dogs. This evidence was obtained using ELISA and indirect fluorescence immunoassay (IFA) kits, and a seroprevalence of 1.7% (38/2,215) was identified (15).

The present study aimed to investigate the molecular and serological prevalence of B. gibsoni in Korean domestic dogs, as well as to understand the clinical occurrence of the infection through the classification and evaluation of patients according to different courses of the disease. The results of this study may help in elucidating the common clinical presentation, geographical distribution, and risk factors associated with canine babesiosis in the ROK.

Sampling methodology and patient data collection

A total of 400 whole blood samples were collected from companion dogs at different veterinary hospitals and clinical centers located in seven metropolitan cities and nine provinces within the ROK (Fig. 1). The samples were then sent to the Laboratory of Veterinary Internal Medicine, Seoul National University, ROK, between April 2019 and December 2020. The samples were collected in sterile blood collection tubes containing EDTA anticoagulant and submitted to the laboratory in an icepack container. The criteria for referring the sample to the laboratory was based on the presumptive diagnosis of tick-borne disease given by a veterinarian. Such diagnoses were based on clinical symptoms alongside the hematological and biochemical abnormalities presented by the patient on the day of its visit to the veterinary hospital or clinic. Along with the whole blood sample, the patient data, its clinical records, and the hematological and blood chemistry results were provided to the laboratory team for further analysis and interpretation.

Figure 1. Map of the Republic of Korea showing the sampled sites for the detection of B. gibsoni infection in companion dogs in 2019-2020. The gray color indicates sampled provinces, and the purple color represents sampled metropolitan cities. The number of samples analyzed per site are shown in circles.

DNA extraction and PCR amplification

Genomic DNA was extracted from 200 µL of the whole blood sample using the commercial LaboPass DNA Purification Kit (Cosmo Genetech, Seoul, ROK) according to the manufacturer’s instructions. The extracted DNA was stored at –20°C until further analysis. For the detection of Babesia species, a single PCR assay was conducted to amplify its partial 18S rRNA gene (641 bp) using the primer pair BabgenF 5’-GAAACTGCGAATGGCTCATTA-3’ and Babesiarev1 5’-CCATGCTGAAGTATTCAAGAC-3’ (22). These reactions were performed in a total volume of 30 µL, containing 10 pmol of each primer, 15 µL of 2x Taq PCR Pre-mix (BioFACT, Daejeon, ROK), and 50 to 100 ng of genomic DNA samples. The reactions were conducted in a SimpliAmp Thermal Cycler (Thermo Fisher Scientific; Foster City, CA) under optimal cycling conditions. The cycling conditions used in this study for successful amplification consisted of 30 cycles, each of 1 min at 94°C for denaturation, followed by annealing for 1 min at 55°C and extension for 1 min at 72°C. The PCR products were visualized under UV light after 1.5% agarose gel electrophoresis using a 100 bp ladder (Sizer-100 DNA Marker Solution, iNtRON Biotechnology, Seongnam, ROK) as a DNA size marker.

Rapid antibody test for B. gibsoni

The SensPERT Canine Babesia gibsoni Ab Test Kit (sensitivity of 91.8% and specificity of 93.5% vs. IFA) (VetAll Laboratories^®, Goyang, ROK) was used to detect seroreactive dogs. The serological test procedure consisted of adding one drop (10 µL) of whole blood to the specimen well using a dropper as a pipette. Two drops (80 µL) of buffer were dispensed onto the whole blood drop until it was completely absorbed. The results were interpreted 10 min later according to the manufacturer’s instructions.

DNA sequencing and phylogenetic analysis

Positive PCR products were purified using the DNA Gel Extraction S & V Kit (BIONICS, Daejeon, ROK) and sequenced using an Applied Biosystems 3730xl capillary DNA Analyzer (Thermo Fisher Scientific; Foster City, CA). Nucleotide sequences were evaluated and confirmed using Chromas software. They were then compared with reference sequences downloaded from the National Center of Biotechnology Information (NCBI) to determine the accuracy of the PCR. Multiple sequence alignment was performed with the program ClustalX 2.1 and corrected with BioEdit software (v 7.2.5). Finally, a similarity matrix was constructed to determine the percentage of similarity between sequences. Phylogenetic analysis was performed based on the maximum likelihood method in MEGA 6.06 software. The stability of the obtained tree was estimated by bootstrap analysis with 1,000 replicates.

Nucleotide sequence accession numbers

The representative sequences obtained in this study of the B. gibsoni partial 18S rRNA gene have been submitted and deposited in the GenBank database under accession numbers MW485704 to MW485725.

Statistical analysis

The statistical analysis was performed using the analytical software package GraphPad Prism (v 5.04; GraphPad Software, Inc., La Jolla, CA). To analyze the significant differences that might exist between regions, genders, age groups, and breeds of dogs, the Chi-square test or Fisher’s exact test was performed. Differences were considered statistically significant if the p value was less than 0.05.

Ethics approval and consent to participate

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Seoul National University Animal Care and Use Committee (No. SNU-190524-2-1). Informed consent was obtained from the owners of all animals involved in the study.

Molecular and serological prevalence

Out of the 400 dogs, 36 (9.0%) were PCR-positive, while only 24 (6.0%) were B. gibsoni seropositive. Dogs from the metropolitan cities of Busan, Incheon, and Seoul were not only PCR-positive, but also antibody-positive, with prevalence rates of 40.0% vs. 13.3%, 12.5% vs. 12.5%, and 11.5% vs. 7.6%, respectively (Table 1). The same pattern of results was observed in Gyeongsangnam-do and Gyeonggi-do provinces, where animals tested positive in both the PCR and antibody tests (Table 1). The highest Babesia spp. infection rate was identified in male dogs (8.9%, 17/192), whereas female dogs were found to be the most seroreactive to B. gibsoni (6.8%, 12/176) (Table 1). The age group classification revealed the highest infection rate in dogs aged 2-4 years (12.9%, 15/116), whereas the highest seroprevalence rate was identified in dogs within the age range of 5-7 years (8.9%, 8/90). Out of the 44 dog breeds, 17 were PCR-positive and 11 were antibody-positive, as shown in Supplementary Table 1. Despite the differences observed between the molecular and serological prevalence analyzed per risk factor, the differences were not statistically significant (Table 1, Supplementary Table 1).

Table 1 . Comparison between antigen and antibody prevalence of Babesia gibsoni in companion dogs in 2019-2020 in the Republic of Korea.

Risk factor	No. tested		PCR positive				Seropositive
			No.	IR (%)	p-value		No.	IR (%)	p-value
Region					0.9693				0.6922
Metropolitan cities
Seoul	131		15	11.5			10	7.6
Incheon	8		1	12.5			1	12.5
Daejeon	7		0	0			0	0
Daegu	31		0	0			2	6.5
Ulsan	8		0	0			1	12.5
Busan	15		6	40.0			2	13.3
Gwangju	3		0	0			0	0
Provinces
Gyeonggi-do	125		10	8.0			6	4.8
Gangwon-do	9		0	0			0	0
Chungbuk-do	10		0	0			0	0
Chungnam-do	10		1	10.0			0	0
Gyeongsangbuk-do	1		0	0			0	0
Gyeongsangnam-do	14		2	14.3			2	13.3
Jeollabuk-do	19		0	0			0	0
Jeollanam-do	8		0	0			0	0
Jeju-do	1		1	100.0			0	0
Total	400		36	9.0			24	6.0
Sex					0.5852				0.6607
Male	192		17	8.9			10	5.2
Female	176		15	8.5			12	6.8
NS	32		4	12.5			2	6.3
Total	400		36	9.0			24	6.0
Age					0.1375				0.4628
≤1 year old	51		2	3.9			2	3.9
2-4 years old	116		15	12.9			7	6.0
5-7 years old	90		8	8.9			8	8.9
8-10 years old	57		6	10.5			5	8.8
≥11 years old	49		1	2.0			1	2.0
NS	37		4	2.7			1	2.7
Total	400		36	9.0			24	6.0

IR, Infection Rate; NS, not specified (sex and age not specified in the clinical record)..

p ≤ 0.05 considered statistically significant..

Sequencing and phylogenetic analysis

The alignment of the Babesia spp. 18S rRNA nucleotide sequences generated in the present study revealed that all sequences had a fragment length of 641 bp and were 100% identical to each other. Through the Basic Local Alignment Search Tool (BLAST) and the phylogenetic analysis based on the 18S rRNA gene fragment of Babesia species, the genetic relationships between our sequences and the reference sequences downloaded from the GenBank database were confirmed. The sequences were 100% identical to previously sequenced B. gibsoni obtained from dogs, cats, and masked palm civets from several countries, as shown in Supplementary Fig. 1 and Supplementary Table 2.

Clinical signs

To evaluate the common clinical presentation of B. gibsoni infection, 34 clinical records out of the 36 B. gibsoni PCR-positive dogs were classified into three different groups according to their clinical signs, as follows: dogs manifesting symptoms of the complicated form of canine babesiosis (Group I – acute infection), dogs with mild to non-specific symptoms (Group II – chronic infection), and apparently healthy dogs (Group III – subclinical infection) (Table 2). Inappetence (61.8%, 21/34), lethargy (50.0%, 17/34), pyrexia (47.1%, 16/34), gastrointestinal symptoms including diarrhea and vomiting (29.4%, 10/34), and hematuria (23.5%, 8/34) were found to be the most common clinical symptoms (Fig. 2). Other clinical complications are shown in Table 2. In terms of tick exposure, only 22 (64.7%) out of 36 PCR-positive dogs had a history of tick bites (Table 2). Considering the three forms of canine babesiosis, 55.8% (19/34) of the dogs presented an acute infection, 32.4% (11/34) a chronic infection, and 11.8% (4/34) a subclinical infection; the latter dogs were considered as carriers of B. gibsoni (Table 2).

Table 2 . Clinical findings observed in dogs infected with B. gibsoni.

No.	Patient data			Tick exposure	Anamnesis	Other complications
	Breed	Age	Sex
Group I. Acute infection (n = 19)
1	Yorkshire Terrier	4 y	F	Yes	Pale gums, lethargy, hematuria	None
2	Yorkshire Terrier	NS	F	Yes	Inappetence, hematuria	None
3	Bichon	2 y	M	No	Pyrexia, lethargy, inappetence	IMHA
4	Pomeranian	NS	M	-	Lethargy, inappetence, splenomegaly	None
5	Great Pyrenees	10 y	F	Yes	Pyrexia, lethargy, inappetence	None
6	Poodle	8 y	M	Yes	Pyrexia, lethargy, inappetence, GS	None
7	Maltese	6 y	F	-	Lethargy, inappetence, hematuria	None
8	Poodle	10 y	NS	-	Pale gums, lethargy, inappetence, weight loss, depression, hematuria	Pancreatitis,hepatic compromise
9	Dachshund	5 y	M	Yes	Pyrexia, GS, splenomegaly, hepatomegaly	Colitis
10	Poodle	10 y	F	No	Eye edema, jaundice, hematuria	None
11	Mixed	6 y	M	-	Pyrexia, lethargy, inappetence, hematuria, splenomegaly	Cholangiohepatitis, urolithiasis
12	Mixed	13 y	M	Yes	Pyrexia, inappetence, weight loss, GS	None
13	Maltese	8 y	M	Yes	Pyrexia, lethargy, inappetence, GS	None
14	Irish Terrier	3 y	M	Yes	Pyrexia, pale gums, lethargy, GS	IMHA
15	Pompitz	3 y	M	Yes	Pyrexia, pale gums, inappetence, GS, jaundice, hematuria, splenomegaly	Pancreatitis
16	Poodle	3 y	M	Yes	Pyrexia, inappetence, hematuria	None
17	Bichon Frise	6 m	M	-	Lethargy, inappetence, splenomegaly	None
18	Mixed	6 y	F	-	Pyrexia, pale gums, lethargy, GS, RS, splenomegaly	Ovarian cysts, chronic bronchitis
19	Miniature Pinscher	3 y	F	Yes	Pyrexia, inappetence, depression	None
Group II. Chronic infection (n = 11)
20	Beagle	3 y	F	Yes	Lethargy, inappetence	None
21	Mixed	6 y	M	Yes	Pyrexia	IMHA
22	Beagle	10 y	F	Yes	Lethargy, inappetence	None
23	Bichon	1 y	M	Yes	Lethargy, inappetence	None
24	Jindo	2 y	NS	Yes	Pyrexia, inappetence	None
25	Italian Greyhound	4 y	M	Yes	Pyrexia	None
26	Poodle	6 y	F	-	Inappetence, GS	None
27	Pomeranian	5 y	F	Yes	Lethargy, inappetence	None
28	Mixed	2 y	M	Yes	GS	None
29	Pomeranian	4 y	M	Yes	Lethargy, inappetence	None
30	Poodle	4 y	M	Yes	Pyrexia, GS	IMHA
Group III. Subclinical infection (n = 4)
31	Mixed	3 y	F	No	Apparently healthy	None
32	Schnauzer	7 y	F	-	Apparently healthy	None
33	Jindo	2 y	F	-	Apparently healthy	None
34	American Pitbull Terrier	2 y	F	Yes	Apparently healthy	None

NS, not specified (age and sex not specified in the clinical record); GS, gastrointestinal symptoms; RS, respiratory symptoms; IMHA, immune-mediated hemolytic anemia; y, years old; m, months; F, female; M, male; Dash, unknown data..

Figure 2. Case of complicated canine babesiosis in the Republic of Korea. (A) A 3-year-old Pompitz (Pomeranian plus Japanese spitz) male dog was presented at the Gyeongsang National University Veterinary Teaching Hospital with a history of pyrexia, jaundice in the (a) skin of the abdomen and at the base of the (b) ears, inappetence, (c) gastrointestinal symptoms such as diarrhea and vomiting, (d) hematuria, pancreatitis, and tick exposure. The hemogram findings were normocytic normochromic regenerative moderate anemia, leukopenia (neutropenia 2.78 10⁹/L, RR: 3.9-8.0), and thrombocytopenia (17 10⁹/L, RR: 148-484). Chemistry results indicated hyperbilirubinemia (1.0 mg/dL, RR: 0.1-0.7). (B) The microscopic examination (1,000×) of the blood smear revealed pleomorphic intraerythrocytic B. gibsoni piroplasms. The clinical record and a whole blood sample from the patient were submitted to the Laboratory of Veterinary Internal Medicine of Seoul National University for further analysis. The patient was (C) PCR-positive to B. gibsoni and (D) seropositive to B. gibsoni antibodies (SensPERT Canine Babesia gibsoni Ab Test Kit, VetAll Laboratories^®). The patient was urgently hospitalized, subjected to blood transfusion treatment, and maintained on oral Atovaquone (13.4 mg/kg three times daily) and Azithromycin (10 mg/kg once daily) for 10 days. Response to treatment was monitored by hematocrit and CBC tests along with repeated PCR testing for B. gibsoni at 30 and 60 days.

Hematologic and biochemical test results

Complete blood count (CBC) and biochemistry test results were available for 26 of the 36 B. gibsoni PCR-positive dogs, including 15 acutely infected, 8 chronically infected, and 3 subclinically infected dogs. When comparing the hematology and blood chemistry test results between the three courses of the disease, it was identified that the acute course commonly tended to include the following abnormalities: thrombocytopenia (93.3%, 14/15), anemia (86.7%, 13/15), hyperbilirubinemia (53.3%, 8/15), hypoalbuminemia (40.0%, 6/15), increased aspartate aminotransferase (AST) enzyme activity (26.7%, 4/15), neutropenia (26.7%, 4/15), lymphocytopenia (26.7%, 4/15), and lymphocytosis (26.7%, 4/15). For both the chronic and subclinical courses, thrombocytopenia and anemia were primarily found (Table 3).

Table 3 . Comparison of hematologic and biochemical test results of B. gibsoni PCR-positive dogs in 2019-2020 in the Republic of Korea.

Blood test	Parameters	Unit	Reference values		Results (No. of dog patients)
			Min	Max	Low						Normal						High
					Disease course				Total		Disease course				Total		Disease course			Total
					A	C	S		No. (%)		A	C	S		No. (%)		A	C	S	No. (%)
Hemogram (n = 26)	PCV	%	37.1	57.0	13	6	2		21 (80.8)		2	2	1		5 (19.2)		0	0	0	0 (0)
	RBC	10 × 12/L	5.7	8.8	11	6	3		20 (76.9)		4	2	0		6 (23.1)		0	0	0	0 (0)
	Hb	g/dL	12.9	18.4	13	6	2		21 (80.8)		2	2	1		5 (19.2)		0	0	0	0 (0)
	RDW	%	11.9	14.5	1	1	0		2 (7.7)		11	5	2		18 (69.2)		3	2	1	6 (23.1)
	PLT	10 × 9/L	148.0	484.0	14	7	2		23 (88.5)		1	0	1		2 (7.7)		0	1	0	1 (3.8)
	WBC	10 × 9/L	5.2	13.9	2	2	0		4 (15.4)		10	5	3		18 (69.2)		3	1	0	4 (15.4)
	NEUT	10 × 9/L	3.9	8.0	4	0	0		4 (15.4)		10	7	2		19 (73.1)		1	1	1	3 (11.5)
	LYMPH	10 × 9/L	1.3	4.1	4	0	0		4 (15.4)		7	6	3		16 (61.5)		4	2	0	6 (23.1)
	MONO	10 × 9/L	0.2	1.1	2	0	0		2 (7.7)		12	6	3		21 (80.8)		1	2	0	3 (11.5)
	EOS	10 × 9/L	0.06	1.23	2	2	0		4 (15.4)		13	5	3		21 (80.8)		0	1	0	1 (3.8)
	BASO	10 × 9/L	0	0.1	0	1	0		1 (3.8)		15	6	3		24 (92.3)		0	1	0	1 (3.8)
Biochemistry (n = 26)	ALT	U/L	10.0	100.0	0	0	0		0 (0)		13	8	2		23 (88.5)		2	0	1	3 (11.5)
	AST	U/L	11.7	42.5	0	0	0		0 (0)		11	7	3		21 (80.8)		4	1	0	5 (19.2)
	BUN	mg/dL	5.0	30.0	0	0	0		0 (0)		14	8	3		25 (96.2)		1	0	0	1 (3.8)
	Creatinine	mg/dL	0.4	1.6	0	0	0		0 (0)		14	8	3		25 (96.2)		1	0	0	1 (3.8)
	Total bilirubin	mg/dL	0.1	0.7	0	0	0		0 (0)		7	6	3		16 (61.5)		8	2	0	10 (38.5)
	Albumin	g/dL	2.3	3.9	6	1	1		8 (30.8)		9	7	2		18 (69.2)		0	0	0	0 (0)
	Total protein	g/dL	4.9	7.2	2	0	0		2 (7.7)		12	7	3		22 (84.6)		1	1	0	2 (7.7)

A, acute (n = 15); C, chronic (n = 8); S, subclinical (n = 3); PCV, packed-cell volume; RBC, red blood cell count; Hb, hemoglobin; RDW, red cell distribution width; PLT, platelet count; WBC, white blood cell count; NEUT, neutrophils; LYMPH, lymphocytes; MONO, monocytes; EOS, eosinophils; BASO, basophils; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen..

In the present investigation, there was a notable difference between the total number of PCR-positive animals and the total number of antibody-positive animals [n = 36 (9.0%) vs. n = 24 (6.0%), respectively]. This pattern of results is consistent with previous studies suggesting that it is perfectly feasible for a dog to be seronegative but positive to Babesia infection, as infections by species such as B. canis and B. gibsoni manifest acutely (26). This idea is further supported by the analysis of the clinical history of each PCR-positive dog, which revealed that a large portion of the dogs exhibited the acute course of the disease.

Regarding host susceptibility, age is one of the factors that may predispose dogs to infection and determine the severity of infection, with young dogs being the most prone to severe babesiosis (1). The findings of the present study highlight this theory. It was found that younger dogs, specifically within the age range of 2-4 years old, are the most affected by B. gibsoni infection, whereas older animals (8-10 years old) are the most seroreactive. Poor humoral response and maternal antibody protection are suspected to directly influence the lower seroprevalence rate observed in younger patients (1). Although the differences observed in the infection rates among the breeds tested in this study were not statistically significant, a breed predisposition has been suggested in the USA (5). Specifically, the American Pitbull Terrier is particularly vulnerable to the development of B. gibsoni infection owing to their frequent use as fighting dogs. No significant sex predisposition to the disease was observed in the current study; however, the proportion of male dogs infected with B. gibsoni was relatively high compared to female dogs. This pattern is consistent with the results obtained in the studies performed by Song et al. (27) and Lee et al. (14), who suggested that male dogs are more likely to be kept outdoors as guard dogs or used as fighting dogs. This also explains why there is a higher risk of tick exposure for male dogs in the ROK.

Additionally, our study demonstrates that the clinical expression of canine babesiosis in the ROK ranges from subclinical to acute, the latter being the most common. This result differs from results reported in the USA, in which the subclinical course was found to be the most predominant (3). In the present investigation, the acute course is characterized by pyrexia, pale gums, lethargy, depression, inappetence, weight loss, vomiting, diarrhea, hematuria, jaundice, splenomegaly, and hepatomegaly. Regarding jaundice, there were only two (5.9%) icteric patients in this study, thus supporting the evidence that clinical icterus is rare in Babesia infection (4). Interestingly, 17.6% of the B. gibsoni-infected dogs presented splenomegaly, which is an abnormality considered as a risk factor for the development of natural and potentially fatal babesiosis (25). Other organ dysfunctions found in the acutely infected patients were pancreatitis, liver failure, and chronic bronchitis. Goris and colleagues considered that multiple organ dysfunction syndrome (MODS) develops as a consequence of the dysregulation of proinflammatory and anti-inflammatory mechanisms, resulting in overwhelming auto-destructive inflammation (7). In addition, another study has also demonstrated a correlation between the number of affected organs and mortality (31). The clinical presentation of the chronic course varied greatly in this study from mild symptoms, such as pyrexia, lethargy, inappetence, diarrhea, and vomiting, to completely asymptomatic cases. In general, subclinical carriers were observed to be apparently healthy; however, it is important to keep in mind that there is the possibility that an individual can remain as an infected carrier for many months, possibly even for life (23). This leads to a potential risk for the rapid spread of the disease.

The most common hematological abnormalities found in the current investigation were anemia (80.8%) and thrombocytopenia (88.5%). The pathogenesis of anemia in canine babesiosis is not dependent on the intensity of parasitism (19). Onishi et al. (19) demonstrated the presence of hemolytic factors in the serum of B. gibsoni-infected dogs and the correlation between the activity of the infection and anemia. Therefore, it seems that the hemolytic factors in the serum of B. gibsoni-infected dogs play an important role in the development of anemia in the infection. Thrombocytopenia is considered as the hallmark symptom associated with canine babesiosis, which may result from immune-mediated platelet destruction, platelet sequestration in the spleen, elevated body temperatures, or disseminated intravascular coagulation (DIC) (2). The severity and rapid recovery of the platelet counts have led to the suggestion that immune-mediated mechanisms are involved (21).

Patients in our study also presented alterations in the white blood cell count, with values occurring above the normal range as well as below the normal range. These findings are further supported by previous research that suggests that the leukocyte count is extremely variable in dogs with babesiosis and can range from leukopenic to leukemoid (16).

Biochemical abnormalities revealed that hepatic enzyme activities, including alanine aminotransferase (ALT) and AST, as well as bile acids and albumin levels could be altered in a patient infected with B. gibsoni. This pattern of results is consistent with previous studies (3,18). Consequently, it is of crucial importance to assess liver biofunction while measuring cholinesterase activity in cases of canine babesiosis (24). Blood urea nitrogen (BUN) and creatine levels were elevated in only one patient. A possible reason for this elevated level in some dogs may be due to the non-renal accumulation of ammonia in serum as a result of hemolytic anemia (17). Azotemia appears to be a common complication of B. microti-like piroplasm (Theileria annae) infection. However, our findings, along with those previously reported in the USA (30), suggest the possibility of also finding cases of canine babesiosis due to B. gibsoni with azotemia and proteinuria.

In conclusion, it can be concluded that B. gibsoni is endemic in the ROK, as PCR-positive and seroreactive animals have been identified across the whole country. In the ROK, the disease occurs in its three courses, with its acute course being the most predominant, thus representing a great challenge for veterinary clinicians due to the severity of clinical signs. The classification of patients according to the clinical presentation of the disease favors the identification of the pattern of symptoms and hematological and biochemical abnormalities found in acutely, chronically, and subclinically infected animals.

This research was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through the Animal Disease Management Technology Development Program, which is funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA) (119053).

The authors wish to thank VetAll Laboratories^® for providing the SensPERT Ab Test Kits for this study. Evelyn Alejandra Miranda expresses her deep gratitude to the National Institute of International Education (NIIED) and the Global Korea Scholarship (GKS) program for the scholarship granted for her graduate studies for 2018-2021.

The authors have no conflicting interests.

Conceptualization: Miranda EA, Chae JS; Data curation: Miranda EA; Formal analysis: Miranda EA; Funding acquisition: Chae JS; Investigation: Miranda EA; Methodology: Miranda EA, Chae JS; Project administration: Chae JS; Resources: Han SW, Rim JM, Cho YK, Yu DH, Choi KS, Chae JS; Software: Miranda EA; Supervision: Han SW, Cho YK, Chae JS; Validation: Miranda EA, Choi KS, Yu DH, Chae JS; Visualization: Miranda EA, Chae JS; Writing-original draft: Miranda EA; Writing-review & editing: Miranda EA.