Discovery of a Novel Species Infecting Goats: Morphological and Molecular Characterization of Babesia aktasi n. sp.

A novel Babesia sp. infecting goats was discovered based on the molecular findings obtained in the current study, which was conducted in the Mediterranean region of Türkiye. The goal of this study was to isolate this species of Babesia (Babesia sp.) infecting goats in vivo and to assess the genetic and morphological characterization of the parasite. To identify the animal naturally infected with Babesia sp. and isolate the parasite from this animal, field studies were conducted first, and genomic DNA were extracted from blood samples taken from goats (n = 50). The Theileria, Babesia, and Anaplasma species were identified using a nested PCR-based reverse line blotting (RLB) method. The study included one goat that was determined to be infected with Babesia sp. (single infection) in RLB for in vivo isolation. A blood smear was prepared to examine the parasite’s morphology, but it was found to be negative microscopically. Following that, a splenectomy operation (to suppress the immune system) was performed to make the parasites visible microscopically in this animal. Parasitemia began after splenectomy, and the maximum parasitemia was determined to be 1.9%. The goat displayed no significant symptoms other than fever, loss of appetite, and depression. During a period when parasitemia was high, blood from this goat was inoculated into another splenectomized goat (Theileria-Babesia-Anaplasma-Mycoplasma spp. free). On the third day of inoculation, 10% parasitemia with high fever was detected in the goat, and on the fourth day, the goat was humanely euthanized due to severe acute babesiosis symptoms. Except for mild subcutaneous jaundice, no lesions were discovered during the necropsy. According to the microscopic measurement results, ring, double pyriform, spectacle-frame-like, and line forms were observed, and it was observed to be between 1.0–2.5 µm (1.38 ± 0.17 to 0.7 ± 0.21-all forms). A phylogenetic analysis and sequence comparison using the 18S rRNA and cox1 genes revealed that this species is distinct from the small ruminant Babesia species (18S rRNA 92–94%, cox1 79–80%) and has the highest similarity to Babesia sp. deer, which has been reported in deer. Furthermore, it was determined to resemble B. venatorum, B. divergens, Babesia sp. FR1 and Babesia sp. MO1 species, all of which are zoonotic. Additional research is needed to clarify the clinical status of this parasite in goats and other hosts (mountain goat, sheep, calf).


Introduction
Shortly after the discovery of Babesia in bovine erythrocytes by Victor Babes in 1888, the same researcher reported that a parasite with similar characteristics also infected sheep [1]. Since then, it has been reported that Babesia ovis, B. motasi, B. crassa, B. taylori and B. foliate cause babesiosis in sheep and goats [2]. Babesiosis is a tick-borne disease caused by the genus Babesia, which is frequently observed in domestic and wild animals in tropical and subtropical regions [2][3][4]. The most pathogenic species is Babesia ovis, which is endemic to southern Europe, Africa, the Middle East, and Asia [5], and causes severe economic losses in sheep and goats [5]. Babesia crassa, which has low pathogenicity, was isolated in Iran, and B. motasi, which includes more than one species and subspecies, was isolated in China and Europe. B. motasi infections can cause mild clinical signs in sheep but can lead to severe Pathogens 2023, 12, 113 2 of 14 anemia and death in goats [6].Human babesiosis caused by B. motasi and B. crassa has also been reported sporadically in Asia [7,8]. There is very little information on B. taylori and B. foliate, which have been reported to infect sheep and goats but for which no molecular data is available [4].
In the last two decades, the use of molecular diagnostic techniques to investigate ticks and tick-borne agents has increased dramatically, and as a result, new species of piroplasm have been discovered [9][10][11][12][13][14]. Using PCR-based reverse line blot (RLB) hybridization, which is used for the detection and identification of piroplasm species, novel Theileria and Babesia species were identified. [15,16]. This study's objective was to perform morphological and molecular characterization of a new species of Babesia previously reported in goats based on molecular data [16].

Study Area and Animal Samples
In 2016, a new Babesia sp. was detected molecularly in goats in Anamur, Mersin province (36 • 01 17 N, 32 • 48 07 E), in the Mediterranean region of Turkiye [16]. In order to investigate more detailed information (morphological and molecular data) about this parasite, blood samples were collected from goats by visiting Mersin province between 2018 and 2020. Mersin has a typical subtropical Mediterranean climate with hot and humid summers and mild and rainy winters. The Taurus Mountains, a mountain complex in southern Türkiye that separates the Mediterranean coastal region from the Central Anatolian Plateau, are located in this region. Sheep and goats are kept in the sheepfold during lambing and at the beginning of milking (January-March), and on pasture during the remainder of the milking season (April-May) in the Taurus Mountains. Additionally, mountain goats inhabit this region [17].

Determination of New Babesia sp. Infected Goat in Field Samples
Blood samples were taken from the vena jugularis of 50 randomly selected goats (apparently healthy) in Mersin province (Anamur, Bozyazi) and placed into EDTA tubes. Animals whose blood samples were taken, were treated with acaricide (Flugon ® 1%, Vetas, Turkiye) and kept in a tick-free environment until the completion of the PCR results. The genomic DNA was extracted from 200 µL of EDTA anticoagulated blood samples from the goats using a kit (PureLink TM Genomic DNA Mini Kit, Invitrogen Corporation, Carlsbad, CA, USA) according to the manufacturer's instructions. For the determination of new Babesia sp., and mixing infection with blood parasites in field samples, a nested PCR was performed to use in an RLB assay for the Anaplasma/Ehrlichia and Theileria/Babesia species, using Ec9/Ec12A [18]-16S8FE/B-GA1B [19] and Nbab1F/Nbab1R [9]-RLBF2/RLBR2 [20] primers, respectively. The nested PCR products were used in reverse line blotting (RLB) to detect the Anaplasma/Ehrlichia and Theileria/Babesia species. Additionally, positive RLB samples were analyzed for hemotropic mycoplasma using nested PCR with 8F/1492R-F2/R2 primers [21,22].
To obtain the near full sequence of the 18S rRNA gene region, PCR was performed using Nbab1F-Nbab1R primers [9] to confirm the determination of new Babesia sp. and to gain further molecular data analysis, and modified nested PCR protocols amplifying cytochrome c oxidase subunit 1 (cox1) gene were performed using the BaFor1/BaRev1 and BaFor2/BaRev2 primers [23]. PCR products were electrophoresed on an agarose gel containing 1.4% agarose, stained with ethidium-bromide, and sequenced by a private company. (BM-Labosis, Turkiye). The primers and probes used in the study are listed in Supplementary Table S1.

Experimental Study and Monitoring Animals
The goat (ID: Manay, 3 year-old female) infected with new Babesia sp., as determined by nested PCR-based RLB and sequence analysis, and another goat (ID: Oglak, 5 monthold male) free of blood parasites, were both brought to the Firat University Veterinary Faculty for experimental research. The animals (Manay and Oglak) were relocated to a separate compartment, and their care and feeding were continued throughout duration of the experiment. Throughout the experimental study, flumethrine 1% (Flugon ® 1%, Vetas) was applied every 21 days to prevent tick infestations. Before the splenectomy, the goat was PCR and RLB tested for Babesia-Theileria-Anaplasma-Mycoplasma spp. Firstly, a splenectomy was performed to suppress the immune system of the goat named oglak and it was examined for blood parasites using the PCR method at certain intervals for about 30 days until the time of the experimental infection [24]. A splenectomy was conducted on the goat, named manay, who tested positive for Babesia sp., and 20 mg of dexamethasone (Vetakort ® 4 mg, Vetas-intramuscular injection) was administered for three days after the operation. The surgical procedures utilized in the splenectomy surgery were carried out exactly as described by Sevinc et al. [25]. Manay was evaluated daily after splenectomy for clinical responses, rectal temperature, and the presence of piroplasm parasites in peripheral blood smears. During the peak of parasitemia, 20 mL of infected blood from this animal was administered to Oglak. Similar to Manay, Oglak was inspected daily, and its parasitemia was determined. In addition, when parasitemia was detected, 20 mg of dexamethasone was administered to Oglak ( Figure 1). To measure parasitemia in both animals, blood smears were taken from the animals' ear tips and stained with Giemsa dye, and parasitemia was estimated using the method published by Luo et al. [26]. After the parasites appeared, as suggested by Uilenberg et al. [27] and Guan et al. [28], measurements were taken with an Olympus microscope BX43 (Olympus, Tokyo, Japan) and photographs were taken with an Olympus DP72 Digital Camera System (Olympus, Tokyo, Japan).

Phylogenetic and Percent Identity Matrix Analyses
Phylogenetic analyzes of new Babesia sp. isolated in this study were carried out using sequences of 18S rRNA and cox1 genes from Babesia species isolated in vertebrate hosts. Two separate phylogenetic trees were constructed for 18S rRNA and cox1 sequences using MEGAX software [29]. The nearly full-length complete sequence of the 18S rRNA (1709 base pairs) and cox1 (~900 bp) genes determined for new Babesia sp. was compared to other targeted Babesia species using Percent Identity Matrix analysis (http://www.ebi.ac.uk/ Tools/msa/clustalo accessed on 6 November 2022).

The Host's Ability to Control Parasitemia Is Diminished by Splenectomy and Pharmacological Immunosuppression with Dexamethasone
The first piroplasm forms of the novel Babesia sp. were seen microscopically in Manay on the tenth day after the splenectomy. Parasitemia reached its peak (1.9%) on the 13th postoperative day, then declined for two more days (14 and 15 days), with no agent observed in the peripheral blood on the 16th. The animal's body temperature was found to be fluctuating. On the seventh postoperative day, there was an increase in body temperature (40.9 °C), followed by a slight decrease for the next two days, and then another increase (40.5 °C) on the tenth day. Fever, anorexia, and depression were observed after splenectomy until the parasitemia disappeared. On the day that the parasite percentage in the peripheral blood reached 1.9%, 20 mL of blood was drawn from manay and inoculated into Oglak. Before inoculation, Oglak was tested for Babesia, Theileria, Anaplasma, and Mycoplasma spp. using PCR-RLB and found to be negative. When the parasite was noticed in blood smears, Oglak was given 20 mg of dexamethasone intramuscularly every day during 4 consecutive days. Piroplasm forms were observed in the Oglak's peripheral blood on the second day after parasite inoculation, parasitemia (10%) and an increase in body temperature (40.7 °C) were observed on the third day ( Figure 1), and the goat was humanely euthanized on the fourth day due to severe acute babesiosis symptoms (fever, low PCV, and anemia). Except for minor icterus under the skin, the necropsy revealed no macroscopic babesiosis signs.

Ethics Statement
This study was carried out according to the regulations of animal and welfare issued by the Turkish legislation for the protection of animals (Animal Experiment Ethic Committee, protocol no: 2018/100).

The Host's Ability to Control Parasitemia Is Diminished by Splenectomy and Pharmacological Immunosuppression with Dexamethasone
The first piroplasm forms of the novel Babesia sp. were seen microscopically in Manay on the tenth day after the splenectomy. Parasitemia reached its peak (1.9%) on the 13th postoperative day, then declined for two more days (14 and 15 days), with no agent observed in the peripheral blood on the 16th. The animal's body temperature was found to be fluctuating. On the seventh postoperative day, there was an increase in body temperature (40.9 • C), followed by a slight decrease for the next two days, and then another increase (40.5 • C) on the tenth day. Fever, anorexia, and depression were observed after splenectomy until the parasitemia disappeared. On the day that the parasite percentage in the peripheral blood reached 1.9%, 20 mL of blood was drawn from manay and inoculated into Oglak.
Before inoculation, Oglak was tested for Babesia, Theileria, Anaplasma, and Mycoplasma spp. using PCR-RLB and found to be negative. When the parasite was noticed in blood smears, Oglak was given 20 mg of dexamethasone intramuscularly every day during 4 consecutive days. Piroplasm forms were observed in the Oglak's peripheral blood on the second day after parasite inoculation, parasitemia (10%) and an increase in body temperature (40.7 • C) were observed on the third day (Figure 1), and the goat was humanely euthanized on the fourth day due to severe acute babesiosis symptoms (fever, low PCV, and anemia). Except for minor icterus under the skin, the necropsy revealed no macroscopic babesiosis signs.
Parasites in infected erythrocytes have been described in various morphological forms including ring, paired pyriform, spectacle frame-like, and line. Except for the ring forms, it was observed that other forms had no transparent and clear cytoplasm.
For the paired pyriform, it was observed that the paired pyriform, unlike the ring forms, did not have a significant cytoplasm. These forms did not appear to be in contact with the two merozoites ( Figure 2, plates 5-8), unlike the double pear forms typically found in many other Babesia species. It was also observed that most of the infected erythrocytes contained only one pair of parasites (Figure 2, plates 5-8), although sometimes it was more than one (Figure 2, plate 21). The size of the piroplasms varied from 0.96 to 1.64 × 0.48 to 1.03 µm, with mean dimensions of 1.26 (±0.18) µm × 0.7 (±0.15) µm.
With regard to the spectacle frame-like type, this form appears microscopically as if they were surrounding the red blood cell like the diameter of a circle. It is called spectacle frame-like because it resembles the shape of a spectacle frame (Figure 2, plates 9-12). In most of the erythrocytes, it was observed that the paired pyriform, which is oval (Figure 2, plates 9, 10, 12) or has a thick line (Figure 2 With regard to the line form, this form is found close to the red blood cell membrane as a thick arc-shaped line. (Figure 2, plates 13-16). The size of the piroplasms varied from 0.95 to 2.12 × 0.28 to 0.68 µm with mean dimensions of 1.64 (±0.31) µm × 0.51 (±0.11) µm.
The new type of Babesia sp. (1.38 ± 0.17 to 0.7 ± 0.21-all forms) is defined as small Babesia because it is located between 1.0-2.5 µm according to the microscopic measurement results. In slides stained with Giemsa, the cytoplasm is not clearly visible in other forms, except for the ring form. Up to four merozoites were seen in an infected erythrocyte (Figure 2, plates 21-24). Furthermore, the different forms of the new Babesia sp. were presented graphically (Figure 2, plates 25).

Sequence Comparisons and Phylogenetic Analysis
The nearly full-length sequence of the 18S rRNA gene of new Babesia sp. was determined from DNA obtained from splenectomized goats (manay and oglak). Sequences were deposited in the EMBL/GenBank databases under accession number OM864353, and MN559399. These two sequences and previously reported sequences (KU714605-KU714606) are 99-100% similar to each other. The sequence identity of (%) between the newly recognized Babesia sequence and other targeted Babesia species is presented in  The partial cox1 sequence of new Babesia sp. obtained in this study was registered with GenBank under accession numbers OM718699 and OM718698. Nucleotide sequence identities showed that our Babesia sp. sequences were highly similar to Babesia sp. deer (MG344869, MG344859), with an identity of 92. 23 B. bovis, B. bigemina, B. major, and B. ovata, respectively (Figure 4). The partial cox1 sequence of new Babesia sp. obtained in this study was registered with GenBank under accession numbers OM718699 and OM718698. Nucleotide sequence identities showed that our Babesia sp. sequences were highly similar to Babesia sp. deer (MG344869, MG344859), with an identity of 92. 23 B. odocoilei, B. capreoli, B. venatorum, B. divergens, B. ovis, B. motasi, Babesia sp. Xinjiang, Babesia sp. Lintan, B. bovis, B. bigemina, B. major, and B. ovata, respectively (Figure 4). A phylogenetic analysis was performed using 18S rRNA and cox1 sequences, including the Babesia sp. sequences identified in this study and other available Babesia sequences from the GenBank. Phylogenetic trees of 18S rRNA and cox1 sequences using the Tamura-Nei model (G+I) [29,31] and the General Time Reversible model (G+I) [29,32] are shown in Figures 5 and 6, respectively. A phylogenetic analysis was performed using 18S rRNA and cox1 sequences, including the Babesia sp. sequences identified in this study and other available Babesia sequences from the GenBank. Phylogenetic trees of 18S rRNA and cox1 sequences using the Tamura-Nei model (G+I) [29,31] and the General Time Reversible model (G+I) [29,32] are shown in Figures 5 and 6, respectively.

Discussion
Babesia species are common in domestic and wild animals in tropical and subtropical regions around the world, including Türkiye, and cause clinical infections with high mortality rates [16,33,34]. Even though the Babesia parasite was discovered approximately 140 years ago, we still only know very little about it. More than 100 Babesia species have been described to date, and new species in vertebrates continue to be discovered in various parts of the world [2][3][4][9][10][11][12][13][14]28]. Molecular tests, which are mostly used for epidemiological studies in a given region, are widely used in research to detect parasites, confirm their presence, and discover new species or genotypes [35]. A molecular survey conducted in 2016 revealed the possibility of a new species in goats [16]. However, we were unable to determine whether this species actually infected goats or was present transiently in these animals. Recent review articles on this topic have stated that it is impossible to identify a new species based solely on gene sequences without observing the organism in question [35,36]. To demonstrate the presence of this parasite in goats, one animal (single infection)

Discussion
Babesia species are common in domestic and wild animals in tropical and subtropical regions around the world, including Türkiye, and cause clinical infections with high mortality rates [16,33,34]. Even though the Babesia parasite was discovered approximately 140 years ago, we still only know very little about it. More than 100 Babesia species have been described to date, and new species in vertebrates continue to be discovered in various parts of the world [2][3][4][9][10][11][12][13][14]28]. Molecular tests, which are mostly used for epidemiological studies in a given region, are widely used in research to detect parasites, confirm their presence, and discover new species or genotypes [35]. A molecular survey conducted in 2016 revealed the possibility of a new species in goats [16]. However, we were unable to determine whether this species actually infected goats or was present transiently in these animals. Recent review articles on this topic have stated that it is impossible to identify a new species based solely on gene sequences without observing the organism in question [35,36]. To demonstrate the presence of this parasite in goats, one animal (single infection) infected with new Babesia sp. was detected in the field study using the RLB method.
Experimental infections have been shown in splenectomized and dexamethasone immunodepression sheep and goats using the Babesia species. Because splenectomy reduces the host's ability to control parasitemia, it allows for the large-scale expansion of previously undetected parasite populations to detectable and, in some cases, clinically significant levels [37,38]. High fever (42 • C), weakness, anorexia, anemia, and hemoglobinuria were observed in sheep with spleen-intact and splenectomized sheep during an experimental infection with B. ovis [39]. In another experiment with Babesia sp. Xinjiang, spleen-intact sheep did not develop parasitemia or clinical signs, whereas the splenectomized group developed fever (41.5 • C), parasitemia, and hemolytic anemia [28]. In a study conducted in China [24], blood from naturally infected sheep with Babesia sp. BQ1 (Ningxian) was inoculated into two splenectomized and spleen-intact sheep [24]. Body temperature increased (41.5 • C) in the splenectomized sheep on the fifth day after parasite inoculation, severe clinical findings with parasitemia developed, and the sheep died on the seventh day. On the fourth day after parasite inoculation, piroplasm forms were seen in the peripheral blood, and body temperature (42 • C) and parasitemia (1.9%) increased, and the sheep with severe clinical findings survived the disease [24]. Three splenectomized sheep were infected with Babesia divergens, the main cause of bovine and human babesiosis in Europe, using an in vitro stabilate, and all sheep developed a high fever and transient parasitemia between 6 and 9 days after infection [40]. The newly identified parasite Theileria haneyi was used in an experiment at the US-Mexico border. Blood from a horse infected with T. haneyi was administered to another spleenectomized horse in order to microscopically identify this novel parasite [14]. In this study, a splenectomy was used to morphologically characterize a new Babesia sp. identified by PCR and RLB. The first piroplasm was observed on the tenth day after splenectomy, and the parasitemia reached 1.9% on the 13th day. The parasitemia lasted 5 days in this goat, with no clinical signs other than high fever, anorexia, and death. Pure infected blood stabilate obtained in vivo from this animal was administered to another goat (Oglak) that was free of blood parasites (Babesia-Theileria-Anaplasma-Mycoplasma spp.). This animal was humanely euthanized after developing severe clinical signs (high fever, anemia), and necropsy revealed mild jaundice and anemia. According to Koch's postulate, re-isolating this parasite from the second goat (Oglak) and demonstrating that it is identical to the original parasite is critical for identifying a new parasite [36].
Babesia parasites are classified into two groups based on their size: large (2.5-5.0 µm long) and small (1.0-2.5 µm long) [41]. The new Babesia sp. described in this study was included in the small group of Babesia. Although morphologically divided into two groups, large and small forms of the same parasite can be found. A sequence analysis of parasites thought to be B. motasi morphologically revealed that they were B. ovis, and that there were large and small forms of this parasite [42]. Babesia crassa, an isolate from Iran and member of the large Babesia group, differs from the others in that it has four parasites in one infected erythrocyte. Babesia sp. Xinjiang, a new type of Babesia isolated in sheep in China, is also included in the large Babesia group.
According to phylogenetic analyses, the Babesia sp. isolated in this study was genetically related to Babesia sp. deer, B. odocoilei, B. venatorum, B. capreoli, and B. divergens. There have been reports of B. capreoli, B. venatorum, and B. divergens in Europe [16,43,44]. Babesia sp. deer has been reported in the Czech Republic as an unnamed species in red (Cervus elaphus) and sika (Cervus nippon) deer [43]. Wapiti/elk, reindeer, and caribou are the natural reservoirs of B. odocoilei. [43,45]. The primary vector of B. odocoilei is Ixodes scapularis (Acari: Ixodidae), which causes fatal infections in cervids [45,46]. Babesia odocoilei has also been detected in I. scapularis ticks collected from domestic dogs and cats in Canada [47].
Babesia venatorum is capable of infecting humans, as well as chamois (Rupicapra rupicapra) and ibex (Capra ibex) in the Alpine region [48]. Babesia venatorum is known to infect humans in Europe, and the roe deer is its reservoir host. Human cases of B. venatorum have been reported in Europe and, more recently, China [49,50]. Babesia venatorum has been found in sheep populations, and it has been suggested that farm animals may play a role in the spread of this parasite [51]. Babesia capreoli has been found in a variety of deer species, with a particularly high prevalence in roe deer [44,52]. There have been no reports of infections in humans. Babesia divergens causes acute babesiosis in cattle and humans [53,54] and has been found in roe, red, and sika deer, chamois, and Alpine ibex (Capra ibex) in Europe [43,48,54]. During field studies, no natural clinical infections caused by new Babesia sp. were detected in domestic goats. In a phylogenetic analysis based on the 18S rRNA gene region, B. venatorum, B. odocoilei, B. capreoli, and B. divergens were included in a clade proposed to be named as Babesids, and the host-specificity of this clade was reported to be lower than that of ungulibabesids (B. bovis, B. bigemina, B. caballi) [55]. The nucleotide sequence analysis revealed that 18S rRNA and cox1 sequences of our Babesia sp. were most similar to those of Babesia sp. deer, sharing 98.36% and 92.23% identity, respectively. In addition, phylogenetic analyses revealed that the new Babesia sp. and Babesia sp. deer formed a sister clade. Although useful for diagnosis, the 18S rRNA cannot distinguish definitively between Babesia species and strains. For example, B. divergens (cattle-human-deer) and B. capreoli (roe deer), which are nearly identical (only three nucleotide differences) according to the 18S rRNA sequence, are distinct species that infect different hosts. It has also been reported that the Babesia sp. FR1 isolate, which was identified in humans in France, differs from B. capreoli by only one nucleotide according to the 18S rRNA sequence [56]. Consequently, the cox1 gene locus has greater genetic diversity than the 18S rRNA gene for determining phylogenetic relationships and distinguishing Babesia species [4,43,57]. The cox1 sequences analysis revealed that the similarity between B. divergens and B. capreoli was 92.23%, as in the new Babesia sp. and Babesia sp. deer species. As a result, despite the fact that the 18S rRNA gene regions of our newly discovered Babesia sp. and Babesia sp. deer were 98.30-98.36% similar, they were classified as two distinct species due to differences in their cox1 sequences and for being identified in different hosts. A~900 bp long mitochondrial cox1 gene alignment of two cox1 sequences from new Babesia sp. with their respective cox1 nucleotide sequences from other piroplasmids confirmed that new Babesia sp. resulted in a strongly supported clade, confirming its identity as a new species.

Conclusions
In this study, the morphological and genetic characteristics of a new Babesia sp. previously described based solely on molecular analyses in goats were determined. There was a genetic relationship between this newly discovered Babesia sp. and other species reported in deer, mountain goats, cattle, and humans at various proportions. It is known that mountain goats inhabit the region where Babesia sp. was defined [17]. It is necessary to conduct additional research on the clinical status and vector competence of this parasite in goats (spleen intact/splenectomy) and other hosts (mountain goats, sheep, calves). Furthermore, the zoonotic significance of this parasite should be investigated, as it shares 97.5% similarity with zoonotic B. venatoum, B. divergens, Babesia sp. FR1, and Babesia sp. MO1 species and genotypes, as determined by the 18S rRNA gene.