Comparative Transcriptome Analysis of Babesia bigemina Attenuated Vaccine and Virulent Strains of Mexican Origin

Bovine babesiosis, caused by the protozoan Babesia bigemina, is one of the most important hemoparasite diseases of cattle in Mexico and the world. An attenuated B. bigemina strain maintained under in vitro culture conditions has been used as a live attenuated vaccine; however, the biological mechanisms involved in attenuation are unknown. The objective of this study was to identify, through a comparative transcriptomics approach, the components of the B. bigemina virulent parasites that are differentially expressed in vivo, as opposed to those expressed by B. bigemina attenuated vaccine parasites when inoculated into naïve cattle. The biological material under study was obtained by inoculating spleen-intact cattle with infected erythrocytes containing either the attenuated strain or a virulent field strain. After RNA extraction, transcriptomic analysis (RNA-seq) was performed, followed by bioinformatic Differential Expression (DE) analysis and Gene Ontology (GO) term enrichment. The high-throughput sequencing results obtained by analyzing three biological replicates for each parasite strain ranged from 9,504,000 to 9,656,000, and 13,400,000 to 15,750,000 reads for the B. bigemina attenuated and virulent strains, respectively. At least 519 differentially expressed genes were identified in the analyzed strains. In addition, GO analysis revealed both similarities and differences across the three categories: cellular components, biological processes, and molecular functions. The attenuated strain of B. bigemina derived from in vitro culture presents global transcriptomic changes when compared to the virulent strain. Moreover, the obtained data provide insights into the potential molecular mechanisms associated with the attenuation or pathogenicity of each analyzed strain, offering molecular markers that might be associated with virulence or potential vaccine candidates.


Introduction
Babesia bigemina is an intraerythrocytic protozoan of the phylum Apicomplexa and is one of the main parasite species of bovine babesiosis in Mexico and the world.The dissemination of this disease is constrained to tropical and subtropical regions, and it is considered one of the foremost vital diseases vectored by Rhipicephalus microplus and Rhipicephalus annulatus ticks [1].In Mexico, the tropical and subtropical regions represent approximately 53% of the national territory, and more than 75% of cattle farming is found in these regions [2].This means that from the national record of approximately 33.5 million cattle, 25.1 million are found in areas of high endemicity.In 2016, an economic estimation for annual losses was ~68,878,694 USD, showing decreased milk production due to the presence of R. microplus, whereas in beef cattle (Bos indicus × Bos taurus) in Mexico, a loss of USD 504,729,382 was only estimated by the vector [3].However, today, there is a worldwide concern about economic losses generated by bovine babesiosis and problems with animal health.As control and prevention strategies, different methods have been used for decades; for instance, tick control with ixodicides, development of genetically resistant cattle [4,5], and inducing an active immune response through available attenuated live vaccines against B. bigemina, resulting in high protection [6][7][8].While it has been shown that there are antigenic similarities and differences between Babesia bigemina and B. bovis [9,10], it is known that a satisfactory cross-immunity between the two species does not happen [11,12].Hence, immunization would be the method that provides the best prospects of preventing and controlling bovine babesiosis [6][7][8]13].In addition, several studies have been published to identify potential vaccine material, some of which are based on lysates of Babesia-infected erythrocytes or Babesia sp.culture supernatants, including Babesia bovis and B. bigemina [7,8,14,15].The use of recombinant subunits and synthetic peptide-based vaccines has also been recently reviewed [16,17].In Australia, the use of live attenuated vaccines obtained by multiple passages in splenectomized calves has been well described [6][7][8]16,17].An experimental live attenuated vaccine has been developed and tested in vaccinated cattle under a variety of controlled needle-challenge and ticktransmitted Babesia natural challenges, showing high efficacy results.The studies carried out by our research group allowed us to show that the B. bigemina strain maintained by continuous in vitro culture behaved as an attenuated and safe parasite population, because that vaccine did not seriously affect hematological values when inoculated into susceptible animals [8,12,13,[18][19][20].An attenuated strain of B. bigemina was tested, inducing protection against heterologous challenge with infected blood or infected ticks in the field.The vaccine strain provides adequate protection without tick infection, which is considered ecologically desirable because it prevents the occurrence of clinical disease caused by the spread of the vaccine strain [8,18].
It has been hypothesized that the absence of selective pressure during the prolonged maintenance of a B. bigemina strain in continuous in vitro culture has induced structural changes in its genome, as evidenced through comparative genomics with a Mexican wild type B. bigemina virulent population [21].However, little is known about the most relevant genes differentially expressed from B. bigemina in vitro culture used as live attenuated vaccine.The aim of this study is to identify, through a comparative transcriptomics approach, the components of the B. bigemina virulent parasites that are differentially expressed in vivo, as opposed to those expressed by B. bigemina attenuated vaccine parasites when inoculated into naïve cattle.A bioinformatics analysis of the differential expression of genes in the two groups of cattle will facilitate a broader understanding of the biology of the virulent strain, particularly with regard to the virulent factors involved in the pathogenicity towards the bovine host.

Sample Collection 2.1.1. Babesia bigemina Virulent Strain
The field isolate of B. bigemina was originally obtained from a clinical case in Mexico [12].It was kept as a stabilate in cryopreservation and maintained by alternate passages in ticks and splenectomized cattle only to reactivate the virulent strain when needed [13,21].

B. bigemina Attenuated Strain
The attenuated strain of B. bigemina is a population of parasites originally derived from the same virulent isolate collected in Mexico during a clinical case of babesiosis.The strain was adapted to in vitro culture using a stationary microaerophilic system [19], and, since then, it has been maintained alternately in continuous culture and cryopreservation [20,22,23].

Experimental Cattle
A total of seven Bos taurus steers free of bovine brucellosis and tuberculosis, as required by the animal diseases zoosanitary campaigns at the national level, but also free of Rhipicephalus microplus, and, therefore, free of bovine babesiosis, were purchased from a beef cattle farm located in the high plateau of central Mexico.Selected animals were confirmed negative for babesiosis by using serological and molecular diagnosis methods such as the Indirect Fluorescent Antibody Test (IFAT) and nested-PCR assay, respectively [20,22].One of the steers was splenectomized to reactivate the virulent strain, which had been kept cryopreserved in liquid nitrogen.After the recovery of the virulent strain, three steers were inoculated with an infected-erythrocyte dose of 1 × 10 8 by the IM route (Group I).The in vitro culture-derived attenuated strain was inoculated into three steers with a similar dose of 1 × 10 8 by the IM route (Group II) (Figure 1).The handling of the experimental animals during the study was carried out following good management practices for animal welfare at CENID-SAI, and with the approval provided by the Institutional Subcommittee for the Care and Use of Experimental Animals (SICUAE.DC-2022/1-3) at FMVZ-UNAM.The attenuated strain of B. bigemina is a population of parasites originally derived from the same virulent isolate collected in Mexico during a clinical case of babesiosis.The strain was adapted to in vitro culture using a stationary microaerophilic system [19], and, since then, it has been maintained alternately in continuous culture and cryopreservation [20,22,23].

Experimental Cattle
A total of seven Bos taurus steers free of bovine brucellosis and tuberculosis, as required by the animal diseases zoosanitary campaigns at the national level, but also free of Rhipicephalus microplus, and, therefore, free of bovine babesiosis, were purchased from a beef cattle farm located in the high plateau of central Mexico.Selected animals were confirmed negative for babesiosis by using serological and molecular diagnosis methods such as the Indirect Fluorescent Antibody Test (IFAT) and nested-PCR assay, respectively [20,22].One of the steers was splenectomized to reactivate the virulent strain, which had been kept cryopreserved in liquid nitrogen.After the recovery of the virulent strain, three steers were inoculated with an infected-erythrocyte dose of 1 × 10 8 by the IM route (Group I).The in vitro culture-derived attenuated strain was inoculated into three steers with a similar dose of 1 × 10 8 by the IM route (Group II) (Figure 1).The handling of the experimental animals during the study was carried out following good management practices for animal welfare at CENID-SAI, and with the approval provided by the Institutional Subcommittee for the Care and Use of Experimental Animals (SICUAE.DC-2022/1-3) at FMVZ-UNAM.

Clinical Monitoring
The B. bigemina-inoculated splenectomized calf as well as the Group I steers were subject to daily clinical monitoring, recording the rectal temperature value and clinical signs associated with B. bigemina infection such as hemoglobinuria [22,23].Blood sampling by coccygeal vein puncture was obtained to determine the Packed Cell Volume (PCV) using the microhematocrit technique.This parameter is essential in monitoring a case of clinical babesiosis, since it reveals the percentage of the volume of erythrocytes in the peripheral blood.When the percentages are low (<25%), and mainly associated with the presence of Babesia bigemina, it is indicative of the clinical severity of the disease due to hemolysis of the erythrocytes.Giemsa-stained blood smears for microscopic examination were implemented to assess the percentage of parasitized erythrocytes (PPE).A PPE ≥3%, fever ≥40 • C, a decrease in microhematocrit ≥20-30% of the baseline value, the presence of hemoglobinuria, anorexia, and animal prostration were parameters indicative that the strain was virulent.After obtaining the virulent parasite population at peak parasitemia, steers were treated with diazoaminodibenzamidine diaceturate and supportive therapy.The steers inoculated with the attenuated vaccinal strain were also clinically monitored as described above; however, as they did not show clinical manifestations of disease, no treatment was initiated, while the steers inoculated with the virulent strain were treated to resolve the clinical disease.

Concentration of Babesia bigemina-Infected Erythrocytes
An iso-osmotic gradient with 68.46% Percoll was used essentially as described before [23]; specifically, by mixing 30 mL of Percoll (SIGMA Chemical) and 4.8 mL of blood from the inoculated steers in VYM solution (V/V) in a transparent polycarbonate tube of 14 × 70 mm, and centrifuging it for 6 min at 30,000× g at 4 • C, with deceleration.Infected erythrocyte-containing fractions were collected to subsequently perform three washes with VYM at 4000 r. p. m. for 15 min at 4 • C [24].Finally, the fractions were separated, and Giemsa-stained smears were made.The pellet containing the concentrated infected erythrocytes was later frozen at −80 • C with RNA for the subsequent extraction of the total RNA.

RNA-Seq Total RNA Extraction
The workflow applied in this study, including the experimental design and the bioinformatics process, is shown in Figure 2. The total RNA of the B. bigemina parasites was extracted using the Rneasy Mini commercial kit (Qiagen ® , Hilden, Germany) with slight modifications (Optional use of DNAse).The obtained RNA molecules were evaluated for integrity by agarose gel electrophoresis to determine quality and abundance [25].Likewise, the RNA samples were quantified using an Implen NanoPhotometer ® (Implen, Munich, Germany).Finally, the RIN (RNA Integrity Number) was determined utilizing a profile generated by the Agilent 2100 equipment (Agilent, Santa Clara, CA, USA) [26,27].

Sequencing
The Illumina platform, MiSeq Next Generation Sequencing Technology (Sanger/Illumina 1.9) with a phred33, was used to sequence the transcripts for both B. bigemina strains.The transcriptomes were generated in the University Unit of Massive Sequencing and Bioinformatics of the Institute of Biotechnology-UNAM, using a minimum of 3 µg of total RNA of the highest possible quality to construct paired and strand-specific read libraries with the TruSeq RNA Library Preparation Kit (Illumina, San Diego CA, USA).

Bioinformatic Analysis
The quality control of the reads was conducted using FastQC v0.11.5 [28].The highquality reads were then mapped to the reference genomes of B. bigemina (GCF_000981445.1)and B. taurus (GCF_002263795.1)using the bwa v0.7.17-r1188 software.Subsequent filtering steps were applied to separate the reads that mapped using Samtools v1.13 and Bamtools v2.3.0 suites.Counting and normalization of the mapped reads were carried out using the Express program (v1.5.1).Finally, the differential expression analysis was performed using the three biological replicates for each of the B. bigemina strains compared in this study; the IDEAMEX web server tool "IDEAmex-Integrated Differential Expression Analysis MultiExperiment (unam.mx)"(accessed on 31 July 2023) was used for this [29].

Sequencing
The Illumina platform, MiSeq Next Generation Sequencing Technology (Sanger/Illumina 1.9) with a phred33, was used to sequence the transcripts for both B. bigemina strains.The transcriptomes were generated in the University Unit of Massive Sequencing and Bioinformatics of the Institute of Biotechnology-UNAM, using a minimum of 3 µg of total RNA of the highest possible quality to construct paired and strand-specific read libraries with the TruSeq RNA Library Preparation Kit (Illumina, San Diego CA, USA).

In Silico Homology Analysis of Genes with Other Apicomplexa Organisms
The upregulated genes in B. bigemina were translated into proteins and used as queries to identify potentially orthologous genes in Apicomplexa organisms, particularly within the genus Babesia.This process was executed using the BLAST aligner software (version 2.2.18) [33] against the non-redundant (nr) protein database from NCBI, BLASTp (protein/protein BLAST, part of the suite of Standard Protein BLAST at NCBI (https: //blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) (accessed on 22 September 2023), and all results underwent manual inspection.

Gene Ontology Enrichment
Using a GO term enrichment analysis of differentially expressed genes, different protein families were identified.This was accomplished using the R program "TopGO" (v2.46.0).GO terms with a p-value < 0.05 were considered significantly enriched (Figure 2) [34].

Splenectomized Calf
The re-activation of the virulent strain was successfully achieved by splenectomizing and later inoculating the calf, obtaining the following results throughout the clinical monitoring: on day 5 post-infection (PI), clinical signs associated with bovine babesiosis were reported as fever 41 • C, hemoglobinuria, PCV value 16% and PPE ≥ 10%.Based on this information, it was decided to obtain biological material to inoculate three corresponding bulls of Group I with a dose of 1 × 10 8 infected erythrocytes (IE).After bleeding, the calf was treated with diasaminodibenzamide diaceturate in addition to supportive care.

Virulent Strain
The three steers inoculated with the virulent dose (recovered from the splenectomized calf) began to present fever (>40 • C) on day 4 PI, and the PCV value decreased from day 5 PI onwards, reaching 11.3% in one of the steers at the end of the monitoring (Supplementary Figures S1 and S2).The presence of the protozoan parasite was determined by light microscopy by examining blood samples on days 5-9 PI.The biological material on one steer was taken at peak parasitemia on day 8 PI, and on day 9 PI on two other steers (estimated PPEs ≥ 8%).The steers were later treated with the same agents as the splenectomized calf to prevent death.

Attenuated Strain
Steers vaccinated with the attenuated strain (G II) did not develop fever during the observation period (Supplementary Figure S1).Regarding the PCV, there was only a slight decrease and moderate clinical signs on PI days 3-7 (Supplementary Figure S2), indicative of animals infected with live attenuated parasites [7,12].It is important to mention that none of the bulls in Group II required treatment against babesiosis; the PCV returned to baseline values on day 9 PI and the PPE assessed was minimal (≤0.1%) on days 6-9 PI.All three GII steers were bled on day 8 PI.This is particularly important since the low percentage of parasite erythrocytes reached was due to the strain's intrinsic lack of virulence.

Concentration of Babesia bigemina-Infected Erythrocytes
To obtain the concentrated B. bigemina-infected erythrocytes, a 68.46%Percoll density gradient protocol was used, which, after high-speed centrifugation, allowed for the recovery of three fractions: the upper thin fraction, an intermediate fraction, and a lower fraction.Microscopic analysis of these three fractions indicated that the thin upper phase contained mainly red cell membranes and some erythrocytes infected with B. bigemina trophozoites, while the middle phase contained most of the erythrocytes infected with B. bigemina trophozoites and merozoites.Finally, in the lower phase of the gradient, there were mostly bovine mononuclear cells (Supplementary Figure S3).

Total RNA Extraction
A total of six RNA extractions were performed, three corresponding to B. bigeminaerythrocytes obtained from steers inoculated with the virulent strain (G I) and three steers inoculated with the attenuated strain (G II), complying with the RNA concentrations and quality parameters required for sequencing.The total RNA samples were found to be free of phenolic contaminants and proteins when quantified with the Nanodrop equipment.Likewise, when analyzed with the Bioanalyzer 2100, RIN numbers were considered adequate for RNA-seq.

Sequencing
A total of six cDNA paired-end libraries, representing three biological replicates for each of the B. bigemina strains analyzed in this study, were built.The sequencing analysis with the Sanger/Illumina 1.9 platform-generated range extends from the lowest to the highest values across different replicates of the same strains, obtaining 9,504,000-9,656,000 and 13,400,000-15,750,000 sequence reads for the attenuated strain replicates and the virulent strain replicates, respectively, with a sequence length of 76 bp and 53% of GC (Table 1).The analysis with the FastQC software (Version 0.12.0)revealedgood sequence quality, with a Phred value of >25.

Bioinformatic Analysis
The RNA-seq reads were mapped against the reference genome of the B. bigemina BOND strain.Concerning the similarity and variation between the biological replicates, the multidimensional scaling (MDS) method reveals a significant similarity, particularly among the triplicates belonging to the virulent strain (Supplementary Figure S4).Additionally, a slightly larger count per million (CPM) was obtained in these than in the attenuated strains, as shown in Supplementary Figure S5.The global assembly of the Babesia bigemina mapped transcripts was used for the differentially expressed gene (DEG) analysis.The DEGs were identified with the criteria of log2 (fold change) > 2 and an adjusted p-value < 0.01 for comparing differential expression patterns between the virulent strain and the attenuated strain of B. bigemina.A total of 3544 genes were detected, with 691 identified using the edgeR software (version 4.0.16),2308 with NOIseq (version 1.42.1), and 545 with DESeq2 (version 1.42.1).The results of the total differentially expressed genes between the two Babesia bigemina strains can be visualized in the volcano plot, represented with red dots (Figure 3).
For our study, we focused on the intersection of the three methods as the most reliable, resulting in a total of 519 differentially expressed genes (see Supplementary Table S1 and Figure 4).The hierarchical grouping of the DEGs illustrated in a heatmap represents the level of expression in each replicate sample when comparing the virulent strain vs. the attenuated strain, allowing for the detection of genes with a similar change in expression (Supplementary Figure S6).This type of map enables the visualization of each gene (rows) for each of the biological triplicates distinguishing between the virulent strain and the attenuated strain (columns).Higher expression levels are represented in yellow, while lower expression is depicted in blue, based on the Z score.By performing a filtering of the differentially expressed genes, the top 100 DE genes were selected, which can be viewed on the heatmap (Supplementary Figure S6).Out of these 100 genes, 41 correspond to Babesia bigemina hypothetical protein, partial mRNA, 29 to B. bigemina ribosomal proteins, 7 to B. bigemina histones, putative partial mRNA, 5 to B. bigemina translation initiation, putative partial mRNA, and the remaining 18 genes to other genes.
in expression (Supplementary Figure S6).This type of map enables the visualization of each gene (rows) for each of the biological triplicates distinguishing between the virulent strain and the attenuated strain (columns).Higher expression levels are represented in yellow, while lower expression is depicted in blue, based on the Z score.By performing a filtering of the differentially expressed genes, the top 100 DE genes were selected, which can be viewed on the heatmap (Supplementary Figure S6).Out of these 100 genes, 41 correspond to Babesia bigemina hypothetical protein, partial mRNA, 29 to B. bigemina ribosomal proteins, 7 to B. bigemina histones, putative partial mRNA, 5 to B. bigemina translation initiation, putative partial mRNA, and the remaining 18 genes to other genes.

In Silico Analysis of Genes with Importance in Apicomplexa Organisms
Of the 100 genes with the highest significance (Supplementary Figure S6), a manua in silico analysis using the NCBI database results showed that 38 up-regulated gene encoding proteins of unknown function were found in the virulent strain and 3 up

In Silico Analysis of Genes with Importance in Apicomplexa Organisms
Of the 100 genes with the highest significance (Supplementary Figure S6), a manual in silico analysis using the NCBI database results showed that 38 up-regulated genes encoding proteins of unknown function were found in the virulent strain and 3 up-regulated genes in the attenuated strain.BlastP homology analysis demonstrated that of the 38 genes up-regulated in the virulent strain and the 3 up-regulated in the attenuated strain, 35 and 3 genes have significant sequence identity with Babesia spp.genes in the virulent and attenuated strains, respectively (Supplementary Table S2).

Gene Ontology Enrichment
Out of the total number of differentially expressed genes, it was possible to identify that 482 correspond to up-regulated genes, and 37 genes are down-regulated, when comparing the virulent strain to the attenuated strain.Regarding the categorization of terms for upregulated genes, 21 terms were associated with Biological Processes, 6 with Cellular Components, and 4 with Molecular Functions (Table 2).A directed acyclic graph (DAG) depicts the results of a GO enrichment analysis of DEGs and indicates the containment relationships between terms (Supplementary Figure S7).Regarding down-regulated genes, the categorization of the gene ontology enrichment classified 16 terms belonging to Biological Processes.The number of DEGs assigned to the Cellular Component category in a GO enrichment analysis was 9, whereas it was 10 in Molecular Functions (Table 3).The category associated with biological processes most frequently found was GO:0006412 translation; however, the genes possibly associated with virulence, following the DAG in the three categories, correspond to XM_012910993.1 Protein kinase domain-containing protein, putative; and XM_012913418.1 60S acidic ribosomal protein, which were down-regulated.On the other hand, genes XM_012913282.1 TBC1 domain family member GTPase-activating protein, putative; XM_012910606.1 hypothetical protein; XP_012770178 RuvB-like 2 DNA helicase, putative; and XM_012914699.1 MORN repeat domain-containing protein, putative, were the overexpressed genes."Annotated" stands for "the total number of times the GO is found in the reference"; "Significant" stands for "the total number of times the GO is found in the list of differentially expressed genes"; "Expected" stands for "the expected value by chance that the GO should be found in the list".
Table 3. Significantly enriched GO terms in the down-regulated genes for the attenuated strain; transcripts ranked according to the p-value < 0.05.1.9 0.0337 "Annotated" stands for "the total number of times the GO is found in the reference"; "Significant" stands for "the total number of times the GO is found in the list of differentially expressed genes"; "Expected" stands for "the expected value by chance that the GO should be found in the list".

Comparative Analysis in Differential Gene Expression of a Virulent Strain vs. an Attenuated Strain of B. bigemina
The results of the differential expression and GO analyses allowed us to identify some of the genes of interest showing gene down-regulation (−1.9 to −3 LogFC) and upregulation (2.4 to 2.9 LogFC), which might be associated with virulent factors in B. bigemina.Worth noting for genes with up-regulation are the 12d3 antigen, putative XM_012910910.1;cAMP-dependent protein kinase regulatory subunit (XM_012910910.1);putative protein kinase domain-containing protein (XM_012910993.1 and XM_012913697.1);putative Ras family protein (XM_012913885.1); and acid phosphatase, putative (XM_012914848.1); which were particularly overexpressed in the virulent strain as opposed to the attenuated strain.By contrast, the down-regulated genes included a hypothetical protein (XM_012910606.1);GTPase-activating protein of the TBC1 domain family member (XM_012913282.1); and protein containing the MORN repeat domain (XM_012914699.1) in the virulent strain (Table 4).

Obtaining Biological Material
To obtain the biological material of the Babesia bigemina virulent strain, a splenectomy was performed on a Bos taurus calf to reactivate the virulent parasite population, which was inoculated with a cryostabilate kept in liquid nitrogen.It is important to mention that this surgical technique is commonly used to recover Babesia parasites that have been maintained in liquid nitrogen for a prolonged period of time [8].Previously, it has also been used as an attenuation procedure in the Babesia field of research.For example, successive passages of B. bovis have been reported to result in progressively less severe signs of disease and reduced virulence in splenectomized calves [35].Likewise, this previously described method has been used to develop a live attenuated vaccine by attenuating Babesia strains in splenectomized calves [1,6,7,35].The results obtained during clinical monitoring showed that the steers inoculated with the virulent strain of Babesia bigemina presented severe clinical disease, as evidenced by the high parasitemia, reaching a maximum PPE of ≥8, a reduced PCV of 15%, and a rectal temperature >41 • C, required specific treatment against bovine babesiosis, in addition to using supportive therapy to reestablish them clinically.On the other hand, cattle inoculated with the attenuated strain, derived from in vitro culture, and previously tested as a live attenuated vaccine, showed moderate clinical signs, with maximum parasitemia reaching a PPE <0.1%, maximum rectal temperature values of 39.5 • C, and a slight decrease in PCV.Previous studies have evaluated the effectiveness of an attenuated vaccine derived from in vitro culture of live Babesia bigemina and Babesia bovis, demonstrating that when the vaccine parasites were established in inoculated cattle and challenged with virulent isolates, they generated adequately protective immunity, either under controlled pen experimental trials or through natural exposure to Babesia-infected ticks in the field [8,12,22,36,37].The aforementioned is essential for this study since it is well-known that the attenuation of Babesia bigemina can be successfully achieved under in vitro culture conditions, leading to its use as a live attenuated vaccine.However, at the level of gene expression, it was not known until now what type of molecular events could be occurring in the B. bigemina virulent parasites as compared to those attenuated after a prolonged time under in vitro culture conditions.Thus, this study presents the first comparison of gene expression, using RNA-seq technology, between a virulent strain and an attenuated strain of Babesia bigemina from Mexico.

Sequencing and Bioinformatic Analysis
RNA Next Generation Sequencing (RNA-seq) provides unprecedented, detailed information about the transcriptional landscape of an organism and enables precise measurement of the expression level of transcripts in a sample [38].The reference genome used in this study to map the obtained reads was that of the B. bigemina BOND (RefSeq: GCF_000981445.1).This reference genome was sequenced to 8x coverage using capillary technology and assembled with Phrap.The genome was manually repaired in Gap4.Sequencing errors were corrected by polymerase chain reaction, giving a B. bigemina genome size of 13.8 Mb, a Contig N50 size of 2.5 Mb, and 5080 genes [39].In our study, despite a theoretical 75X coverage, mapping of the generated reads against the B. bigemina reference genome resulted in a B. bigemina transcriptome containing only 3544 genes of the total B. bigemina gene content.Due to the variation between biological replicates, especially for an attenuated strain on a multidimensional scale, it has been suggested that specific transcriptional responses to parasite infection may be difficult to detect in natural populations with high individual genetic diversity.Thus, the importance of transcriptional variation in response to specific interactions between host and parasite genotypes [40].Furthermore, host-parasite evolutionary dynamics in many systems have led to the emergence and maintenance of different parasite and host genotypes within the same population.Genotypes differ in fundamental characteristics: parasite genotypes differ in their infectivity, host genotypes differ in susceptibility, and the outcome of infection is often a consequence of the genotypic identity of both parties [41].
On the other hand, in a similar study, variability was identified in biological replicates by comparing blood stages of B. bovis in the host vs. kinete stages in the vector, examining their gene expression.Principal component analysis (PCA) showed that the pattern of gene expression in the B. bovis kinete samples was different from the blood phase samples.The first principal component explained 90% of the variation in the data.Furthermore, PCA shows that the transcriptomes of the B. bovis blood stages are similar to each other as compared to the transcripts from the B. bovis kinete samples, as shown by the stronger clustering of the blood stage samples in the second principal component [42].In the bioinformatics analysis performed, at least 519 differentially expressed genes were detected when the transcriptome of the B. bigemina virulent strain was compared against the attenuated strain.Babesia parasites were obtained during the clinical phase from the virulent strain and in the establishment of the vaccine parasites of the attenuated strain (both parasite populations obtained from intact cattle with spleen, and without purposive immunosuppression).Out of the 519 DEGs, 482 corresponded to genes being up-regulated, whereas 37 genes were down-regulated.The variability generated in the reads obtained between the biological triplicates of the virulent strain obtained by RNA-seq analysis could be associated with the fact that some pathogens manage to adapt dynamically and continuously to a susceptible host.This can generate variability in virulence at the population level, which can also vary due to the genetic make-up of the host and the host's immunological status, among other features.

In Silico Homology Analysis of Genes with Other Apicomplexa Organisms
In this study, several up-regulated genes that encode hypothetical proteins were found in the virulent strain.With the in silico analysis performed, a high percentage of sequence identities were identified with annotated genes, especially those of Babesia ovata.It is a bovine Babesia species originally isolated from Japan, transmitted by the ixodid tick Haemaphysalis longicornis, and widely distributed in several East Asian countries [43,44].Clinical signs in cattle infected with B. ovata are usually mild, including fever and anemia [45].In addition to B. ovata, several species of Babesia can infect livestock.Phylogenetic and evolutionary analyses using 18S rRNA sequences show that B. bigemina is the closest species to B. ovata.However, clinical signs are more severe in cattle infected with B. bigemina than in those infected with B. ovata [1,43,46].With regard to the second species, in which a high sequence identity of B. bigemina DEGs was identified, that species is B. caballi.Previous studies have shown that there are at least 263 orthologous genes that have been identified in ten different species of the Apicomplexa (i.e., B. bovis, B. bigemina, B. ovata, B. microti, B. divergens, Babesia sp.Xinjiang, T. equi, P. falciparum, T. gondii and B. caballi).Finally, it is important to note that B. caballi was phylogenetically closer to B. bigemina and B. ovata than to B. bovis [47].

Gene Ontology Enrichment
Bovine babesiosis is a disease that affects cattle in the tropical and subtropical regions of Mexico and around the world [1].Currently, the only prototype of a live attenuated vaccine in Mexico has been developed at CENID-SAI, INIFAP [8].Thus, it was of utmost importance and interest to identify the differentially expressed genes between a virulent parental B. bigemina strain and an attenuated in vitro culture-derived B. bigemina strain, which have been subject to study as a live attenuated vaccine during the last 30 years [8].RNA-seq followed by a bioinformatic differential expression analysis could help identify differences in the total gene expression existent between a virulent parasite population and the attenuated in vitro culture-derived B. bigemina strain.In this way, and taking advantage of the GO classification, a gene enrichment bioinformatic procedure can be used to analyze the performance and categorization of the results obtained in the differential expression analysis.Thus, by performing the GO enrichment analysis, and based on the subgraph produced by the R program "TopGO" (v2.46.0), we selected some enrichment terms and their corresponding genes that could possibly be implicated in the virulent phenotype of a wild type B. bigemina strain, and/or in the attenuation features of the B. bigemina strain used as a live attenuated vaccine.Thus, from the group of up-regulated genes specifically identified in the virulent strain of B. bigemina and with a biological process classification, the term GO:0032147 activation of protein kinase, the putative protein kinase domain-containing protein genes (XM_012913697 and XM_012910993) are highlighted.In this context, a previous study on the genomic comparison of these same virulent and attenuated strains of B. bigemina resulted in the identification of 27 virulence-associated genes, out of which only 5 were definitely identified in the attenuated strain, including a calcium-dependent protein kinase 4 (XM_012911530.1),Calmodulin-domain protein kinase 2 (XM_012910710.1),cAMP-dependent protein kinase (XM_012914446.1), and Casein kinase I (XM_012913338.1), to name a few, enzymes that belong to the kinase family of proteins [21].Biologically, proteins of the kinase family are fundamental in some signaling pathways of apicomplexans, including Babesia spp.Kinases are essential for the invasion of the parasite, in addition to the ligands of the parasite to the cell.Likewise, kinases comprise a class of Apicomplexa-specific serine/threonine enzymes known as calcium-dependent protein kinases (CDPKs), involved in cellular processes such as glidding, as well as invasion, mainly emphasizing the role of phosphorylation and calcium-based signaling.On the other hand, it is important to mention that, in Babesia bovis, 44 protein kinases have been reported [48].This information is very important since, in this study, they are overexpressed in the virulent strain, unlike the attenuated strain.These molecules are important as possible promising pharmacological targets in the future for parasitic diseases caused by apicomplexans, despite the fact that these kinases have structural differences [49].On the other hand, the kinase domain-containing protein has been studied in Plasmodium, where several functions for the PKA (dependent protein kinase) in the pathogenesis of malaria have been defined.The recently described Plasmodium falciparum phosphoproteomes introduced a large volume of phosphopeptide data for both basic research and the identification of new therapeutic targets against malaria.Phosphorylation at sites in the activation cycle could be mediating several processes, from regulating parasite kinase activity to mediating the coupling of other proteins.Important differences between Plasmodium and mammalian PKA isoforms indicate that the parasite kinase is a valid therapeutic target against malaria [50] and, perhaps, in Babesia.
Within the genes that were overexpressed in the category of biological processes, the term GO:0051250, defined as negative regulation of lymphocyte activation, was identified in the virulent strain vs. the attenuated strain.In a study where a comparison of a Babesia bovis virulent strain and an attenuated strain was performed by a microarray analysis with two biological replicas, a total of 78% and 89% detectable transcripts were found, identifying differentially regulated transcripts within each pair of strains [51].These differentially regulated transcripts included VESA1, SmORFs, undefined membrane and hypothetical protein-coding genes.It was reported that the majority of strain-regulated individual specific gene transcripts were not shared between the two strains [51].Other studies with B. bovis have also shown upregulation of calcium-dependent protein kinase 4 (cdpk4), tubulintyrosine ligase (ttl), and methyltransferase (mt) genes in sexually induced parasite stages in vitro and parasite development in its vector tick [52,53].
Another term classified within the biological processes possibly associated with virulence and up-regulated in the B. bigemina virulent strain is GO:0042742 defense response to the bacteria, which manages to find the 12d3 gene, a parasite antigen originally described in B. bovis.This gene has been previously identified as being expressed in B. bigemina parasites derived from in vitro culture [54] and it is a highly conserved 12d3 gene in 20 Mexican isolates of Babesia bovis [55].Within the results of the up-regulated genes identified in this study and related to the defense response to bacterium biological process category, the Ras family protein putative (XP_012769339) is worth highlighting.Ras superfamily signaling depends on the binding of specific effectors.Therefore, small changes in the sequence, structure, and/or cellular regulation of superfamily members affect binding to regulators and, thus, cellular signaling.The Ras superfamily is divided into five main families: Ras, Rho, Arf/Sar, Ran, and Rab [56].Members of the Ras family function as signaling hubs activated by various extracellular mechanisms that stimulate and regulate intracellular signaling.Rab genes code for small GTP-binding proteins of the Ras superfamily.These proteins contribute to the targeting and fusion of transport vesicles in the secretory and endocytic pathways [56,57].A family of Rab GTPases has been identified in Plasmodium.Additionally, most Apicomplexa parasites share common signal peptides of the secretory pathway proteins.This signaling ultimately controls gene transcription, which in turn affects fundamental processes such as cell growth and differentiation [58].There is information on a phylogenetic analysis that indicated a core set of essential Rabs in Apicomplexan parasites, with Babesia bovis encoding 9 Rabs, as well as Theileria annulata and T. parva [58].
The last gene selected from the biological processes category term is an acid phosphatase putative (XP_012770302).The site of acid phosphatase activity is known to occur in the erythrocytic phase of Plasmodium gallinaceum and P. berghei.Acid phosphatase activity has been demonstrated in the food tubes and endoplasmic reticulum of both parasites [59].This study identified the food vacuole as the site of digestion in the host cell cytoplasm.In addition, enzyme activity has been observed in compartments or structures that are located in infected host cells.Apparently, this mechanism does not exist in B. bigemina, as the parasite does not generate a vacuole similar to that found in Plasmodium.However, in B. microti, while it lacks a parasitophorous vacuole, there is evidence that after the invasion of the host erythrocyte, it undergoes an important morphogenetic change during which it produces an intertwining of vesicles, representing a new mechanism for the delivery of parasitic factors to the host in the phylum Apicomplexa [60].
Within the classification of cellular components, the term GO:0005952 includes genes that encode for proteins such as the cAMP-dependent protein kinase complex; cAMPdependent protein kinase regulatory subunit, putative (XP_012766364), and protein kinase domain-containing protein, putative (XP_012766447), and those characterized by the AGC family of serine-threonine protein kinases as the effector kinase of cAMP signaling.In this manner, protein kinase A (PKA) is involved in the regulation of a variety of cellular processes, including metabolism, cell development, gene expression, and apoptosis.The cAMP-dependent PKA signaling pathway plays a vital part amid the pathogenesis and virulence of various pathogens.Since cAMP flux is involved in numerous intracellular processes, the PKA signaling pathway may affect various pathological inflammatory processes.In addition, cAMP-PKA signaling pathways have been identified that are relevant to Plasmodium falciparum infection of erythrocytes, as well as a kinase anchor protein (AKAP) targeting PKA in PGE2 signaling through the prostaglandin receptor for PTGER4 (EP4)-PKA prostaglandin signaling in Toxoplasma [61].
With regard to the gene TBC1 domain family member GTPase-activating protein putative (XM_012913282), data on GTPases from apicomplexans are available, indicating that they are involved in the infection process of intracellular parasites.Three GTPases, Rac1, Cdc42 and Arf6, have been found to be involved in the invasion of host cells by Trypanosoma cruzi.During invasion, these GTPases promote parasite penetration by regulating the actin cytoskeleton at the site of parasite invasion.A similar mechanism has been observed in the invasion of the intracellular parasite Leishmania donovani, where the Rac1 and Arf6 genes are activated when the parasite enters macrophages and mediates phagocytosis.After invasion, Rac1 localizes to the phagosome where L. donovani resides, where it interacts with Cdc42 to form an F-actin sheath around the phagosome, thereby inhibiting phagosome maturation [62].
Recently, in studies on the avian influenza virus (IAV), where a multi-omic analysis was performed, a subset of IFN-dependent and independent cellular defense mechanisms that inhibit IAV replication have been identified.Among them, the autophagy regulator TBC1 domain family member 5 (TBC1D5) is highlighted.TBC1D5 binds to Rab7 to allow autophagosome-lysosome fusion, regulating IAV replication in vitro and in vivo and promoting IAV lysosomal replication by targeting the M2 protein of the influenza virus [63].Likewise, the eukaryotic protein kinase G PknG promotes the induction of macroautophagy/autophagy but inhibits the maturation of the autophagosome, as reported in Mycobacterium tuberculosis, generating the effect of blocking the flow of autophagy and increasing intracellular survival of the pathogen [64].PknG inhibits the activation of AKT (AKT serine/threonine kinase) by competitively binding to its pleckstrin (PH) homology domain, causing the induction of autophagy.PknG can also inhibit autophagosome maturation to inhibit autophagic flux by targeting RAB14, a host small GTPase.Importantly, PknG directly interacts with RAB14 and inhibits RAB14-GTP hydrolysis.In addition, PknG inhibits GTPases by phosphorylating TBC1D4/AS160 (TBC1 domain family member 4).Taken together, these results reveal a dual-function bacterial effector that tightly regulates host autophagy flux to benefit intracellular pathogen survival [65].Whether this or a similar mechanism might be occurring in Babesia bigemina remains to be tested.
Finally, the term GO:0016308, 1-phosphatidylinositol-4-phosphate 5-kinase activity, with a classification based on molecular function, selected the MORN repeat domaincontaining protein, putative gene (XP_012770153).Currently, not much is known about this protein in apicomplexan organisms; studies performed with Toxoplasma gondii identified proteins with multiple membrane localization and assembly (MORN) motifs.MORN1 appears to be conserved among apicomplexans.Interestingly, MORN1 is specific to centrosomes, specialized nuclear structures that form the spindle and ring structures at the apical and posterior ends of the endomembrane complex [66].BEWA_033160 was annotated as a MORN repeat domain-containing protein in the genome of Theileria equi and was identified as a member of a cluster of genes orthologous to Plasmodium, Theileria, B. bovis, Toxoplasma gondii, and Cryptosporidium parvum proteins [67].
No doubt further experimental work needs to be done in B. bigemina to demonstrate that the regulated gene transcripts identified in this study are involved in some sort of mechanism participating in the virulence/attenuation expression phenotype of the B. bigemina parasite populations analyzed.In addition, steers were used in this particular study due to their availability and cost.One-year-old steers are as susceptible as older animals.Previous studies, where the attenuated strain (live attenuated vaccine) was used, have included steers, bulls, cows and pregnant cows [8].In all cases, 80 to 100% efficacy has been obtained when the attenuated Babesia parasites are used as a vaccine.Further studies are required to determine if the DEGs identified in this study would also be found differentially expressed in heifers, cows and/or older bulls infected with virulent B. bigemina parasites.

Conclusions
The findings presented in this study offer a pioneering comparison of the transcriptomes between a virulent and an attenuated Babesia bigemina strain of Mexican origin using RNA-seq through Illumina sequencing.The data generated provides essential information to better understand the biological processes that exist for each of the Babesia bigemina strains used in this study.This will eventually allow for the identification and targeting of specific genes to design precise intervention strategies, such as the development of new diagnostic tools, vaccines, and innovative pharmaceutical drugs for the control of bovine babesiosis caused by B. bigemina.The potential impact of these findings holds promise for advancing both scientific knowledge and practical solutions in the field.between samples and conditions reflects their similarity, for each biological sample of the attenuated and virulent strain of B. bigemina; Figure S5: CPM Plot.A bar plot for each sample is generated where Counts Per Million for each gene in each biological sample are represented in the attenuated and virulent strains of B. bigemina; Figure S6: Heatmap of the differentially expressed genes in the virulent strain vs. the attenuated strain of Babesia bigemina.A higher level of expression can be observed in yellow and a lower expression in blue, according to a Z score (right legend of the map); Figure S7: Directed acyclic graph (DAG) depicting the results of a GO enrichment analysis of DEGs; Table S1: Differentially expressed genes identified between the virulent strain and the attenuated strain of Babesia bigemina in Mexico; Table S2: B. bigemina virulent strain Up-regulated genes.

Figure 1 .
Figure 1.Illustration of the experimental design to obtain biological material for the research study.(1A) Activation of the B. bigemina virulent strain in a splenectomized calf.(1B) In vitro culture to obtain the B. bigemina attenuated (vaccine) strain.(2A,2B) inoculation of steers by deep intramuscular route, with the specified dose for each of the B. bigemina strains (n = 3, for both groups).(3) Obtaining the biological material from each of the inoculated steers.(4) Infected erythrocyte concentration performed with a 68.46%Percoll density gradient.(5) Concentration of infected erythrocytes.(6) Extraction of total RNA from each of the samples obtained (3 biological replicates for each B. bigemina strain).

Figure 1 .
Figure 1.Illustration of the experimental design to obtain biological material for the research study.(1A) Activation of the B. bigemina virulent strain in a splenectomized calf.(1B) In vitro culture to obtain the B. bigemina attenuated (vaccine) strain.(2A,2B) inoculation of steers by deep intramuscular route, with the specified dose for each of the B. bigemina strains (n = 3, for both groups).(3) Obtaining the biological material from each of the inoculated steers.(4) Infected erythrocyte concentration performed with a 68.46%Percoll density gradient.(5) Concentration of infected erythrocytes.(6) Extraction of total RNA from each of the samples obtained (3 biological replicates for each B. bigemina strain).

Vaccines 2024 , 21 Figure 2 .
Figure 2. Workflow for the RNA-seq analysis of B. bigemina attenuated (vaccine) and virulent strains.(A) Sample preparation (total RNA).(B) Sequencing with the Illumina MiSeq Next Generation Sequencing Technology platform (Sanger/Illumina 1.9), obtaining the sequence "reads" in fastq formats.(C) Primary analysis using the FastQC program (version 0.11.5).(D) Cleanup of sequence reads by filtering out reads that mapped to the Bos taurus genome (GCF_002263795.1),and alignment of the filtered reads by mapping to the B. bigemina reference genome (GCF_000981445.1).(E) Quantitation and normalization of data.(F) Differential expression analysis with the IDEAMEX platform, using the edgeR, DESeq2 and NOISeq programs.(G) Functional annotation performed by assigning the gene ontology (GO) categories, with the R program "TopGO" (v2.46.0).(Partial images supported by BioRender.com)(accessed on 2 November 2023).

Figure 2 .
Figure 2. Workflow for the RNA-seq analysis of B. bigemina attenuated (vaccine) and virulent strains.(A) Sample preparation (total RNA).(B) Sequencing with the Illumina MiSeq Next Generation Sequencing Technology platform (Sanger/Illumina 1.9), obtaining the sequence "reads" in fastq formats.(C) Primary analysis using the FastQC program (version 0.11.5).(D) Cleanup of sequence reads by filtering out reads that mapped to the Bos taurus genome (GCF_002263795.1),and alignment of the filtered reads by mapping to the B. bigemina reference genome (GCF_000981445.1).(E) Quantitation and normalization of data.(F) Differential expression analysis with the IDEAMEX platform, using the edgeR, DESeq2 and NOISeq programs.(G) Functional annotation performed by assigning the gene ontology (GO) categories, with the R program "TopGO" (v2.46.0).(Partial images supported by BioRender.com)(accessed on 2 November 2023).

Figure 3 .
Figure 3. Volcano plot of differentially expressed genes (DEGs) between virulent and attenuated strains of Babesia bigemina.The X-axis is the logarithmic scale 2 of the fold change in gene expression (log2 (fold change)).The Y-axis is the log10-adjusted p-value, indicating the level of significant difference in expression.Black dots represent genes without differential expression; red dots represent differentially increased genes based on p-adj and logFC cut-off values.

Figure 3 . 2 Figure 4 .
Figure 3. Volcano plot of differentially expressed genes (DEGs) between virulent and attenuated strains of Babesia bigemina.The X-axis is the logarithmic scale 2 of the fold change in gene expression (log2 (fold change)).The Y-axis is the log10-adjusted p-value, indicating the level of significant difference in expression.Black dots represent genes without differential expression; red dots represent differentially increased genes based on p-adj and logFC cut-off values.Vaccines 2024, 12, x FOR PEER REVIEW 9 of 2

Figure 4 .
Figure 4. Venn diagram.Integration summary with the intersection for DE genes identified with NOISeq, edgeR, and DESeq2.

Table 1 .
Summary of the sequencing results for three biological replicates of the Mexican Babesia bigemina virulent and attenuated strains, with paired-end readings.

Table 2 .
Significantly enriched GO terms in the up-regulated transcripts for the attenuated strain ranked according to the p-value < 0.05.

Table 4 .
Up-regulated and down-regulated genes of attenuated versus virulent strains of B. bigemina, with possible association with one or more virulent factors.Cut-off is log 2-fold change in expression, p-value ≤ 0.01, (n = 3).