The First Records of Canine Babesiosis in Dogs from Dermacentor reticulatus—Free Zone in Poland

Tick-borne microorganisms belong to important etiological agents of many infectious diseases affecting humans and animals. Among them, there are haemoprotozoans of the Babesia genus, which infect erythrocytes of a host and may cause many clinical symptoms. Canine babesiosis is an emerging tick-borne disease in Southern and Central Europe. In this study, we report two cases of symptomatic canine babesiosis caused by Babesia canis in domestic dogs from the Silesian Voivodeship, Poland, as well as the presence of Dermacentor reticulatus ticks detected on one of the Babesia-infected dogs (D. reticulatus-free zone). The molecular analysis confirmed the presence of Babesia canis in the dogs’ blood, and the sequencing analysis showed that the obtained sequence is 100% identical to the sequence of Babesia canis isolate 3469 (sequence ID: KX712122.1). Our findings should raise awareness of B. canis infection among dog owners and veterinarians in the region where B. canis was not previously reported in residential, non-traveling dogs, as well as ensuring that adequate diagnostic methods are available.


Introduction
Tick-borne microorganisms are significant etiological agents of many infectious diseases affecting humans and animals worldwide. Some of the tick-borne pathogens (TBP) are protozoans of the genus Babesia, parasites that infect erythrocytes of the host and cause many clinical manifestations [1][2][3].
Canine babesiosis is an emerging tick-borne disease affecting companion animals in countries of Southern and Central Europe, e.g., England, France, Portugal, Hungary, Croatia [10,11], Germany [12], Austria [13], Slovakia [14,15], as well as Poland [16,17]. Dog infections caused by European strains of B. canis may exhibit mild to severe symptoms. The clinical manifestations of acute clinical symptoms include lethargy, weakness, apathy, elevated temperature, dehydration, anorexia, icterus, splenomegaly, color change in mucous membranes and urine, hemoglobinuria, renal failure, vomiting, diarrhea, and tachycardia. A complete blood count usually presents many abnormalities, such as hemolytic Pathogens 2022, 11, 1329 2 of 10 anemia, leucopenia, neutropenia, thrombocytopenia, and lymphopenia [11,18]. Babesia infection is diagnosed by microscopic examination of May-Grünwald-Giemsa or Diff-Quick stained peripheral blood smears, with the finding of large pyriform parasites within the infected erythrocytes. Furthermore, polymerase chain reaction (PCR) analysis is a significant diagnostic tool for confirmation of the presence of the Babesia subspecies in dogs [19].
Different species of Babesia protozoans are transmitted to domestic and wild animals, as well as humans, mainly through tick bites. The occurrence of B. canis in dogs is closely related to the geographical range of the ornate dog tick-Dermacentor reticulatus (Fabricius, 1794), which is the main vector of this haemoprotozoan species [20,21]. In Poland, D. reticulatus commonly occurs in Eastern, Western and Central regions, and a similar pattern of canine babesiosis cases was recorded in this country, especially in Eastern and Central parts [22]. The occurrence of canine babesiosis reflects the seasonal activity of adult D. reticulatus ticks in this country, with two peaks of activity, the first in March-April (the spring peak of activity) and the second in September-October (the autumn peak of activity) [22,23]. Due to scientific reports, there is a confirmed D. reticulatus-free zone in Poland (so-called gap zone) which spreads from West Pomerania and Pomerania Voivodeships in Northern Poland to Opole, Silesia, Lesser Poland, and Subcarpatia Voivodeships in Southern Poland [24].
Our findings indicate that B. canis infection in domestic dogs, as well as D. reticulatus, is present in the previously mentioned D. reticulatus-free zone in Poland. Such information gives new light on the current scientific knowledge in this field and is especially valuable to veterinarians as well as dog owners.

Dogs-Clinical Examination
Veterinary diagnostic procedures of an English Cocker Spaniel male (Dog 1) from Zawiercie (Silesia, Poland) included a detailed anamnesis, as well as a clinical examination with complete blood count (CBC) (automatic hematology analyzer Mindray BC-30 Vet, Stamar, Dabrowa Gornicza, Poland) and biochemical blood analyses (biochemical analyzer VETSCAN VS2, ZOETIS, Warsaw, Poland). The thin blood smears were prepared from the EDTA-treated blood samples taken from the cephalic vein and a Diff-Quik staining method (Hemavet Kit, KOLCHEM, Lodz, Poland). Blood smears allowed microscopic evaluation of Babesia protozoan's size and shape in order to classify them as large or small forms (Nikon ECLIPSE E 200, Tokio, Japan).
A clinical examination of a Labrador Retriever female (Dog 2) from Zawiercie (Silesia, Poland) included an X-ray of the abdomen, and similarly as in the first case, an anamnesis, a complete blood count, a biochemical blood analysis, and a blood smear examination.

Blood and Ticks Analyses
Blood samples used in our study consisted of surplus material, which was collected for routine diagnosis from dogs brought to the veterinary clinic. Therefore, no formal ethical approval was needed. In order to use the surplus material for additional diagnostic tests, oral consent from the dog owners was obtained. Genomic DNA was extracted from 200 µL of EDTA-blood samples using a commercial Quick Blood DNA Purification Kit (EURx, Gdańsk, Poland) according to the manufacturer's protocol. The concentration of DNA was measured spectrophotometrically at 260/280 wavelength in the Nanospectophotometer PEARL (Implen, Munich, Germany). DNA extracts were stored at −20 • C until use. Babesia spp. were detected in dogs' blood by PCR method. To detect this protozoan, a pair of primers specific to the 18S rRNA gene was used [25]. Then, the amplification product was separated electrophoretically in 2% ethidium bromide-stained gel and visualized under ultraviolet light. The PCR product was then isolated from the gel with the use of an Agarose-Out Kit (EURx, Gdańsk, Poland) according to the manufacturer protocol. Then, the samples were sequenced (Genomed, Warsaw, Poland), and the obtained sequences were compared with the sequences found in the GenBank database. Two ticks were removed from Dog 1 by tick-tweezers during the clinical exam at the veterinary clinic. Both of them were placed separately in marked plastic tubes with 70% ethanol and transported to the Department of Parasitology (Medical University of Silesia in Katowice, Poland) in order to identify species and life stages of ticks, as well as molecular analysis. Ticks were identified under the stereomicroscope (Zeiss Stemi 2000C, Warsaw, Poland) using the identification keys [26,27]. Then, DNA was isolated from two ticks using the ammonium hydroxide method, according to previously described protocols [28], and the concentration of DNA was measured spectrophotometrically by the Nanospectrophometer PEARL (Implen, Munich, Germany). To detect Babesia spp. in ticks, a pair of primers specific to the 18S rRNA gene was used [25]. In turn, to detect A. phagocytophilum, B. burgdorferi s.l., Rickettsia spp., and Bartonella spp., the primers specific to the 16S rRNA gene, flagellin gene, gltA gene, and rpoB gene were used respectively [29][30][31][32]. The amplification products were separated electrophoretically in 2% ethidium bromidestained gels and visualized under ultraviolet light.

Dog 1-Clinical Examination and Molecular Analysis
In March 2021, a 2.5-years old male English Cocker Spaniel from Zawiercie (Silesia, Poland) was presented to the veterinary clinic for many clinical symptoms, such as weakness, lethargy, appetite disorder, and others (Table 1). During the examination, two engorged ticks were detected and removed from dogs' skin. The medical history of this dog revealed no travel history in the last 2 months before the symptoms occurred. It had no anti-tick prophylaxis regardless of the fact that it was probably infected with canine babesiosis before, as a one-year-old puppy (information from the owners, Dog 1 was not a patient of this veterinary clinic before). Table 1. "Dog 1" and "Dog 2"-a compilation of clinical symptoms.

Dog 1 Dog 2
Presence of ticks The complete blood count of the dog revealed many abnormalities. First of all, severe hemolytic anemia with a low number of red blood cells (RBC), low values of hematocrit (HCT) and hemoglobin (HGB), as well as severe thrombocytopenia (PLT) and leucopenia (WBC) were reported at the beginning of the infection. Towards day 4 the CBC worsened. The number of WBC increased, while the values of HCT decreased ( Table 2, abnormalities are shown in bold). The biochemical parameters worsened as well. There was a significant fall in the levels of albumins, while there were slight changes in creatinine level (fall) and bilirubin concentration (increase). There were no abnormalities in levels of alanine and asparagine aminotransferases, lipase, and urea. Blood smears prepared from the EDTA-treated blood samples showed the presence of large-sized Babesia protozoans (Figure 1a). No morulae of Anaplasma phagocytophilum were detected in the analyzed blood smears. Blood smears prepared from the EDTA-treated blood samples showed the presence of large-sized Babesia protozoans (Figure 1a). No morulae of Anaplasma phagocytophilum were detected in the analyzed blood smears.
The molecular analysis confirmed the presence of Babesia canis in the dogs' blood. The sequencing analysis showed that the obtained sequence was 100% identical to the sequence of Babesia canis isolate 3469 (sequence ID: KX712122.1), a European B. canis strain belonging to the type B (B. canis 18S rRNA-B).
After confirmation of babesiosis (day 2), the dog was treated with imidocarb dipropionate (Imizol, 6.6 mg/kg, intramuscularly) initially and then again in two weeks. Despite the treatment, the dog's clinical condition, as well as blood results, deteriorated, so supportive therapy was needed. The dog received: a blood transfusion, intravenous fluids, and an oral antibiotic (doxycycline 200 mg per day for 14 days). Ten days after admission, health status and all blood parameters improved.

Dog 2-Clinical Examination and Molecular Analysis.
In October 2021, a 5-years old female Labrador Retriever from Zawiercie (Silesia, Poland) was presented to the veterinary clinic for dyspepsia manifestations. During the The molecular analysis confirmed the presence of Babesia canis in the dogs' blood. The sequencing analysis showed that the obtained sequence was 100% identical to the sequence of Babesia canis isolate 3469 (sequence ID: KX712122.1), a European B. canis strain belonging to the type B (B. canis 18S rRNA-B).
After confirmation of babesiosis (day 2), the dog was treated with imidocarb dipropionate (Imizol, 6.6 mg/kg, intramuscularly) initially and then again in two weeks. Despite the treatment, the dog's clinical condition, as well as blood results, deteriorated, so supportive therapy was needed. The dog received: a blood transfusion, intravenous fluids, and an oral antibiotic (doxycycline 200 mg per day for 14 days). Ten days after admission, health status and all blood parameters improved.

Dog 2-Clinical Examination and Molecular Analysis
In October 2021, a 5-years old female Labrador Retriever from Zawiercie (Silesia, Poland) was presented to the veterinary clinic for dyspepsia manifestations. During the clinical examination, other unspecific symptoms were noted, such as malaise, lethargy, and weakness (Table 1). A detailed anamnesis brought the information that the dog has not left Pathogens 2022, 11, 1329 5 of 10 the Silesian Voivodeship since it was born, had no anti-tick prophylaxis, and it has had recent tick bites (presence of skin lesions after tick bites).
The CBC showed no abnormalities except severe thrombocytopenia (PLT) ( Table 3). The biochemical analysis showed mild hyperglycemia, which may be an effect of a stress induction during a visit to the veterinary clinic, as well as a slightly lowered level of total proteins. Other biochemical parameters (alanine and asparagine aminotransferases, lipase, creatinine, and urea) were normal. Table 3. "Dog 2"-a compilation of the complete blood count (CBC) parameters. Blood smears showed the presence of pear-shaped trophozoites and merozoites of Babesia spp. inside red blood cells (Figure 1b).

Parameters CBC
Like in the first case, the molecular analysis confirmed the presence of Babesia canis in the blood of the dog. The sequencing analysis showed that the obtained sequence was 100% identical to the sequence of B. canis isolate 3469 (sequence ID: KX712122.1).
After confirmation of babesiosis (day 2), the dog was treated with imidocarb dipropionate (Imizol, 6.6 mg/kg, intramuscularly) initially and then again in two weeks. This dog also received supportive therapy (intravenous fluids and oral antibiotic-doxycycline 400 mg per day for 14 days). Seven days after admission, health status and all blood parameters improved (Table 3).

Ticks-Identification and Molecular Detection of Pathogens
Two ticks were removed from Dog 1 at the veterinary clinic in Zawiercie, Poland. Based on their morphological traits, both ticks were identified as adult ticks (one female and one male) of the species Dermacentor reticulatus. Both ticks were fully engorged ( Figure 2). None of the D. reticulatus ticks was positive for Babesia spp., A. phagocytophilum, Borrelia burgdorferi sensu lato, Rickettsia spp., or Bartonella spp.

Discussion
In the last years, the number of canine babesiosis incidents in Poland and in other Central European countries has increased [6,7,12,14]. This situation is probably a result of the rapid development of tourism with companion animals, such as domestic dogs and cats, and the wider spreading of ticks and B. canis vectors to new locations [33,34]. Furthermore, the use of sensitive detection techniques, such as PCR-based diagnostic tools, has contributed to a higher detection rate of this parasite in veterinary clinics [19,35].
So far, there have been many publications concerning the epidemiology of tick-borne diseases in domestic dogs in Europe , as well as the prevalence of tick-borne pathogens in ticks collected from pets in this area [42][43][44]. Nevertheless, the current study adds interesting new data to this subject. It is the first one to report B. canis infection in dogs with acute canine babesiosis symptoms from a new location in Poland, which was considered B. canis-free until now. Moreover, this study also presents D. reticulatus ticks feeding on one of the two Babesia-infected dogs, a dog with no travel history in the last 8 weeks before the first visit to a veterinary clinic.
In Poland, canine babesiosis is an endemic disease, which is mainly diagnosed in regions located to the east of the Vistula River (Lubelskie, Podlaskie, and Masovian Voivodships) [6,45]. Moreover, there are single reports regarding the presence of this disease in animals from western voivodships, which may be a natural consequence of the expansion of the main vector of B. canis-D. reticulatus in these areas [22,[46][47][48]. Our study presents two cases of B. canis in dogs from the Silesia Voivodeship, a region of Poland, where B. canis was not previously detected, except for one case in the city of Żywiec, where a Babesia-positive dog that originally came from the vicinity of Warsaw was diagnosed [49].
Both examined dogs were thoroughly diagnosed, according to the applicable diagnostic guidelines for veterinarians, based on the clinical examinations, as well as

Discussion
In the last years, the number of canine babesiosis incidents in Poland and in other Central European countries has increased [6,7,12,14]. This situation is probably a result of the rapid development of tourism with companion animals, such as domestic dogs and cats, and the wider spreading of ticks and B. canis vectors to new locations [33,34]. Furthermore, the use of sensitive detection techniques, such as PCR-based diagnostic tools, has contributed to a higher detection rate of this parasite in veterinary clinics [19,35].
So far, there have been many publications concerning the epidemiology of tickborne diseases in domestic dogs in Europe , as well as the prevalence of tick-borne pathogens in ticks collected from pets in this area [42][43][44]. Nevertheless, the current study adds interesting new data to this subject. It is the first one to report B. canis infection in dogs with acute canine babesiosis symptoms from a new location in Poland, which was considered B. canis-free until now. Moreover, this study also presents D. reticulatus ticks feeding on one of the two Babesia-infected dogs, a dog with no travel history in the last 8 weeks before the first visit to a veterinary clinic.
In Poland, canine babesiosis is an endemic disease, which is mainly diagnosed in regions located to the east of the Vistula River (Lubelskie, Podlaskie, and Masovian Voivodships) [6,45]. Moreover, there are single reports regarding the presence of this disease in animals from western voivodships, which may be a natural consequence of the expansion of the main vector of B. canis-D. reticulatus in these areas [22,[46][47][48]. Our study presents two cases of B. canis in dogs from the Silesia Voivodeship, a region of Poland, where B. canis was not previously detected, except for one case in the city ofŻywiec, where a Babesia-positive dog that originally came from the vicinity of Warsaw was diagnosed [49].
Both examined dogs were thoroughly diagnosed, according to the applicable diagnostic guidelines for veterinarians, based on the clinical examinations, as well as microscopic and molecular analyses, which confirmed the presence of B. canis infection. In both cases, the Babesia infected dogs had symptoms of acute canine babesiosis with severe thrombocytopenia, elevated temperature, weakness, anemia, hemoglobinuria, vomiting, anorexia, apathy, and lethargy, which are typical symptoms of the infection caused by European B. canis strain belonging to the type B (B. canis 18S rRNA-B) [50][51][52]. In the current study, both B. canis sequences identified in the blood of the dogs were identical to the sequence of the more virulent type B, which was previously noted in symptomatic dogs from endemic regions of Poland, as well as other countries in Central Europe [7,16,35]. Due to the lack of information on previously reported symptomatic cases of canine babesiosis in the Silesian Voivodeship in Poland, the presence of clinical manifestations in two dogs caused the launch of other potential diagnoses (intoxication or neoplastic diseases), but no other diseases were confirmed. Detailed anamnesis showed the information that Dog 1 suffered from babesiosis in the past. There is no information about the circumstances of the previous Babesia infection and the travel history of this dog because it became a patient of our veterinary clinic at the beginning of 2021. Therefore, we could not exclude that the owners changed their place of residence from the area in Poland, where canine babesiosis is endemic. On the other hand, we cannot be confident that the previous diagnosis was correct because we have no information about the diagnostic procedures that were used. The studies conducted by Adaszek et al. 2011 [6] and Neelawala et al. 2021 [53] confirmed that dogs with recurrent babesiosis are noted to be prone to develop systemic complications. The case of Dog 1 was severe, with blood transfusion and strongly expressed symptoms. Therefore, relapsing babesiosis should not be ruled out. The obtained results indicate that infections with B. canis in dogs from the Silesian Voivodeship should be taken into account during differential diagnosis of tick-borne diseases.
In Central Europe, canine babesiosis is mostly caused by B. canis, and as we previously mentioned, its prevalence is strictly dependent on the presence of D. reticulatus ticks in the environment [54]. This tick species prefers rather humid habitats, which may positively correspond with the location of Zawiercie (50 • 29 15 N, 19 • 24 59 E), in the basin of the Warta and Odra rivers, among the deciduous and coniferous forests. So far, only one study described the presence of three D. reticulatus ticks collected from domestic dogs in two cities of Silesian Voivodeship (Racibórz andŻywiec), where the ticks from Racibórz were detected on a residential dog [49]. In order to estimate the local risk of B. canis infection, the prevalence rate should be determined by questing D. reticulatus ticks from the Silesian Voivodeship, although this species has not been recorded in the environment at this location yet.
In our study, both of the collected D. reticulatus ticks from Dog 1 with canine babesiosis were Babesia spp. negative. This result confirms that the infection status of the dog may not necessarily reflect the infection rate of ticks [20]. The role of Ixodes ricinus, the most common tick species in Poland, as a vector for B. canis should not be excluded, especially in the case of B. canis type A (B. canis 18S rRNA-A), which is responsible for a milder course of the canine babesiosis in domestic dogs [7,16,55,56]. There are studies from northern Poland [57], as well as from Croatia, Slovakia, and the Czech Republic [58,59], which show the presence of B. canis DNA in this tick species. I. ricinus is involved in the transmission of a large variety of pathogens of medical and veterinary importance, but its role in transmitting this haemoprotozoan species is unclear and needs to be further investigated.
Our study shows the occurrence of acute symptomatic canine babesiosis in dogs from the Silesian Voivodeship in Poland. These are the first reported cases of B. canis infections in dogs from this location, where D. reticulatus ticks have never been found in their natural habitat, apart from the host. Our findings should raise awareness of B. canis infection among dog owners and veterinarians in this region and ensure that adequate diagnostic methods are available. Funding: This research and the APC were funded by the Medical University of Silesia in Katowice, the grant number: PCN-2-081/N/1/I; Title: "The pilot study of companion animals' blood for detection of vector-borne pathogens and estimation of potential human exposure to zoonoses". Institutional Review Board Statement: Ethical review and approval were waived for this study due to blood samples used in our study consisting of surplus material, which was collected for routine diagnosis from dogs brought to the local veterinary clinic. The samples were not collected for study reasons. We declare that all testing methods have been carried out in accordance with the relevant guidelines and regulations, as well as all of the experimental protocols have been approved by the Medical University of Silesia in Katowice.

Informed Consent Statement:
Each dog owner has agreed to use the material (ticks/blood-surplus material used for routine diagnosis at the veterinary clinic) from their animals and give informed consent to publish the results of the collected material.
Data Availability Statement: Not applicable.