Introduction

Tick-borne protozoan Babesia ovis is the causative agent of ovine babesiosis, and is considered the most economically significant pathogen affecting sheep in endemic regions, including Northern Africa, Southern Europe, the Middle East, and Asia (1-6). Babesia ovis is widespread in sheep across nearly all regions of Türkiye and is known for its high pathogenicity (7). Sheep infected with B. ovis develop severe clinical infection, characterized by high fever, anemia, icterus, hemoglobinuria, and in some cases, death (8,9). The biology of Babesia protozoa, which consists of three main stages (gamogony, sporogony, and merogony), is completed between ixodid ticks and vertebrate hosts (10-12). Babesia ovis is transmitted by Rhipicephalus bursa through both transovarial (from parent ticks to their offspring via eggs) and transstadial (from one developmental stage to another) routes (6-17). The development of the parasite in adult female Rhipicephalus bursa ticks begins when the tick feeds on an infected sheep and ingests the piroplasms. Sexual development occurs in the lumen of the female tick’s midgut. Then, the kinetes are released from the midgut into the hemolymph and subsequently invade the ovaries (6,16,18). As a result, the parasite is transmitted to the larvae that hatch from the eggs of the adult female ticks. Pre-adult stages of R. bursa, such as larvae and nymphs, do not directly transmit B. ovis; instead, the kinetes are passed on to the adult stage. Infectious sporozoites develop in the salivary glands of the adult tick and are transmitted to susceptible sheep hosts during feeding (15,17). Although the mechanisms that allow parasites to persist within tick populations are not fully understood, transovarial transmission has been suggested to play a significant role in maintaining the parasite within endemic regions (14,15).

Despite this, the efficiency of tick acquisition of B. ovis and its transovarial transmission to subsequent generations remains insufficiently characterized. In this study, we aimed to evaluate the transovarial transmission capacity of R. bursa for B. ovis and to determine the rate of transmission of infection from engorged adult females to their larval progeny.

Materials and methods

Tick and parasite

The sterile, laboratory-reared adult R. bursa ticks used in this study were obtained from a colony maintained in the laboratory, as described previously (19). The B. ovis-free colony was established at our department (Parasitology, Veterinary Faculty, Fırat University, Elazığ, Türkiye). In this study, the B. ovis-Alacakaya stabilate was used as the source of infection to obtain infected adult Rhipicephalus bursa ticks. The isolation and molecular characterization of the stabilate have been described in previously published studies (9,17,20,21). All experimental procedures involving animals were approved by the Ethics Committee of Fırat University (protocol no. 2023/03-02).

Selection of the lamb

An 8-month-old tick-borne pathogens-free Akkaraman lamb was used for the experimental infection. To ensure that the lamb was free of infections caused by Babesia, Theileria, and Anaplasma species, nested polymerase chain reaction (PCR) was performed as previously described (20). In addition, the presence of antibodies against B. ovis in the lamb was assessed by an indirect ELISA using the recombinant B. ovis spherical body protein 4 (rBoSBP4) as the antigen, as previously reported (22). As no ELISA kits are available for ovine tick-borne pathogens (Theileria spp. and Anaplasma spp.), serology was not conducted.

Experimental infection of the splenectomized lamb

In previous studies aimed at determining the vector competence of blood parasites such as Babesia and Theileria, splenectomy has been commonly used to induce high parasitemia and thereby enhance vector infection efficiency (20,23). In this study, the lamb was also splenectomized at the Fırat University Animal Hospital under general anesthesia using standard surgical techniques before experimental infection. The cryopreserved B. ovis-Alacakaya stabilate containing 5% parasitized erythrocytes (PPE) was thawed in a 37°C water bath, and 15 ml was intravenously injected into the lamb.

The acquisition of B. ovis by adult R. bursa from the experimentally infected lamb

Before the experimental infection, a feeding capsule made of EVA foam was attached to the shaved posterior thoracic area of the lamb (19,24,25). Twenty-five unfed adult R. bursa (10 females and 15 males) were placed in the feeding capsule. Following tick infestation, the lamb was monitored daily for clinical findings of babesiosis, as well as the duration of tick engorgement and detachment from the host. All fully engorged ticks that dropped into the capsule were collected in a plastic container. Fully engorged female ticks were maintained under controlled conditions at 27 ± 1 °C with 70-80% Relative Humidity (RH) to facilitate oviposition. The carcasses of females that had completed oviposition were retrieved from the incubator. They were aseptically bisected using a sterile scalpel. Each female tick carcass was placed into an individual Eppendorf tube and kept at -20 °C for subsequent use in DNA isolation and nested polymerase chain reaction (nested PCR). When the color of the eggs began to change, five separate egg pools, each consisting of 100 eggs, were prepared from the egg batches derived from individual engorged female ticks. After larval emergence was complete from these egg pools, the larval pools were kept at -20 °C until DNA isolation.

Feeding of R. bursa Larvae on the rabbit and obtaining unfed adults

One of the larval batches confirmed by nested PCR to be infected with Babesia ovis was randomly selected and used to experimentally infect a New Zealand rabbit. A total of ~ 2.000 viable larvae were introduced into EVA foam capsules affixed to the rabbit, following the methodology previously described (24) and further refined in recent studies (19,25,26). All engorged nymphs collected from the rabbit were maintained under controlled environmental conditions (27 ± 1 °C and 70 ± 10% RH) and allowed to molt into unfed adult ticks. Engorged nymphs kept in the incubator were monitored daily. Upon completion of molting, the total number of unfed adults was recorded, and they were sorted into females and males. Of these, 150 unfed adults (75 males, 75 females) were randomly selected and stored in a deep freezer for subsequent DNA extraction and molecular analysis.

Microscopic detection of B. ovis

Thin blood smears were methanol-fixed and subsequently stained using a 10% Giemsa solution. The prepared slides were then examined under a light microscope at 1000X magnification to detect intraerythrocytic forms of Babesia spp. To determine the percentage of parasitized erythrocytes (PPE), at least 20 fields of view were assessed, following the methodology described in an earlier study (27).

DNA isolation and nested PCR

Eight engorged female ticks, a total of 40 separate larval pools (n = approximately 400 larvae; 5 larval pools from each of the 8 engorged female ticks), and 150 unfed adults (75 males, 75 females) were individually homogenized in liquid nitrogen using sterile pestles and subsequently processed for genomic DNA extraction (27,28). Genomic DNA from blood and tick specimens was isolated using the PureLink™ Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) according to the kit protocol. The extracted DNA was utilized as a template in nested PCR assays to detect the 18S rRNA gene of B. ovis using two primer sets and the protocol previously reported in the literature (29,30). For the amplification of B. ovis, the first PCR was performed using the Nbab1F/Nbab1R primers (30), followed by a second nested PCR using the Bbo-F/ Bbo-R primers (29). Each PCR reaction included both positive (genomic DNA of B. ovis confirmed by DNA sequencing, GenBank accession no. EF092454) and negative (DNase/RNase-free water) controls. PCR amplifications were performed using a thermal cycler (Labcycler Gradient, Göttingen, Germany). Ten microliters of each PCR product were separated by electrophoresis on a 1.4% agarose gel for 30 minutes and then visualized with a Quantum Vilber Lourmat gel documentation system (Marne-la-Vallée, France). To confirm the presence of R. bursa DNA, PCR was performed using the 16S +1 and 16S -1 primers (31).

Results

Experimental infection of B. ovis in the lamb

Following the inoculation of B. ovis-Alacakaya stabilate, clinical and parasitological monitoring revealed a response consistent with acute babesiosis characterized by high fever, anemia, jaundice, hemoglobinuria, anorexia, and abdominal breathing. Intracellular piroplasms of the parasite were first detected in peripheral blood smears on day 6 post-inoculation. This observation coincided with a marked rise in rectal temperature, reaching 42.3°C. Parasitemia progressively increased, reaching a maximum level of 10% on day 15 post-infection (Figure 1). The prepatent period overlaps with the active tick attachment and feeding phase (days 6 to 15), which is considered the most favorable timeframe for the efficient acquisition of B. ovis by adult R. bursa ticks. Due to the severity of clinical signs and the poor prognosis, the animal was humanely euthanized on day 15, after the collection of all engorged female ticks. PCR analysis followed by DNA sequencing confirmed that the lamb was infected with B. ovis.

Figure 1: Progression of clinical infection and adult R. bursa infestation in the lamb. a) Progression of parasitemia (percentage of parasitized erythrocytes, PPE) and rectal temperature (°C) throughout the course of infection in the lamb (the lamb was euthanized on day 15 post-infection due to the development of acute babesiosis). b) Timeline of tick infestation in the lamb (GraphPad Prism v8 (GraphPad Software, San Diego, CA) was used to create time-course and scatter plots).

Acquisition of B. ovis by adult R. bursa fed on the clinically infected lamb.

Out of 25 adult R. bursa ticks (comprising 10 females and 15 males) applied to the lamb, a total of 8 females and 12 male ticks successfully completed their feeding. They were recovered as engorged individuals between the 6^th and 9^th days post-infestation (Figure 1b). Following incubation, engorged females began oviposition on day 5. Egg hatching began between days 28 and 34 post-oviposition and continued for 6 to 13 days. Post-oviposition, all engorged female tick carcasses tested positive for B. ovis via nested PCR analysis (Figure 2, lanes 1-8). Likewise, parasite DNA was detected in all larval pools derived from these females (Figure 2, lanes 9-10). These findings confirm that female adult R. bursa ticks acquired B. ovis during feeding on the infected lamb, and were capable of transmitting the pathogen to their offspring through transovarial transmission.

Figure 2: Gel electrophoresis image illustrating nested PCR amplification of B. ovis DNA (549 bp) from engorged female ticks, larval pools, and unfed adult Rhipicephalus bursa collected from a clinically infected lamb. M: 100 bp marker, N: negative control (distilled water), P: positive control (B. ovis confirmed by DNA sequence analysis, GenBank accession no. EF092454), 1-8: Engorged female carcasses, 9-10: representative positive larval pools, 11-12: representative positive unfed adults.

Transovarial transmission efficiency of B. ovis in R. bursa

The attached larvae, having derived from engorged female R. bursa ticks that imbibed B. ovis-containing blood from the infected lamb, completed their feeding and detached from the rabbit as engorged nymphs between the 12^th and 14^th days post-infestation. A total of 372 engorged nymphs were collected from the rabbit and incubated at 27±1°C with 70–80% RH to allow molting into the adult stage, resulting in 370 (173 females, 197 males) unfed adult R. bursa (Figure 3).

Figure 3: Unfed adults R. bursa infected with B. ovis, male (a) and female (b)

From this population, a representative subset of 150 unfed adult ticks (75 females and 75 males) was randomly selected and screened for the evidence of B. ovis DNA using nested PCR (Table 1). The results showed that B. ovis DNA was amplified in 135 of 150 ticks tested, corresponding to an overall infection rate of 90%. This finding indicates that adult female R. bursa ticks acquired B. ovis during the feeding period on the infected lamb and transmitted it transovarially to the subsequent generation. These findings demonstrate a high efficiency of transovarial transmission of B. ovis in R. bursa. Specifically, the infection prevalence was 90.6% in female and 89.3% in male ticks (Table 1).

Table 1: PCR analysis outcomes demonstrating the acquisition and transovarial retention efficiency of Babesia ovis in Rhipicephalus bursa ticks fed on the experimentally infected lamb

* Pool tick sample (approximately 100 larvae in each pool)

Discussion

Variation in tick-borne diseases is influenced by multiple factors, including global climate change, urbanization, and human activity (16). In addition, key biological mechanisms, such as transovarial transmission by tick vectors and the competence of reservoir hosts, also play crucial roles (32). Research on the transovarial transmission of pathogens by ixodid ticks helps elucidate mechanisms underlying the circulation, persistence, and epidemiology of tick-borne microorganisms in endemic foci (16,33). Babesia ovis is a tick-transmitted protozoan parasite that causes ovine babesiosis, a disease of significant economic impact in tropical and subtropical areas (1,34). Despite Rhipicephalus bursa being a known biological vector of B. ovis, the dynamics of vertical (transovarial) transmission are poorly understood. This study provides compelling experimental evidence supporting the transovarial transmission of B. ovis by R. bursa under controlled laboratory conditions. The successful acquisition of B. ovis by adult female ticks, followed by the detection of parasite DNA in their progeny (larvae and unfed adults), underscores the vectorial competence of R. bursa for this economically significant hemoparasite of sheep.

The experimental infection model employed in a splenectomized lamb effectively induced a high parasitemia, peaking at 10% parasitized erythrocytes. The experimental model used in the present study is consistent with previous studies, which used splenectomy to enhance parasite amplification and thereby optimize conditions for vector infection (20,23,35). The overlap between the peak parasitemia phase and the active feeding period of adult R. bursa likely facilitated the efficient acquisition of the parasite. Our findings revealed that 8 engorged female R. bursa ticks tested positive for B. ovis by nested PCR, and more importantly, parasite DNA was also detected in their larval progeny. These results confirm not only the successful acquisition but also the vertical transmission of B. ovis through the transovarial pathway. This is consistent with earlier reports suggesting the possibility of vertical transmission of Babesia spp. in particular tick species (13,17,36,37).

Furthermore, larvae derived from PCR-positive females were able to develop through the nymph stage and molt into adult ticks, with a high percentage (90%) of these unfed adults testing positive for B. ovis. This is a particularly significant observation, as it demonstrates R. bursa's capacity not only to transmit B. ovis transovarially but also to maintain the infection across developmental stages (transstadial persistence). The observed infection rates were slightly higher in female ticks (90.6%) compared to males (89.3%). However, the difference was not statistically significant, suggesting that both sexes are competent carriers in the absence of differential susceptibility. These results are consistent with previous findings (15), which reported that both male and female R. bursa play an essential role in the transmission of B. ovis.

The high transovarial transmission efficiency observed in this study suggests that R. bursa populations could serve not only as vectors but also as long-term reservoirs of B. ovis, thereby contributing to the persistence of infection in the absence of active transmission cycles involving infected hosts. This is particularly relevant in endemic foci where seasonal fluctuations in tick activity and host availability may interrupt horizontal transmission dynamics. Previous findings indicated that while up to 95% of vertically infected female ticks were capable of producing infected eggs, less than 30% of these eggs were actually infected (38). In contrast, our study demonstrated a significantly higher infection rate of 90% in unfed adult ticks, suggesting substantially greater vertical transmission efficiency under our experimental conditions. This notable difference may be attributed to variations in parasite strains, the genetic background of the tick populations, or methodological approaches. Overall, our results provide strong evidence for the effective and stable maintenance of B. ovis through vertical transmission, even in the absence of selective pressure.

Conclusion

This study confirms that R. bursa can acquire B. ovis from an infected lamb and transmit the parasite transovarially to its offspring with high efficiency. These results highlight the potential role of R. bursa as both a vector and a reservoir of B. ovis, emphasizing the need to include this species in integrated control strategies targeting ovine babesiosis in endemic areas. It is also noteworthy that molecular confirmation of B. ovis was achieved using a sensitive and specific nested PCR targeting the 18S rRNA gene. The use of multiple life stages (engorged female, larvae, and unfed adults) enhanced the reliability of the findings. However, detecting parasite DNA does not necessarily confirm the viability or infectivity of the organisms, and future studies should include attempts to isolate viable parasites from progeny or to demonstrate transmission to susceptible hosts.

Funding

This study was financially supported by the TUBITAK Grant Program (Project no. 222O123).

Acknowledgments

We gratefully acknowledge the language editing support provided by ChatGPT-4o, developed by OpenAI.