https://orcid.org/0000-0002-4492-143X
Figures
Abstract
Small ruminant babesiosis remains a neglected disease despite causing significant economic losses to sheep and goat herds in many regions around the world. The pathogenesis and clinical manifestations of ovine babesiosis are well-known, but there is a lack of information regarding caprine babesiosis. Since the discovery of the first Babesia spp. in 1888, several species/subspecies/genotypes, including Babesia aktasi, have been described. Our recent molecular survey revealed that the parasite is highly prevalent (22.5%) in indigenous goats from Mediterranean region of Türkiye. The aim of this experimental study was to determine the pathogenicity and virulence of B. aktasi in immunosuppressed (n = 5) and immunocompetent (n = 7) purebred Saanen goats. The goats were experimentally infected with fresh B. aktasi infected blood, and examined for clinical, parasitological, hematological, and serum biochemical findings throughout the infection. Following the parasite inoculation, intra-erythrocytic parasites were detected from the 1st day post-infection, followed by an increase in rectal temperature and parasitemia. The parasitemia was detected ranging from 4.3% to 33.5% in the immunosuppressed group, while it was 2.1% to 7.6% in the immunocompetent. Severe clinical symptoms characterized by anemia, jaundice, and hemoglobinuria developed in both groups. A statistically significant inverse correlation was observed between the increase in parasitemia and RBC, WBC, HCT, and Hb values in the goats compared to pre-infection levels. Values of AST, ALT, GGT, Total bilirubin, and Albumin showed a significant increase, with all the immunosuppressed goats dying on the 4th and 7th days post-infection, while four out of seven immunocompetent goats died on between 6-8th days. Severe edema in the lungs, frothy fluid in the trachea, jaundice in the subcutaneous and mesenteric fat, and dark red urine were detected in necropsy. The results obtained in this study demonstrated that B. aktasi was highly pathogenic to purebred Saanen goats. Current work assures valuable insights into the pathogenesis and virulence of B. aktasi and serves as a foundation for future studies to develop effective control strategies against caprine babesiosis.
Author summary
Advancements in molecular parasitology have led to the discovery of novel Babesia species/genotypes, including Babesia aktasi, identified in goats in the Mediterranean region of Türkiye. Host resistance against blood protozoa such as Babesia and Theileria exhibits different behaviors, but there is a lack of information about host resistance against Babesia spp. in goats and sheep. The pathogenicity of B. aktasi in local breeds has been revealed in previous studies. There may be differences in susceptibility to Babesia parasites between different breeds of the same host species. To better understand the biology of B. aktasi, its pathogenesis and virulence were revealed by experimental infection in immunosuppressed and immunocompetent purebred Saanen goats. Saanen goats in both groups were infected with fresh blood infected with B. aktasi and a rapid increase in parasitemia was observed. Both groups experienced severe clinical infection, resulting in mortality. Significant changes in hematological and biochemical parameters compared to pre-infection levels were noted in both groups, revealing that B. aktasi is highly pathogenic for purebred Saanen goats. It is a step forward in elucidating the biology of B. aktasi and provides a basis for future studies to develop effective control strategies against caprine babesiosis.
Citation: Ulucesme MC, Ozubek S, Aktas M (2024) Experimental infection of purebred Saanen goats high pathogenicity and virulence of Babesia aktasi. PLoS Negl Trop Dis 18(12): e0012705. https://doi.org/10.1371/journal.pntd.0012705
Editor: Rana Nagarkatti, Center for Biologics Evaluation and Research, Food and Drug Administration, UNITED STATES OF AMERICA
Received: May 16, 2024; Accepted: November 18, 2024; Published: December 2, 2024
Copyright: © 2024 Ulucesme et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.
Funding: This work was supported by the Scientific and Technological Council of Türkiye (TUBITAK) Grant Program (project number: 221O119). MCU, SO, and MA received the grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Babesia is a genus of intra-erythrocytic protozoan parasites in various parts of the world, where the responsible vector ticks are prevalent [1–3]. Since the first discovery of Babesia in 1888, many species, subspecies or genotypes have been described that infect domestic and wild mammals, some of which also have zoonotic character [4]. Typically, Babesia parasites cause serious clinical signs characterized by high fever, hemolytic anemia (destruction of red blood cells), icterus, hemoglobinuria, and in severe cases, death [5]. Babesia ovis, B. motasi and B. crassa are the main Babesia spp. responsible for small ruminant babesiosis [3,6,7]. Of these, B. ovis is one of the first identified Babesia spp. caused by clinical infection with high mortality in tropical and subtropical countries [8–10].
In light of advances in molecular parasitology in recent years, novel species or genotypes have been added to the blood protozoans belonging to the Babesia genus. In this context, Babesia lengau-like, Babesia sp. Xinjiang and B. motasi-like complex have been reported to cause babesiosis in small ruminants [6,11–13]. Studies based on apicoplast and mitochondrial genome analyses revealed that the parasites in the B. motasi-like group were divided into two different branches, one branch includes Babesia sp. Lintan and Babesia sp. Tianzhu, while the other includes Babesia sp. Hebei and Babesia sp. Ningxian [14,15]. In a recent study based on molecular methods, we have identified a novel Babesia sp. that is distinct from other Babesia pathogens [16]. According to Robert Koch’s postulates [17], then, in vivo isolation of the newly identified Babesia sp. was made from a naturally infected goat, and named Babesia aktasi [18]. In our large molecular survey carried out in the Mediterranean region of Türkiye, it was detected that 22.5% of the goats (113/503) were infected with the parasite [19]. Then, an experimental study performed on the immunocompetent goats revealed that B. aktasi did not cause typical clinical findings of babesiosis (anemia, icterus, hemoglobinuria) except for increased body temperature [20].
Türkiye serves as a vast land bridge between European and Asian countries, where ovine babesiosis is significant due to the favorable geographical and climatic conditions that support the maintenance of vector ticks [21]. This disease is the most common parasitic infection that seriously affects the health of sheep and goats in Türkiye, leading to substantial economic losses because of the costs associated with control and treatment [7,9]. The recent nationwide epidemiological study conducted in Türkiye indicates that the distribution and prevalence of ovine babesiosis can vary regionally [22]. In particular, the disease is reported to be more prevalent in the Central and Southeastern Anatolia regions of Türkiye [22]. Babesia ovis, B. crassa, and B. motasi have been molecularly identified in small ruminant and ixodid ticks in Türkiye [19,23–26].
Host resistance to blood protozoans such as Babesia and Theileria involves a complex interplay of various immune responses [27,28]. The host resistance to these parasites can vary among different host species and even among individuals within a species [29]. The pathogenicity of these parasites is expected to be higher in purebred breeds than in native breeds. It has been reported that purebred Bos indicus cattle show higher levels of resistance to babesiosis than Bos taurus [30]. The pathogenicity of B. aktasi in purebred Saanen goats is unknown. It is important to conduct pathogenicity studies to comprehensively understand the ability of B. aktasi to cause clinical disease in purebred goats. Therefore, an experimental study using fresh B. aktasi-infected blood was performed to assess pathogenicity and virulence of the parasite in both immunosuppressed and immunocompetent purebred Saanen goats.
Materials and methods
Ethics statement
This study was approved by the Fırat University Animal Experiments Local Ethics Committee (session number: 2021/12).
Research material
The stabilate of B. aktasi, as previously described by Ozubek et al. [20], was used in this study. An indigenous goat was experimentally infected with the stabilate. When parasitemia (PPE) reached 9.2–11.5%, jugular venous blood was collected into vacutainer tubes coated with EDTA. Infected red blood cells were cryopreserved with 10% dimethyl sulfoxide (DMSO) in 5 mL aliquots and frozen in liquid nitrogen. In this study, these batches of stabilates were used for the experimental infection of indigenous donors to obtain fresh blood infected with B. aktasi.
Selection of purebred Saanen goats for experimental infection
The sample size required to detect a significant difference between two independent groups was determined through a priori power analysis using the G*Power program (Version 3.1.9.3) before starting the experiments [31]. A one-tailed t-test was selected with an effect size (d) of 1.5 and a significance level (α) of 0.05. The required sample size was determined to be 7 animals per group, for a total of 14 animals. The actual power achieved with this sample size was 0.841.
The representative design of the experimental infection conducted in this study is shown in Fig 1.
a) Selection of tick-borne pathogen-free (Babesia spp. Theileria spp., Anaplasma spp.) purebred Saanen and indigenous goats; b) Infection of the immunosuppressed indigenous donors with B. aktasi stabilates (PPE 9.2–11.5%) to obtain the fresh infected blood for the infection of immunosuppressed and immunocompetent Saanen goats. Fig 1 was created with BioRender.com (www.biorender.com).
Prior to experimental infection, 5 mL of whole blood were collected in vacutainer tubes containing EDTA from 5-month-old purebred Saanen (n = 22) and indigenous male goats (n = 15) from B. aktasi-free breeding farms in Elazig province. A small amount of peripheral blood was taken from the ear vein of each goat using a sterile needle to prepare thin blood smears. The samples were brought to the laboratory at the Fırat University Faculty of Veterinary Medicine, Department of Parasitology. Thin blood smears and DNA isolated from EDTA blood samples were screened for the presence of Babesia spp., Theileria spp., and Anaplasma spp. using microscopy and nested PCR [19,24]. Seventeen male goats (14 purebred Saanen, 3 indigenous goats) that tested negative for the selected pathogens were purchased and maintained at the Ministry of Agriculture, Elazığ Veterinary Control Institute, small ruminant unit. It was ensured that the goats were of similar age, weight, and sex to minimize variables affecting on the results of experimental study. Each of the goats maintained at the unit were raised in a separate tick-free pen, and kept in quarantine for 3 weeks. In order to eliminate tick exposure, the goats were treated with Flumethrin 1% pour-on (Ba-tick, BaVET, 10 mg Flumetrin, 100 mL, Türkiye) every 21 days during the study. Following the quarantine period, microscopy and nested PCR were repeated, and it was confirmed that they were free of Babesia spp., Theileria spp., and Anaplasma spp.
Splenectomy and post-operative care
In this study, a randomized controlled experimental infections were performed with two groups of purebred Saanen goats, one consisting of immunosuppressed (splenectomy + dexamethasone administered) goats (n = 7) and the other consisting of immunocompetent (spleen-intact + not dexamethasone applied) individuals (n = 7). The goats in the immunosuppressed group underwent splenectomy using standard anesthesia, analgesic and surgical techniques at Fırat University Animal Hospital before the experimental infection [20,32]. Splenectomized goats were maintained in separate pens throughout the experiment. As post-operative care, the goats were stabilized by administering intravenous 0.9% physiological saline (250 mL) and 5% dextrose (250 mL) once a day for 2 days. In order to prevent possible bacterial infections, a 5-day course of intramuscular antibiotic treatment was administered after splenectomy. Additionally, antibiotic spray (Neocaf, MSD, Oxytetracycline, aerosol spray suspension, 200 mL, USA) was applied to the surgical wound every day for a week. The rectal temperature and clinical symptoms were monitored every two days. The goats were also examined by microscopy and nested PCR for the presence of Babesia spp., Theileria spp. and Anaplasma spp. twice a week.
Experimental infection of immunosuppressed indigenous goats with B. aktasi stabilate
In our previous study, we demonstrated that the parasite has a high level of pathogenic effect in immunosuppressed indigenous goats [20]. Therefore, an immunosuppressed indigenous goats were used as donors to obtain a source of fresh blood infected with the parasite in this study. As seen in Fig 1, the B. aktasi stabilates with 9.2–11.5% PPE kept in the liquid nitrogen was thawed at 37°C, and 15 mL was intravenously inoculated to the donor goats (#Donor-1, #Donor-2, and #Donor-3). Following the parasite inoculation, dexamethasone was also intramuscularly injected to the donors as previously described [11,20]. Subsequent, the donors were monitored daily for the clinical and parasitological findings. When the PPE reached approximately 9.2% and 13.5% on the 12th day post-infection (DPI) in #Donor-1 and #Donor-2, respectively, and 12.3% on the 8th DPI in #Donor-3, venous blood was collected from the donors, and utilized for the experimental infection of the Saanen goats. Then, the donors received treatment with imidocarb dipropionate (0.1 mg/kg body weight IM, İmicarp, Teknovet, Türkiye) and oxytetracycline (10 mg/kg bodyweight IM once daily for 5 days, Primamycin/LA, Zoetis, USA).
Experimental infection of purebred Saanen goats with fresh blood infected with B. aktasi
Three immunosuppressed Saanen goats (#Saanen-1, #Saanen-6 and #Saanen-7), and two immunocompetent (#Saanen-2, and #Saanen-3) were injected intravenously with 15 mL and 30 mL, respectively, of fresh blood infected with B. aktasi from the indigenous #Donor-1 (PPE 9.2%). Two immunocompetent Saanen goats (#Saanen-4, #Saanen-5) were each injected with 30 mL of fresh infected blood from the #Donor-2 (PPE 13.5%) (Fig 1). Similarly, two immunosuppressed Saanen goats (#Saanen-8 and #Saanen-9) and three immunocompetent Saanen goats (#Saanen-10, #Saanen-11, and #Saanen-12) were injected with 15 mL and 30 mL, respectively, of fresh blood infected with B. aktasi from #Donor-3 (PPE 12.3%) (Fig 1). Post-injection of the infected blood, rectal temperature, general physical condition and the presence of specific clinical signs of babesiosis (anemia, jaundice, and hemoglobinuria) were assessed daily for the duration of the experimental infections. Additionally, peripheral blood smears from the goats were daily prepared for microscopic examination. Blood samples from the jugular venous of each goat were also collected in EDTA and clot activator vacutainer tubes. The anticoagulated blood samples were utilized for hematological analysis and DNA extraction, while the sera were used for serum biochemical profiles.
Microscopic examination of peripheral blood smears
Thin blood smears from the peripheral blood were fixed with absolute methanol (Scharlau, Spain) for 5 minutes, then allowed to dry and stained with 10% Giemsa solution (Carlo Erba Reagents, France) for 30 minutes. The stained blood smears prepared from the goats were examined for the presence of intra-erythrocytic stages of Babesia spp., Theileria spp. and Anaplasma spp. under BX43 light microscopy (Olympus, Tokyo, Japan) with 100X objective (x1000 magnification). Twenty microscope fields randomly selected from each of blood smear were scanned, and PPE was calculated as previously reported [20,32].
DNA isolation and Polymerase Chain Reaction (PCR)
Genomic DNA was extracted from 200 μl whole-blood samples using a commercial DNA isolation kit (PureLink Genomic DNA Mini Kit, Invitrogen Corporation, Carlsbad, USA) according to the manufacturer’s instructions. The DNA was stored at -20°C until PCR amplification. Babesia/Theileria [33,34], and Anaplasma [35,36] genus specific nested PCR protocols were used to amplify 18S rRNA and 16S rRNA genes, respectively. The PCR reaction and thermal cycling conditions were as described by Ozubek et al. [20]. In each PCR mixture, reference positive control DNAs for B. aktasi (OQ120434), T. ovis (EF092452), B. ovis (EF092454) and A. ovis (MG693754) available in our laboratory stocks and previously confirmed by sequencing were used. Distilled water and genomic DNA isolated from Babesia spp., Theileria spp. and Anaplasma spp. free 1-month-old goat was used as negative controls. Ten μl of the PCR products were run on 1.4% agarose gel for 30 minutes. After electrophoresis, the agarose gel was stained with Ethidium Bromide (Sigma-Aldrich, USA) for 20 minutes and examined on the Quantum Vilber Lourmat (France) gel imaging system.
Hematology and serum biochemistry
Hemogram and serum biochemistry analyzes were performed at before the experiment and from appearance to disappearance of intracellular parasites in blood smears. The following parameters were examined for hematological analysis: Red blood cell count (RBC), hemoglobin level (Hb), hematocrit level (HCT), white blood cell count (WBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width-coefficient of variation (RDW-CV), and red cell distribution width-standard deviation (RDW-SD). The separated serum samples were analysed for estimation of biochemical parameters of Creatinine, Total protein, Albumin, Total bilirubin, Glucose, Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), and Gamma glutamyl aminotransferase (GGT). The hematology and serum biochemistry analyses were conducted at Fırat University Animal Hospital using Mindray BC-5000 Vet and NX500 Auto Haematology Serum Biyochemistry Analysers (Bio-Medical Electronics Co. Ltd., Shenzhen, China), respectively. Hematobiochemical changes in the experimentally infected goats were compared with pre-infection levels.
Statistical analysis
A comparison of hemogram and serum biochemistry values was conducted between the immunosuppressed and immunocompetent Saanen goats. The SPSS package program (IBM SPSS Statistics Version 21) was used for statistical analyses. The appropriateness of the data for parametric or non-parametric tests was assessed using the Shapiro-Wilk test, while the homogeneity of variances was evaluated using the Bartlett test. For parameters subjected to pairwise comparisons and exhibiting a normal distribution, independent samples Student t-test was employed, whereas dependent samples t-test was used for dependent variables. Statistical analyses were conducted using the Mann-Whitney U test for samples not conforming to normal distribution. Data were presented as mean and standard error. The statistical significance level was set at p<0.05.
Results
Pathogenicity of B. aktasi in immunosuppressed Saanen goats
After the injection of fresh blood infected with B. aktasi, all immunosuppressed goats developed severe clinical infection. Correlated with the appearance of intracellular parasites, an increase in rectal temperature up to 42.2°C was measured in the goats. Characteristic signs of clinical disease (anemia, jaundice and evident hemoglobinuria) were detected in all Saanen goats, however, it is noteworthy that the signs of anemia and jaundice were mild. In addition to these specific findings, the Saanen goats also exhibited general clinical signs such as lethargy, loss of appetite, teeth grinding, trembling, moaning, rapid breathing, immobility, and leaning their heads on the ground. The infection progressed rapidly, and all immunosuppressed Saanen goats died on the 4th day post-inoculation, except for one (#Saanen-9) that died on the 7th day. High PPE levels, ranging from 4.3% to 33.5%, were detected in the blood smears (Fig 2). Seven Saanen goats were initially selected for the immunosuppressed group. However, four goats (#Saanen-1, #Saanen-6, #Saanen-7, and #Saanen-8) died on the 4th day post-infection (DPI), and one goat (#Saanen-9) died on the 7th DPI due to severe clinical signs of babesiosis (Table 1). Following the deaths of these five animals, the remaining two goats were not experimentally infected for ethical reasons. This decision was made to minimize the number of animals used in accordance with ethical guidelines, while ensuring that sufficient data were obtained to address the study objectives.
High Virulence of B. aktasi to immunocompetent Saanen goats
After the inoculation, serious clinical infections resulting in death occurred in the immunocompetent Saanen goats. With the appearance of the intracellular parasite, an increase in rectal temperature up to 41.8°C was measured (Table 1). Specific clinical signs of babesiosis (anemia, jaundice, and hemoglobinuria) were observed in all the immunocompetent Saanen goats. Additionally, symptoms such as lethargy, loss of appetite, teeth grinding, tremors, rapid breathing, and inability to stand were observed in the goats. In the #Saanen-2, #Saanen-3, and #Saanen-12, improvement in clinical symptoms was observed along with the disappearance of parasites from peripheral blood on the 6th and 7th DPI, and the goats survived the acute disease. However, #Saanen-10 died on the 6th post-infection, followed by #Saanen-11 on the 7th, and both #Saanen-4 and #Saanen-5 on the 8th day, due to severe clinical infection. Peak PPE, ranging from 2.1% to 7.6%, was detected on the 3th DPI (Table 1).
Hematological and serum biochemical findings
The pre-infection and post-infection hematological and serum biochemical values in immunosuppressed and immunocompetent Saanen goats are summarized in Table 2.
Statistical significance is indicated as follows: **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05.
Significant changes in hematological parameters were observed in both experimental groups compared to pre-infection levels (Table 2 and Fig 3). The increase or decrease in hematological values was associated with the rise in PPE. A statistically significant decrease in RBC, WBC, HCT, and Hb values was observed in the both Saanen groups. An increase in MCV value was not statistically significant in immunosuppressed goats, whereas in the immunocompetent group was found to be significant. MCH value showed non-significant increase in the both groups. In the immunosuppressed group, a non-significant changes in MCHC and RDW-CV values was detected while significant increase was found in the immunocompetent goats. RDV-SD value showed significant increase in the both groups (Table 2 and Fig 3).
Comparison of hematological values between pre-infection and post-infection in immunosuppressed (A) and immunocompetent (B) Saanen goats. Statistical significance is indicated as follows: **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05, ns: not significant.
Intra-erythrocytic stages of the parasite were first detected from the 1st DPI in all experimentally infected Saanen goats (Table 1 and Figs 4 and 5). An increase in rectal temperature ranging from 41.2°C to 42.2°C was observed in correlation with the appearance of the parasites. In all of the immunosuppressed Saanen goats, the peak PPE was observed on the day of death, while it was observed within 4 days following parasite inoculation in the immunocompetent goats. In both groups, a decrease in HCT, RBC, and Hb values was observed as PPE increased. However, as the infection duration was longer in immunocompetent goats, the decrease in HCT, RBC, and Hb values reached their lowest levels when PPE decreased to a level almost undetectable by light microscopy (Fig 5). In the goats that survived the acute disease (#Saanen-2, #Saanen-3, and #Saanen-12), an increase in HCT, RBC, and Hb values began from the 8th and 9th days after parasite inoculation. Due to the death of other goats, an increases in these values (HCT, RBC, Hb) could not be determined. (Figs 4 and 5).
There was a statistically significant increase in AST, ALT, GGT and total bilirubin values in the both infected groups compared to their pre-infection levels. However, albumin value exhibited a statistically significant reduction in the both infected groups. The decrease in total protein value and the increase in creatinine value were not significant in the immunosuppressed group, whereas but these changes were statistically significant in the immunocompetent group. The differences in glucose was not significant for any group (Table 2 and Fig 6).
Statistical significance is indicated as follows: **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05, ns: not significant).
Necropsy
Three out of 5 dead goats (#Saanen-1, #Saanen-4, and #Saanen-5) were submitted for necropsy. During necropsy, severe pneumonia associated with edema in the lungs, accumulations of frothy exudate in the trachea, jaundice in subcutaneous and mesenteric adipose tissue and dark red urine (hemoglobinuria) in the urinary bladder were detected in the dead goats (Fig 7A, 7B and 7C).
A, pulmonary edema, frothy fluid in the lung and trachea; B, jaundice in subcutaneous and mesenteric adipose tissue; C, dark red urine in the bladder (hemoglobinuria).
Discussion
Small ruminant babesiosis causes substantial economic losses in sheep and goat herds worldwide and remains classified as a neglected disease [3,32,33]. The primary causative agents of small ruminant babesiosis include B. ovis, B. motasi, and B. crassa [4]. One of the initially identified Babesia spp., B. ovis has been reported to cause clinical infections with high mortality in sheep under field conditions in the Southern Europe, Middle East, African and Asian countries [9,37–39]. Babesia crassa is non-pathogenic in sheep and goats, whereas B. motasi is considered moderately pathogenic [40]. The pathogenesis and clinical manifestations of ovine babesiosis caused by B ovis are well-known, but there is a lack of information on caprine babesiosis [3,39,41,42]. In here, clinical, parasitological, hemotological and serum biochemical data from immunosuppressed and immunocompetent purebred Saanen goats were assessed to create a clear picture of the pathogenicity and virulence of newly identified B. aktasi. Our findings indicated that the novel B. aktasi was highly pathogenic to both immunosuppressed and immunocompetent purebred Saanen goats.
Studies on host resistance against bovine Babesia spp. have shown that host genotype plays a significant role [27,28]. However, detailed information regarding genetic resistance to ovine Babesia spp. is not available [29]. In a previous study carried out by Malandrin et al. [43], B. divergens infection model in sheep was established. The study indicated that the sensitivity of sheep erythrocytes to B. divergens varied among different sheep breeds, and concluded that if erythrocytes were resistant to this parasite in vitro, it was difficult for sheep to become infected with B. divergens. An in vitro study conducted in China demonstrated that the Chinese Tan mutton breed sheep erythrocytes were highly sensitive to Babesia sp. BQ1 (Lintan), while French Vendéen breed sheep were not susceptible to the parasite [29]. These findings suggest that different breeds of the same host species may vary in their susceptibility to Babesia parasites. In our previous study investigating the pathogenicity of B. aktasi in indigenous goats [20], the parasite caused mild fever but did not induce clinical infection in immunocompetent goats. However, it is not known whether B. aktasi will exhibit different behavior in terms of pathogenicity and virulence in purebred Saanen goats. In the current experimental study using fresh blood infected with B. aktasi, this question was addressed in purebred Saanen goats. The findings obtained in this experiment indicated that B. aktasi exhibited variable pathogenic effect among different breeds of the same host species, and induced severe clinical infections resulting in death in purebred Saanen goats.
Experimental studies indicated that the spleen plays a significant role in host defense against Babesia spp. After recovering of acute babesiosis, PPE rapidly decreases, but the parasite persists in the host for an extended period (even at levels undetectable under the microscope). When the spleen is removed in these carrier hosts, PPE increases, and clinical infection occurs, leading to the death of the host [11,19,44]. This situation allows for the in vivo isolation of Babesia parasites and facilitates its pathogenicity studies. It has been reported that ovine Babesia spp. have different pathogenicity and exhibit variations in their response to splenectomy. Experimental studies with B. ovis demonstrated that all splenectomized sheep died due to severe clinical infection [45,46]. On the other hand, experimental studies with B. motasi and B. crassa demonstrated that splenectomized sheep did not develop clinical infection resulting in mortality [47–48]. When blood obtained from a naturally infected sheep with Babesia sp. BQ1 (Ningxian) was inoculated into two sheep, one with splenectomized and the other with spleen-intact, the splenectomized sheep exhibited severe clinical symptoms resulting in death including an increase temperature to 41.5°C. In the same study, similar findings were observed in the spleen-intact sheep, but the sheep recovered from the disease [49]. In our previous study, serious clinical infection resulting in death was observed in immunosuppressed indigenous breed goats infected with B. aktasi [20]. Furthermore, it has been reported that in experimental infections conducted on immunosuppressed sheeps, both clinical and microscopic, as well as molecular examinations, indicated that it did not cause infection [50]. In the current study, serious clinical infection was also occurred in immunosuppressed Saanen goats, and four out of five goats died on the 4th day post-fresh infected blood injection due to severe clinical symptoms, while one goat died on the 7th day.
It has been reported that splenectomy and dexamethasone are the most suitable approaches for the experimental studies conducted on Babesia ovata, Babesia bigemina and Babesia sp. Xinjiang to suppress the immune system [11,51,52]. In an experimental study conducted in China, splenectomized sheep infected with Babesia sp. Xinjiang survived acute infection, but death occurred in the sheep that received splenectomy combined with dexamethasone [11]. In an experimental study with Babesia sp. BQ1 (Lintan), it was reported that clinical infection did not develop in splenectomized goats. However, when dexamethasone was injected to the same goats, clinical signs of the disease and a high PPE reaching up to 85% were observed [53]. In a subsequent experimental study using the same isolate, Babesia sp. BQ1 (Lintan) did not induce clinical infection in spleen-intact sheep and goats [54]. In our previous study, dexamethasone was injected to the splenectomized indigenous goats to fully reveal the effects of B. aktasi, and this combined approach increased the occurrence of clinical signs of babesiosis [20]. It is not known whether B. aktasi has a pathogenic effect on purebred Saanen goats. Therefore, in the current study, dexamethasone was injected to the splenectomized goats to achieve complete suppression of the immune system, consistent with previous studies [11,20,51,52,54]. Thus, we obtained more convincing data regarding the pathogenicity and virulence of B. aktasi on purebred Saanen goats.
In this study, both immunosuppressed and immunocompetent Saanen goats exhibited severe clinical findings such as high fever, anemia, jaundice, and hemoglobinuria. In both groups, the severe clinical infection led to the death of the goats. These findings are consistent with previous experimental studies conducted on lambs infected with B. ovis [45,46]. Babesia motasi Ameland strain did not cause PPE or clinical symptoms in splenectomized goats [48], on the contrary, B. crassa induced a mild fever and PPE [55]. In our previous study conducted on indigenous goats, B. aktasi induced severe clinical symptoms in the immune suppressed goats [20]. These findings indicate that different Babesia spp. may cause varying clinical symptoms depending on the presence or absence of the spleen [56–59]. It is well-known that distinct Babesia spp. can lead to different clinical signs. For instance, B. motasi Wales strain caused mild fever, anemia, and PPE up to 1% in spleen-intact goats [48]. In our previous study [20], we determined that B. aktasi has a non-pathogenic effect on the immunocompetent indigenous goats. Interestingly, in the current study, B. aktasi induced serious clinical babesiosis leading to death in purebred immunocompetent Saanen goats. These results indicated that goat breed plays a vital role in the severity of B. aktasi infection, and the parasite poses a significant health risk for purebred Saanen goats.
The main hematological measurements considered in the determination of anemia are total RBC, HCT and Hb values. In our study, a statistically significant decrease in HCT, RBC, WBC, and Hb values was observed (Table 2 and Fig 3), and the decrease of HCT, RBC and Hb were inversely associated with the increase in PPE (Figs 4 and 5). Similar results have been obtained in previous studies, suggesting that this decline in the aforementioned blood parameters may be attributed to the destruction of erythrocytes [9,20,60–63]. Our previous experimental study conducted on indigenous goats using B. aktasi revealed that MCV was normal in both the immunosuppressed and immunocompetent groups, while there was a statistically significant decrease in MCHC value in the immunosuppressed group, indicating a decrease in hemoglobin concentration within RBCs [20]. In contrast to our previous findings [20], the current study observed a significant increase in MCV values and a decrease in MCHC values in the immunocompetent group. Additionally, it was determined that these values remained within the normal range in the immunosuppressed group. These findings indicated that the anemia was macrocytic-hypochromic character in the immunocompetent goats. These findings showed differences from the results obtained in previous studies on B. bovis and B. bigemina in cattle [64,65]. A recent study conducted on sheep infected with B. ovis reported that the RDW (red cell distribution width) value was within the reference range before and after treatment [9]. In the current study, no significant change in RDW-CD and RDW-SD values was detected in the immunosuppressed group, but a significant increase was observed in the spleen intact group (Table 2 and Fig 3). This result indicates a significant reticulocytosis (an increase in young red blood cells) in the spleen intact group, suggesting that the infection lasted longer in this group. Also this finding suggests that spleen function may play an important role in controlling Babesia infection [66].
It has been reported that Babesia parasites cause cellular destruction resulting in hemolysis in the vascular system, inflammatory lesions and anoxia, particularly in certain organs such as liver and kidneys inducing nephrotoxic effects causing to an elevated AST, ALT, BUN and creatinine levels [9,67]. In this study, along with the increase in PPE, a statistical significant increase in ALT and AST levels was observed in both experimental groups compared to pre-infection levels. Our results agree with the previous studies conducted on indigenous goats experimentally infected with B. aktasi [20] and sheep naturally infected with B. ovis [9]. An increase in ALT and AST values indicating hepatic dysfunction has also been observed in cattle infected with B. bovis [68]. ALT elevation has been previously reported in animals with babesiosis, suggesting that this increase may be due to alterations in liver function associated with the disease [9,69]. Recent studies indicated that certain Babesia spp. can cause damage to liver tissue, potentially suppressing the overall liver function [9,69,70].
The pathology of babesiosis is related to the haemolytic anaemia, as a consequence of the intravascular destruction of erythrocytes either by the immune system or by the direct damage caused by the parasite itself [67]. In this study, postmortem changes observed in the goats subjected to necropsy were similar to those previously observed in sheep experimentally infected with B. ovis [71,72]. Serious pulmonary edema suggest that emphysema may lead to a respiratory failure and could provide direct evidence for death [47,72,73]. Therefore, it can be argued that babesiosis is a respiratory tract disease.
Experimental studies conducted on different goat breeds will allow for a better understanding of the pathogenicity and virulence of B. aktasi. In contrast, purebred Saanen goats, which have been selectively bred for traits such as milk production, may be more susceptible to infections like B. aktasi. Studies have shown that while purebred Saanen goats are highly susceptible to trypanosome infections, Saanen crossbreeds exhibit greater resilience [74]. This breed-specific susceptibility underscores the importance of considering breed-specific responses when evaluating the pathogenicity of infectious agents.
In conclusion, in both groups, besides specific signs of acute clinical babesiosis (high fever, anemia, jaundice, hemoglobinuria), general symptoms such as lethargy, anorexia, teeth grinding, trembling, groaning, rapid respiration, lying down with the head on the ground, and immobility were also observed. Shortly after parasite inoculation, PPE was detected at rates ranging from 4.3% to 33.5% in the immunosuppressed group, while it was determined to be 2.1% to 7.6% in the immunocompetent individuals. In the dead goats due to severe clinical babesiosis, edema in the lungs, frothy fluid in the lung and trachea, jaundice in the subcutaneous fat tissue and mesenteric fat, and dark red urine in the urinary bladder were observed. These findings suggest that B. aktasi is a pathogenic species causing acute infections that result in mortality in purebred Saanen goats. Additionally, considering the abundance of wild goats in the region where the parasite is prevalent, further research is needed to identify the natural reservoirs of B. aktasi. These further studies will provide valuable information regarding the biology, epidemiology, and ecology of the parasite.
Acknowledgments
We would like to express our gratitude to Aleyna Karoglu for her exceptional technical and administrative assistance, to Cemal George Orhan for his contributions in preparing the statistical data, to Ali Risvanli, Tarık Safak, Burak Fatih Yüksel, Mert Turanli for performing the splenectomy procedures, and to Burak Karabulut for conducting the necropsies. This work was part of a PhD thesis produced by Mehmet Can ULUCESME.
References
- 1. Krause PJ. Human babesiosis. Int J Parasitol. 2019;49: 165–174. pmid:30690090
- 2. Ozubek S, Bastos RG, Alzan HF, Inci A, Aktas M, Suarez CE. Bovine babesiosis in Turkey: Impact, current gaps, and opportunities for intervention. Pathogens. 2020;9: 1041. pmid:33322637
- 3. Schnittger L, Ganzinelli S, Bhoora R, Omondi D, Nijhof AM, Florin-Christensen M. The Piroplasmida Babesia, Cytauxzoon, and Theileria in farm and companion animals: Species compilation, molecular phylogeny, and evolutionary insights. Parasitol Res. 2022;121: 1207–1245. pmid:35098377
- 4. Schnittger L, Rodriguez AE, Florin-Christensen M, Morrison DA. Babesia: A world emerging. Infect Genet Evol. 2012;12: 1788–1809. pmid:22871652
- 5.
Friedhoff KT. Transmission of Babesia. Babesiosis of Domestic Animals and Man. CRC Press; Boca Raton, FL, USA 1988.
- 6. Liu AH, Yin H, Guan GQ, Schnittger L, Liu ZJ, Ma ML, et al. At least two genetically distinct large Babesia species infective to sheep and goats in China. Vet Parasitol. 2007;147: 246–251. pmid:17531391
- 7. Ozubek S, Aktas M. Molecular and parasitological survey of ovine piroplasmosis, including the first report of Theileria annulata (Apicomplexa: Theileridae) in sheep and goats from Turkey. J Med Entomol. 2017;54: 212–220. pmid:28082649
- 8. Ranjbar-Bahadori S, Eckert B, Omidian Z, Shirazi NS, Shayan P. Babesia ovis as the main causative agent of sheep babesiosis in Iran. Parasitol Res. 2012;110: 1531–1536. pmid:21975684
- 9. Sevinc F, Sevinc M, Ekici OD, Yildiz R, Isik N, Aydogdu U. Babesia ovis infections: Detailed clinical and laboratory observations in the pre- and post-treatment periods of 97 field cases. Vet Parasitol. 2013;191: 35–43. pmid:22889552
- 10. Rjeibi MR, Gharbi M, Mhadhbi M, Mabrouk W, Ayari B, Nasfi I, et al. Prevalence of piroplasms in small ruminants in North-West Tunisia and the first genetic characterisation of Babesia ovis in Africa. Parasite. 2014;21: 23. pmid:24849588
- 11. Guan G, Ma M, Moreau E, Liu J, Lu B, Bai Q, et al. A new ovine Babesia species transmitted by Hyalomma anatolicum anatolicum. Exp Parasitol. 2009;122: 261–267. pmid:19460377
- 12. Giadinis ND, Chochlakis D, Kritsepi-Konstantinou M, Makridaki E, Tselentis Y, Kostopoulou D, et al. Haemolytic disease in sheep attributed to a Babesia lengau -like organism. Vet Rec. 2012;170: 155–155. pmid:22238200
- 13. Niu Q, Liu Z, Yang J, Yu P, Pan Y, Zhai B, et al. Genetic diversity and molecular characterization of Babesia motasi-like in small ruminants and ixodid ticks from China. Infect Genet Evol. 2016;41: 8–15. pmid:26976477
- 14. Wang X, Wang J, Liu J, Liu A, He X, Xu J, et al. Comparative analysis of apicoplast genomes of Babesia infective to small ruminants in China. Parasit Vectors. 2019;12: 312. pmid:31234937
- 15. Wang X, Wang J, Liu J, Liu A, He X, Xiang Q, et al. Insights into the phylogenetic relationships and drug targets of Babesia isolates infective to small ruminants from the mitochondrial genomes. Parasit Vectors. 2020;13: 378. pmid:32727571
- 16. Ozubek S, Aktas M. Molecular evidence of a new Babesia sp. in goats. Vet Parasitol. 2017;233: 1–8. pmid:28043378
- 17. Uilenberg G, Gray J, Kahl O. Research on Piroplasmorida and other tick-borne agents: Are we going the right way? Ticks Tick Borne Dis. 2018;9: 860–863. pmid:29567148
- 18. Ozubek S, Ulucesme MC, Aktas M. Discovery of a novel species infecting goats: Morphological and molecular characterization of Babesia aktasi n. sp. Pathogens. 2023;12: 113. pmid:36678461
- 19. Ulucesme MC, Ozubek S, Karoglu A, Turk ZI, Olmus I, Irehan B, et al. Small ruminant piroplasmosis: High prevalence of Babesia aktasi n. sp. in goats in Türkiye. Pathogens. 2023;12: 514. pmid:37111400
- 20. Ozubek S, Ulucesme MC, Bastos RG, Alzan HF, Laughery JM, Suarez CE, et al. Experimental infection of non-immunosuppressed and immunosuppressed goats reveals differential pathogenesis of Babesia aktasi n. sp. Front Cell Infect Microbiol. 2023;13. pmid:38029260
- 21. Inci A, Yildirim A, Duzlu O, Doganay M, Aksoy S. Tick-borne diseases in Turkey: A review based on one health perspective. PLoS Negl Trop Dis. 2016;10: e0005021. pmid:27977689
- 22. Ceylan O, Sevinc F. Endemic instability of ovine babesiosis in Turkey: A country-wide sero-epidemiological study. Vet Parasitol. 2020;278: 109034. pmid:31991285
- 23. Altay K, Aktas M, Dumanli N. Detection of Babesia ovis by PCR in Rhipicephalus bursa collected from naturally infested sheep and goats. Res Vet Sci. 2008;85: 116–119.
- 24. Aydin MF, Aktas M, Dumanli N. Molecular identification of Theileria and Babesia in sheep and goats in the Black Sea Region in Turkey. Parasitol Res. 2013;112: 2817–2824. pmid:23689604
- 25. Bilgic HB, Hacilarlioglu S, Bakirci S, Kose O, Unlu AH, Aksulu A, et al. Comparison of protectiveness of recombinant Babesia ovis apical membrane antigen 1 and B. ovis-infected cell line as vaccines against ovine babesiosis. Ticks Tick Borne Dis. 2020;11: 101280. pmid:31506224
- 26. Kose O, Bilgic HB, Bakirci S, Karagenc T, Adanir R, Yukari BA, et al. Prevalence of Theileria/Babesia species in ruminants in Burdur Province of Turkey. Acta Parasit. 2022;67: 723–731. pmid:35032244
- 27. Bock RE, de VoS A, Kingston TG, McLellan DJ. Effect of breed of cattle on innate resistance to infection with Babesia bovis, B bigemina and Anaplasma marginale. Aust Vet J. 1997;75: 337–340. pmid:9196820
- 28. Benavides MV, Sacco AMS. Differential Bos taurus cattle response to Babesia bovis infection. Vet Parasitol. 2007;150: 54–64. pmid:17919816
- 29. Guan G, Moreau E, Brisseau N, Luo J, Yin H, Chauvin A. Determination of erythrocyte susceptibility of Chinese sheep (Tan mutton breed) and French sheep (Vendéen breed) to Babesia sp. BQ1 (Lintan) by in vitro culture. Vet Parasitol. 2010;170: 37–43. pmid:20223592
- 30. Bock RE, Kingston TG, de VoS A. Effect of breed of cattle on transmission rate and innate resistance to infection with Babesia bovis and B bigemina transmitted by Boophilus microplus. Aust Vet J. 1999;77: 461–464. pmid:10451733
- 31. Erdfelder E, Faul F, Buchner A. GPOWER: A general power analysis program. Behav Res Methods, Instruments, Comput. 1996; 28: 1–11.
- 32. Sevinc F, Turgut K, Sevinc M, Ekici OD, Coskun A, Koc Y, et al. Therapeutic and prophylactic efficacy of imidocarb dipropionate on experimental Babesia ovis infection of lambs. Vet Parasitol. 2007;149: 65–71. pmid:17709209
- 33. Georges K, Loria GR, Riili S, Greco A, Caracappa S, Jongejan F, et al. Detection of haemoparasites in cattle by reverse line blot hybridisation with a note on the distribution of ticks in Sicily. Vet Parasitol. 2001;99: 273–286. pmid:11511414
- 34. Oosthuizen MC, Zweygarth E, Collins NE, Troskie M, Penzhorn BL. Identification of a novel Babesia sp. from a sable antelope (Hippotragus niger Harris, 1838). J Clin Microbiol. 2008;46: 2247–2251. pmid:18508943
- 35. Bekker CPJ, de Vos S, Taoufik A, Sparagano OAE, Jongejan F. Simultaneous detection of Anaplasma and Ehrlichia species in ruminants and detection of Ehrlichia ruminantium in Amblyomma variegatum ticks by reverse line blot hybridization. Vet Microbiol. 2002;89: 223–238. pmid:12243899
- 36. Kawahara M, Rikihisa Y, Lin Q, Isogai E, Tahara K, Itagaki A, et al. Novel genetic variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a novel Ehrlichia sp. in wild deer and ticks on two major islands in Japan. Appl Environ Microbiol. 2006;72: 1102–1109.
- 37. Hashemi-Fesharki R. Tick-borne diseases of sheep and goats and their related vectors in Iran. Parassitologia. 1997;39: 115–117. pmid:9530694
- 38. Iqbal F, Fatima M, Shahnawaz S, Naeem M, Shaikh RS, Ali M, et al. A study on the determination of risk factors associated with babesiosis and prevalence of Babesia sp., by PCR amplification, in small ruminants from Southern Punjab (Pakistan). Parasite. 2011;18: 229–234. pmid:21894263
- 39. Galon EM, Zafar I, Ji S, Li H, Ma Z, Xuan X. Molecular reports of ruminant Babesia in Southeast Asia. Pathogens. 2022;11: 915. pmid:36015035
- 40. Uilenberg G. Babesia—A historical overview. Vet Parasitol. 2006;138: 3–10. pmid:16513280
- 41. Stuen S. Haemoparasites—Challenging and wasting infections in small ruminants: A review. Animals. 2020;10: 2179. pmid:33266365
- 42. Stevanović O, Radalj A, Subić I, Jovanović NM, Sladojević Ž, Amović M, et al. The presence of malignant ovine babesiosis in Bosnia and Herzegovina indicates a possible emerging risk for Balkan region. Comp Immun Microbiol Infect Dis. 2022;90–91: 101893. pmid:36240662
- 43. Malandrin L, Jouglin M, Moreau E, Chauvin A. Individual heterogeneity in erythrocyte susceptibility to Babesia divergens is a critical factor for the outcome of experimental spleen-intact sheep infections. Vet Res. 2009;40: 25. pmid:19245784
- 44. Knowles DP, Kappmeyer LS, Haney D, Herndon DR, Fry LM, Munro JB, et al. Discovery of a novel species, Theileria haneyi n. sp., infective to equids, highlights exceptional genomic diversity within the genus Theileria: implications for apicomplexan parasite surveillance. Int J Parasitol. 2018;48: 679–690. pmid:29885436
- 45. Habela M, Reina D, Nieto C, Navarrete I. Antibody response and duration of latent infection in sheep following experimental infection with Babesia ovis. Vet Parasitol. 1990;35: 1–10. pmid:2188415
- 46. Rahbari S, Nabian S, Khaki Z, Alidadi N, Ashrafihelan J. Clinical, haematologic and pathologic aspects of experimental ovine babesiosis in Iran. Iran J Vet Res. 2008;9: 59–64
- 47. Uilenberg G, Rombach MC, Perié NM, Zwart D. Blood parasites of sheep in the Netherlands. II. Babesia motasi (Sporozoa, Babesiidae). Vet Q. 1980;2: 3–14. pmid:22039853
- 48. Lewis D, Holman MR, Purnell RE, Young ER, Herbert IV, Bevan WJ. Investigations on Babesia motasi isolated from Wales. Res Vet Sci. 1981;3: 239–243.
- 49. Bai Q, Liu G, Liu D, Ren J, Li X. Isolation and preliminary characterization of a large Babesia sp. from sheep and goats in the eastern part of Gansu Province, China. Parasitol Res. 2002;88: S16–S21. pmid:12051600
- 50. Ulucesme MC, Ozubek S, Aktas M. (2024). Sheep displayed no clinical and parasitological signs upon experimental infection with Babesia aktasi. Vet Sci. 2024;11(8): 359. pmid:39195813
- 51. Fujinaga T. Effect of splenectomy and dexamethasone administration on cattle experimentally infected with Babesia ovata. Nihon Juigaku Zasshi. 1982;44: 71–79. pmid:7098242
- 52. Ravindran R, Mishra AK, Rao JR. Methodology for raising high parasitaemic Babesia bigemina infection in donor bovine calves. J Appl Anim Res. 2006;29: 59–60.
- 53. Guan G, Yin H, Luo J, Lu W, Zhang Q, Gao Y, et al. Transmission of Babesia sp to sheep with field-collected Haemaphysalis qinghaiensis. Parasitol Res. 2002;88: S22–S24. pmid:12051601
- 54. Guan G, Moreau E, Liu J, Hao X, Ma M, Luo J, et al. Babesia sp. BQ1 (Lintan): Molecular evidence of experimental transmission to sheep by Haemaphysalis qinghaiensis and Haemaphysalis longicornis. Parasitol Int. 2010;59: 265–267. pmid:20026243
- 55. Hashemi-Fesharki R, Uilenberg G. Babesia crassa n. sp. (Sporozoa, Babesiidae) of domestic sheep in Iran. Vet Q. 1981;3: 1–8
- 56. Bach O, Baier M, Pullwitt A, Fosiko N, Chagaluka G, Kalima M, et al. Falciparum malaria after splenectomy: a prospective controlled study of 33 previously splenectomized Malawian adults. Trans R Soc Trop Med Hyg. 2005;99: 861–867. pmid:16099487
- 57. Buffet PA, Safeukui I, Deplaine G, Brousse V, Prendki V, Thellier M, et al. The pathogenesis of Plasmodium falciparum malaria in humans: insights from splenic physiology. Blood. 2011;117: 381–392. pmid:20852127
- 58. Lewis SM, Williams A, Eisenbarth SC. Structure and function of the immune system in the spleen. Sci Immunol. 2019;4: eaau6085. pmid:30824527
- 59. Sears KP, Knowles DP, Fry LM. Clinical progression of Theileria haneyi in splenectomized horses reveals decreased virulence compared to Theileria equi. Pathogens. 2022;11: 254. pmid:35215197
- 60. Esmaeilnejad B, Tavassoli M, Asri-Rezaei S. Investigation of hematological and biochemical parameters in small ruminants naturally infected with Babesia ovis. Vet Res Forum. 2012;3: 31.
- 61. Sevinc F, Sevinc M, Koc Y, Alkan F, Ekici OD, Yildiz R, et al. The effect of 12 successive blood passages on the virulence of Babesia ovis in splenectomized lambs: A preliminary study. Small Rumin Res. 2014;116: 66–70.
- 62. Salem NY, Farag HS. Clinical, hematologic, and molecular findings in naturally occurring Babesia canis vogeli in Egyptian Dogs. Vet Med Int. 2014;2014: e270345. pmid:24693460
- 63. Kage S, Mamatha GS, Lakkundi JN, Shivashankar BP, D’Souza PE. Detection of incidence of Babesia spp. in sheep and goats by parasitological diagnostic techniques. J Parasit Dis. 2019;43: 452–457. pmid:31406410
- 64. Mahmoud MS, Kandil OM, Nasr SM, Hendawy SHM, Habeeb SM, Mabrouk DM, et al. Serological and molecular diagnostic surveys combined with examining hematological profiles suggests increased levels of infection and hematological response of cattle to babesiosis infections compared to native buffaloes in Egypt. Parasit Vectors. 2015;8: 319. pmid:26062684
- 65. Salem NY, Yehia SG, Farag HS, Elkhiat MA. Clinical, hemato-biochemical alterations and oxidant–antioxidant biomarkers in Babesia-infected calves. Int J Vet Sci Med. 2016;4: 17–22. pmid:30255034
- 66.
Brockus CW. Andreasen CB. Erythrocytes. In Duncan and Prasse’s Veterinary Laboratory Medicine: Clinical Pathology; Latimer K.S., Mahaffey E.A., Prasse K.W., Eds.; Iowa State Press: Ames, IA, USA, 2011; pp. 3–45.
- 67. Villanueva-Saz S, Borobia M, Fernández A, Jiménez C, Yzuel A, Verde MT, et al. Anaemia in sheep caused by Babesia and Theileria haemoparasites. Animals. 2022;12: 3341. pmid:36496866
- 68. Zulfiqar S, Shahnawaz S, Ali M, Bhutta AM, Iqbal S, Hayat S, et al. Detection of Babesia bovis in blood samples and its effect on the hematological and serum biochemical profile in large ruminants from Southern Punjab. Asian Pac J Trop Biomed. 2012;2: 104–108. pmid:23569878
- 69. Schneider DA, Yan H, Bastos RG, Johnson WC, Gavin PR, Allen AJ, et al. Dynamics of bovine spleen cell populations during the acute response to Babesia bovis infection: an immunohistological study. Parasite Immunol. 2011;33: 34–44. pmid:21155841
- 70. Esmaeilnejad B, Dalir-Naghadeh B, Tavassoli M, Asri-Rezaei S, Mahmoudi S, Rajabi S, et al. Assessment of hepatic oxidative damage, paraoxonase-1 activity, and lipid profile in cattle naturally infected with Babesia bigemina. Trop Anim Health Prod. 2021;53: 219. pmid:33751256
- 71. Habela MA, Reina D, Navarrete I, Redondo E, Hernández S. Histopathological changes in sheep experimentally infected with Babesia ovis. Vet Parasitol. 1991;38: 1–12. pmid:2024425
- 72. Ceylan O, Xuan X, Sevinc F. Primary tick-borne protozoan and rickettsial infections of animals in Turkey. Pathogens. 2021;10: 231. pmid:33669573
- 73. Suarez CE, Alzan HF, Silva MG, Rathinasamy V, Poole WA, Cooke BM. Unravelling the cellular and molecular pathogenesis of bovine babesiosis: is the sky the limit? Int J Parasitol. 2019;49: 183–197. pmid:30690089
- 74. Geerts S, Osaer S, Goossens B, Faye D. Trypanotolerance in small ruminants of sub-Saharan Africa. Trends Parasitol. 2009;25: 132–138. pmid:19200783