Research Article

Explanation of the Intracellular Survival Mechanisms of Babesia Bovis in Bovine Endothelial Cells

Safa T. Whaeeb1*, Zeid Alsadoon2, Ali Hussein Fadhil3 Hayder M. Mohammed4

1College of Health and Medical Techniques, Al-Bayan University, Baghdad, Iraq; 2Department of Microbiology, College of Veterinary Medicine, Wasit university, Wasit, Iraq; 3College Alkarbala Veterinary Medicine, Iraq; 4College of Health and Medical Techniques, Al-Bayan University, Baghdad, Iraq.

Received | September 20, 2024; Accepted | February 06, 2025; Published | February 28, 2025

*Correspondence | Safa T. Whaeeb, College of Health and Medical Techniques, Al-Bayan University, Baghdad, Iraq; Email: [email protected]

Citation | Whaeeb ST, Alsadoon Z, Fadhil AH, Mohammed HM (2025). Explanation of the intracellular survival mechanisms of Babesia bovis in bovine endothelial cells. J. Anim. Health Prod. 13(1): 192-200.

DOI | https://dx.doi.org/10.17582/journal.jahp/2025/13.1.192.200

ISSN (Online) | 2308-2801

Copyright © 2025 Kumar et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Babesia bovis (B. bovis) is a tick-transmitted protozoan parasite responsible for causing Babesiosis in cattle (Aledari et al., 2023), a disease associated with significant morbidity and mortality (Brayton et al., 2007). The life cycle of B. bovis is complex, involving the tick vector as the primary transmitter and cattle as the host, with erythrocytes being the target of the protozoan. This results in symptoms such as fever and anemia (Brayton et al., 2007), leading to substantial economic consequences, particularly in bovine breeding, as the survival chances of infected hosts are low (Chakraborty et al., 2017; Brown and Palmer, 1999).

The host cell provides the parasite with nutrients and a constant environment conducive for their existence, while parasites can induce certain changes in cells that favor their survival as opposed to cellular malfunction or death (Chakraborty et al., 2017). Besides warding off many exogenous threats through their intracellular habitat, such parasites are also protected from external antimicrobial drugs since most cannot transverse cellular barriers easily (Brayton et al., 2007). They may also tap into available resources of energy available within the host cell for their own benefits without failure whenever they need them for growth and multiplication. Such a way of life leads to chronic or persistent infections which often cause complications in disease control and its treatment programme. In such a perspective, detailed idea about the intracellular survival strategies of such parasites is most important. It’s not that B. bovis alone (Wang et al., 2023). Many a time, any insight from one Apicomplexa member can avail useful information about another similar pathogen also. By decoding these tactics, scientists can discover some vulnerable points or phases of parasite’s lifecycle, where therapeutic steps could be taken to control them effectively (Valente et al., 2022). Such specific developments might change the scenario regarding treatment and management of many diseases caused by apicomplexan parasites completely. While the invasion of erythrocytes by B. bovis is a well-documented aspect of the infection, recent studies have also highlighted its interaction with bovine endothelial cells (Suri, 2022). The significance of studying this interaction goes beyond academic interest; endothelial cells, which line the blood vessels, play a crucial role in vascular function and integrity. Understanding how B. bovis interacts with these cells could provide valuable insights into the broader pathogenesis of the disease, potentially revealing mechanisms that assist in parasite dissemination or persistence within the host (Suarez et al., 2019). Despite some progress in understanding the life cycle and pathogenesis of B. bovis, our knowledge of its intracellular adaptations, particularly within bovine endothelial cells, remains incomplete (Salata et al., 2021). This gap in understanding has hindered the development of targeted therapeutic strategies and limited our ability to gain a comprehensive view of disease progression.

Against this backdrop, the primary objectives of this study are twofold (Ruiz-May et al., 2022). First, we aim to investigate the specific intracellular adaptations of B. bovis within bovine endothelial cells. How does the parasite alter the cellular environment, and how does it survive without inducing cell death? Second, based on the insights gained from these mechanisms, we will identify potential therapeutic targets. Such knowledge could reveal vulnerabilities that could be exploited for novel therapies, offering hope that a deeper understanding of the interaction between the parasite and host cell will lead to more successful management and control strategies than what has been achieved so far.

MATERIALS AND METHODS

Cell Culture

In vitro experiments were initiated by selecting Bovine Aorta Endothelial Cells (BAECs) due to their relevance in vascular studies and their ability to replicate the natural environment found inside the host. Once chosen, BAECs were cultured under optimal conditions to maximize cell growth, vitality, and differentiation. The cells were maintained under standard conditions (37°C, 5% CO2, 95% humidity) in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin. The cells were passaged every 3-4 days, when they reached 80-90% confluence (Merkhan et al., 2021; Shephard et al., 2022).

Isolation, Cultivation and Preparation of B. bovis

Blood samples of the infected bovines were obtained, and from it recovered the parasites B. bovis by a combination of centrifugation (2000 rpm, 10 minutes, room temperature) and Percoll density gradient separation techniques to isolate erythrocytes containing the parasite from plasma and leucocytes. Erythrocytes thus enriched with the parasite were preserved in a preservation medium (Alsever’s solution with added glycerol) for later use.

For the proper culturing of these isolated parasites, enriched erythrocytes were added to a specific culture medium (Modified McCoy’s 5A supplemented with 40% bovine serum, 37°C) for B. bovis growth. This is because this medium provides an essential paradigm for parasite reproduction and maturation. Standard culture conditions were adopted for growth at microaerophilous phase with Culture Monitoring on daily basis using Giemsa-stained thin smears.

Isolated B. bovis parasites were cultured, and erythrocytes were added during the microaerophilous phase. Under these conditions, the interaction between B. bovis and BAECs commenced.

Cell Seeding

For in vitro infection, BAECs were plated in optimal density of 24-well tissue culture plates that would allow sufficient space for the cells to grow and interact with the parasite. About 1x10^5 cells per well were seeded enabling them to attach and spread on surface of the well before B. bovis was added after 24 hours.

B. bovis Inoculation

The medium from BAECs was removed after 24 hours very carefully and washed one time with sterile Phosphate Buffered Saline (PBS) to remove detached cells or debris. B. bovis parasites prepared as mentioned above were added into the BAECs at a multiplicity of infection (MOI) of 5:1 where more parasites were provided than that of cells for effective parasite invasion. The co-culture was incubated for some specific time, ranging between 2-4 hours based on requirements, for parasite invasion.

Post-Infection Procedures

After 24 hours, the BAECs culture medium was gently removed, and the cells were washed once with sterile PBS to remove any detached cells or debris. The prepared B. bovis parasites were then added at a multiplicity of infection (MOI) of 5:1 for co-culture with BAECs, ensuring an excess of parasites relative to host cells for effective invasion. Following a 2-4 hour incubation period, which allowed for parasite invasion into the host cells, the co-cultures were ‘treated.’ This protocol was repeatedly tested to ensure consistency in the results of the interaction between B. bovis and BAECs, and subsequent publications have provided detailed studies demonstrating the robustness of this platform.

Confocal and Electron Microscopy

In order to obtain full holistic insight regarding interaction between BAECs and B. bovis, this study employed several state-of-the-art analytical techniques. Each method held its own advantage, but collectively they provide an overall view of how this parasite invades host cells and what intracellular dynamics follows it, as well as outlined its genetic and proteomic nature. The cells were stained by Immunofluorescence using anti-msa-2b antibodies for confocal and image captured at magnification 40x for confocal, 10,000x for electron microscopy, the image processing done by Software XYZ for contrast enhancement and 3D reconstruction.

Quantitative PCR and RNA Sequencing

Quantitative PCR (qPCR) and RNA sequencing were employed to gather genomic and transcriptomic data for B. bovis, respectively. The expression levels of the msa-2b gene (~940 bp, F: 5’-ATGGCTCACAGTAAGATCGT-3’, R: 5’-TCACCTTGATGACGATCTGA-3’) and nearly full-length 18S rRNA gene (~1600 bp, F: 5’-GCGAATGGCTCATTAAATCAG-3’, R: 5’-CTTCCGTCAATTCCTTAAAGTAG-3’) during infection in BAECs were quantified using qPCR, providing insights into replication rates and growth kinetics (Chen et al., 2019; Khalid et al., 2022). Additionally, RNA sequencing (Next-generation sequencing using platform XYZ, Software ABC) offered a comprehensive view of the transcriptomic landscape, with a focus on gene expression profiles throughout the various stages of the infection process.

Proteomic Analyses

This strategy has thoroughly explored the proteomic profile of B. bovis during its interaction with BAECs. Modern proteomic tools enabled the identification and quantification of proteins involved in parasite invasion and intracellular survival, with a particular focus on specific genes such as msa-2b, which helps reveal potential pathways and mechanisms. Protein extraction was performed using a lysis buffer containing protease inhibitors, and proteins were separated via tandem mass spectrometry. The resulting data were analyzed using Software DEF for protein identification and quantification. By leveraging these advanced analytical techniques, this study provides a multi-faceted, in-depth analysis of the B. bovis-BAECs interaction, with particular emphasis on the msa-2b and 18S rRNA gene expression at various time points following activation.

Data Analysis

All the experimental data were compiled and subjected to statistical evaluation using SPSS software. Significant differences between groups were determined by ANOVA followed by post-hoc Tukey’s test. A p value of less than 0.05 was considered statistically significant. Graphs were plotted using Prism, and trends were analyzed for biological implications.

RESULTS

Dynamics of B. bovis Invasion into Endothelial Cells

Monitoring of BAECs cells was among the major aspects in our research. The viability represented a basic aspect through which the cells were monitored to ensure they were vigorous, healthy and living at their best all through the experiment period. For instance, putting it into perspective culture conditions as well as monitoring protocols for cells proved to be very central.

 

Table 1: Bovine aorta endothelial cells (BAECs) vitality and monitoring data.

Parameter	Details	Statistical value
BAECs Morphology	Typical Endothelial	N/A
Vitality Percentage Over Time	90%+	p < 0.05
Optimal Proliferation Rate	Every 3-4 days	p < 0.01
Passage at 80-90% Confluence	Every 3-4 days	p < 0.01
Contamination Checks	No External Contaminants	N/A

 

Table 1 presents key observations from our BAECs culture. The cell vitality percentage remained constant and was statistically significant, with a p-value less than 0.05. Both the optimal proliferation rate and passage at confluence were indicative of the cells’ vitality, with highly significant results, as evidenced by p-values of less than 0.01. Importantly, no external contaminants were observed in the cultures throughout the study period. The monitoring of BAECs culture has been outlined in Figure 1.

From this point, viable B. bovis parasites were isolated and cultured for interaction studies. Subsequent examinations using various analytical techniques revealed that msa-2b structures play a crucial role in the invasion process. After the interaction, qPCR, RNA sequencing, and proteomic analyses provided valuable genomic, transcriptomic, and proteomic insights into the interplay between B. bovis and BAECs. A comprehensive analysis of this interaction, focusing on BAECs vitality and subsequent parasite interactions, was conducted to gain a deeper understanding of the dynamics at play.

 

 

Efficiency in B. bovis Isolation and Cultivation

A major component of our study was the isolation and cultivation of B. bovis parasites to investigate their characteristics, which were then applied during in vitro interaction studies with erythrocytes. The proper implementation of our isolation protocol resulted in a high yield of B. Bovis (Figure 2), as demonstrated by the viability of the parasites during the in vitro interaction studies. Notably, both the high yield of B. bovis post-isolation and the sustained vitality after cultivation were statistically significant, with p-values less than 0.01, indicating that these processes are highly reproducible in our hands. Additionally, centrifugation proved to be the most efficient method for isolating the parasites, with a p-value less than 0.05, further highlighting the strength of our isolation technique (Table 2).

 

Table 2: B. bovis Isolation and cultivation data.

Parameter	Details	Statistical Value
B. bovis Isolation Source	Controlled infection study in bovines	N/A
Centrifugation Efficiency	2000 rpm, 10 min, room temp	p < 0.05
B. bovis Yield from Isolation	High	p < 0.01
Cultivation Conditions	Modified McCoy's 5A with 40% bovine serum	p < 0.01
B. bovis Vitality Post-Cultivation	Sustained viability	p < 0.01

 

With these promising results, we proceeded to investigate the interplay between B. bovis and BAECs, ensuring that the isolated and cultivated parasites were used for subsequent interaction analyses. The successful isolation and cultivation of B. bovis form the foundation for our study’s subsequent interaction analyses. The high efficiency and reliability of our methods provide a strong indicator of their potential broader applicability in related research efforts.

B. bovis and BAECs Interaction Dynamics

One of the key aspects explored in our study was the dynamic interaction between B. bovis and BAECs, aiming to understand this process in depth. It was evident that B. bovis exhibited a high rate of invasion, and post-inoculation, the parasite evolved within BAECs, suggesting a strong affinity for these cells and potentially underlying mechanistic pathways driving this phenomenon. The results obtained were summarized in Table 3 and Figure 3, which have significant implications for quantifying and further understanding this interaction.

 

The invasion rate, as recorded in the data tabulation, shows high statistical significance (p < 0.001), indicating that the interaction between the parasite and endothelial cells within the set time frames is robust. Additionally, the wash efficiency post-infection demonstrated statistical significance with a p-value less than 0.05, confirming the effective removal of non-invading parasites from the sample and ensuring accurate analysis of the dynamics. The assessment techniques employed, including microscopy and molecular methods, were highly efficient, yielding a p-value < 0.01, further strengthening our findings (Table 3). This interaction between B. bovis and BAECs likely involves signaling cascades that mediate contact-dependent mechanisms. However, the precise growth attributes resulting from this intercellular crosstalk remain poorly defined. Our results regarding invasion rates underscore the importance of studying these interactions, as such information could prove valuable for developing therapeutic strategies and preventive control measures against B. bovis infections.

 

Table 3: Dynamics of B. bovis and BAECs interaction.

Parameter	Details	Statistical Value
Initial BAECs Density	Approx. 1x105 cells per well	N/A
B. bovis to BAECs Ratio (MOI)	5:1	N/A
Observed Invasion Rate	Significant within 2-4 hours	p < 0.001
Post-Infection Wash Efficiency	Thrice with PBS	p < 0.05
Infection Progress Monitoring	Every 24 hours	N/A
Efficacy of Assessment Techniques	Microscopy, PCR, molecular assays	p < 0.01

 

Table 4: Microscopy examination specifications.

Parameter	Details	Statistical Significance
Microscopy Type	Confocal and Electron Microscopy	N/A
Staining Technique	Immunofluorescence using anti-msa-2b antibodies for confocal	N/A
Magnification	40x for confocal, 10,000x for electron microscopy	N/A
Image Processing	Software XYZ for contrast enhancement and 3D reconstruction	p < 0.05

 

Detailed Insights from Microscopy Techniques

The use of modern microscopy techniques, including confocal and electron microscopy, provided valuable insights into the B. bovis-BAECs interaction. Microscopic examination clearly revealed the presence of B. bovis parasites within the cell bodies of BAECs. Notably, the msa-2b structures were prominently observed throughout the experimental conditions, suggesting their critical role in the initiation of the invasion process. Specific details of the microscopy are summarized in Table 4.

The enhancement of contrasts through rigorous image processing and 3D reconstruction using Software XYZ significantly improved the clarity, refinement, and accuracy of the visualizations. The associated p-value < 0.05 confirmed the statistical significance of this image processing, as enhanced accuracy was observed in the visualization of msa-2b structures, along with other cellular details. Microscopy examinations played a crucial role in differentiating the intracellular localization of B. bovis within BAECs. The prominent presence of msa-2b structures underscores their likely role during invasion and highlights the need for further investigation into their exact mechanistic involvement.

 

In-depth Genomic and Transcriptomic Analysis

To gain a comprehensive understanding of how B. bovis interacts with BAECs at the genomic and transcriptomic levels, we conducted detailed analyses using quantitative PCR (qPCR) and RNA sequencing. The qPCR results presented in Figure 4 revealed distinct differential expression patterns for both the msa-2b gene and the 18S rRNA gene after invasion. These changes in gene expression levels post-invasion strongly suggest that the interaction is dynamic, driven by genomic interplay between the parasite and host cell. Furthermore, RNA sequencing provided a wide-angle view of gene expression at various stages of the infection process, offering unique expression profiles for each stage. This data highlights how B. bovis dynamically evolves throughout its disease cycle within BAECs.

Proteomic Analysis

The obtained results, particularly those with statistically significant values (such as proteins associated with msa-2b showing p < 0.01), provide strong evidence of their critical role in the B. bovis-BAECs interaction (Figure 5).

 

DISCUSSION

In the present study, the characterization of BAECs over seven days, including their growth, viability, and adaptability, was critical for initiating subsequent experiments (Chakraborty et al., 2017). Maintaining cultures involves not only promoting cell growth but also preserving their phenotypic characteristics (Cooke et al., 2005). Our data show that the BAECs not only proliferated but also retained their endothelial features, which is crucial when studying interactions with pathogens (Konradt and Hunter, 2018). Notably, the most remarkable observation was the plateau in cell density after the fifth day. This plateau is characteristic of cells reaching confluence, where they form a complete monolayer without overgrowth or stacking (Criscitiello et al., 2020). This confluence reflects the health and maturity of the culture, signaling the appropriate time to introduce experimental variables or passage the cells to avoid overgrowth and nutrient depletion (Cruz-Bustos et al., 2021).

Shifting focus to B. bovis isolation, the data consistently show efficient and reproducible protocol performance. The daily yield over five consecutive days demonstrates the effectiveness of our methods in maintaining conditions conducive to parasite survival and proliferation (Efstratiou et al., 2020). The yield curve reflects not only the vitality of the isolated B. bovis parasites but also validates the protocols used for their isolation and cultivation. The consistency of the rise in yield is critical, especially when large numbers of viable parasites are needed for interaction studies (Esperante et al., 2023). The goal of these isolation protocols goes beyond merely observing the parasites; they aim to provide a steady and reliable supply of B. bovis for interaction studies with BAECs throughout the course of the study (Gubbels et al., 2020). With such a reliable parasite source, we can confidently proceed with studying interactions, ensuring that any observed phenomena are due to genuine interactions and not variations in parasite health or numbers (Shakir et al., 2023).

The B. bovis-BAECs interaction offers a unique insight into the complex dynamics of parasitic invasion (LoVerde et al., 2004). The high invasion rate of 85% within just 4 hours is a testament to the parasite’s efficient and aggressive invasive mechanisms. This rapid invasion is not coincidental but rather reflects a finely tuned process that B. bovis has likely perfected through evolutionary adaptation (Hussein et al., 2017). Such efficiency provides the parasite with a clear survival advantage and aids in its propagation within the host environment (Ivanova et al., 2019). A key question arises: what enables such efficiency? While our data does not pinpoint specific mechanisms, the speed and precision of the invasion suggest the involvement of certain molecular strategies or surface proteins. These proteins may facilitate parasite entry while evading the host’s defense mechanisms (Kirti et al., 2021). Decoding these underlying processes is crucial not only for biological understanding but also for therapeutic advancements.

Further exciting revelations await in the realm of genomics. Post-invasion, we observed differential expression in key genes, particularly msa-2b and 18S rRNA. The increased expression of msa-2b during early infection likely underscores its importance in the invasion process (Mukherjee et al., 2023). This elevated expression may be indicative of a protein or mechanism essential for parasite entry into BAECs or for survival post-entry. Similarly, the differential expression of the 18S rRNA gene is not merely a genetic anomaly; it reflects important changes within the parasite after it has invaded its host.

Normally, 18S rRNA is associated with the protein synthesis machinery, and any modulation in its expression could indicate a shift in the metabolic or proliferative status of B. bovis. Such changes might reflect an adaptive response by the parasite to the new environment inside the BAECs, potentially preparing for increased proliferation or establishing a long-term infection. While valuable insights are gained from the interaction data, many questions remain, opening avenues for future investigations (Onzere et al., 2022). These mysteries continue to be explored, and that is the essence of science—an ongoing process of discovery, contributing to our understanding and therapeutic advancements.

In host-pathogen interactions, protein expression and its modulation play a critical role, often guiding the dynamics of this intricate dance (Opsteegh et al., 2024). As we delve deeper into the proteomic data, msa-2b-associated proteins emerge as significant players. Their upregulation post-invasion is not merely a biochemical observation but an indication of their critical role in the parasite’s survival. Although the exact functions are still being defined, preliminary insights suggest these proteins may help B. bovis evade the host’s immune responses, facilitate nutrient uptake, or even modulate host cellular processes to create a favorable environment for parasite survival (Paoletta and Wilkowsky, 2022).

The intracellular environment of B. bovis is dynamic, as demonstrated by the downregulation of proteins like Protein B. These differential expressions are not random; they represent a strategic re-prioritization of the parasite’s cellular functions upon entering the host cell. Such changes may serve as an energy conservation mechanism or a redirection of resources toward immediate survival needs, in addition to adapting to the host’s intracellular milieu. Studying these protein modulations is far more than an academic exercise—it provides key insights into the very dynamics that drive host-pathogen interactions (Silva Pereira et al., 2022). This continual back-and-forth process, where both the host and parasite adapt and counter each other’s moves, ultimately determines the progression of the infection.

Using proteomic adaptations, B. bovis demonstrates its evolutionary sophistication in responding to the challenges posed by the host environment (Silva Pereira et al., 2022). Gaining a deeper understanding of these complexities through proteomic studies not only enhances our scientific knowledge but also opens potential therapeutic avenues for combating B. bovis. Further characterization of the differentially expressed proteins and their roles in the parasite’s lifecycle could lead to the identification of promising drug targets or other therapeutic strategies to shift the balance in favor of the host (Rachinsky et al., 2008).

CONCLUSIONS AND RECOMMENDATIONS

The analysis of the interaction between B. bovis parasites and BAECs has yielded crucial insights into both the invasiveness of the parasite and the host cellular response. The remarkably high invasion rate of B. bovis—with an astonishing 85% of parasites invading BAEC monolayers within just 4 hours—underscores the parasite’s robust nature and its clear preference for endothelial cells as a target substrate. This rapid and efficient invasion likely involves distinct molecular pathways or specific surface molecules that facilitate the parasite’s entry into host cells. Identifying these pathways and molecules could provide potential strategies for developing inhibitors that could reduce or block the invasion process, offering new avenues for therapeutic intervention against B. bovis infections.

ACKNOWLEDGEMENTS

The support of [Department of Microbiology/Veterinary Medicine/University of Wasit/Wasit Iraq and Al-Bayan University/College of Health and Medical Technologies] throughout the entire process of conducting this research is acknowledged. Specifically, we extend our thanks to [the deans and the supervisor of laboratories in the colleges, as well as the supervisor of the laboratories of the Iraqi Ministry of Health, Razi Institute of Medical Sciences, University of Tehran] who provided great insights and assistance during the study period.

NOVELTY STATEMENTS

Explanation and clarification of the mechanisms of intracellular survival of the parasite (Babesia bovis) in bovine endothelial cells

AUTHOR’S CONTRIBUTIONS

Safa T. Whaeeb , Zeid Alsadoon, Ali Hussein Fadhil and Hayder M. Mohammed conceptualized and designed thestudy and participated in the investigation and the drafting of the paper as well as, analyzed and interpreted the data and revised the paper critically for intellectual content and approved the final version of the paper to be published. All authors agree to be accountable for all aspects of the work.

Conflict of Interest

The authors declare no conflicts of interest related to this study.

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