“Expression of a recombinant chimeric capsid-nucleocapsid protein from bovine leukemia virus genotype 1 for the diagnosis of enzootic bovine leukosis”

Abstract

Bovine Leukemia Virus (BLV) is the causative agent of enzootic bovine leukosis (EBL), a neoplastic disease responsible for significant economic losses in the cattle industry. While diagnostic tests primarily target the capsid (p24) and envelope (gp51) proteins, the immunodiagnostic potential of the nucleocapsid protein (p12) remains underexplored. This study aimed to express a recombinant chimeric capsid-nucleocapsid protein (p24-p12) from BLV genotype 1 and evaluate its diagnostic performance. Following in silico epitope prediction, an 851 bp fragment of the gag gene encoding the chimeric protein (RCP24-12) was cloned, expressed in E. coli, and used to develop an indirect ELISA. The assay was used to test 680 plasma samples from Holstein Friesian cattle and compared to a commercial gp51-based ELISA. Discordant results were resolved by Western blot (WB) using RCP24-12 as antigen. The RCP24-12 ELISA detected antibodies in samples negative by the commercial test. Using a composite reference standard (commercial ELISA + WB), the in-house assay demonstrated a sensitivity of 88.8% and a specificity of 79.2%. These findings indicate that the recombinant chimeric protein p24-p12 is a promising antigen for the serological diagnosis of BLV genotype 1 infection, offering improved detection capability over a single-protein (gp51) antigen.

Introduction

Enzootic bovine leukosis (EBL) is a worldwide distributed, infectious lymphoproliferative disease of cattle. The causative agent is Deltaretrovirus bovleu (Bovine Leukemia Virus, BLV), family Retroviridae, closely related to Deltaretrovirus priTlym1 (Primate T-lymphotropic virus, PTLV) (Polat 2017; ICTV 2025). Most BLV infections are subclinical; however, cattle over three years of age may develop persistent lymphocytosis (30% of infected animals), and some individuals present lymphosarcomas in various organs (Sandev 2004; Gillet 2007). Like other retroviruses, the BLV genome comprises four structural genes (gag, pol, pro, env) and accessory genes within the pX region (tax, rex, G4, R3). The gag and env genes encode the major structural proteins. The gag gene gives rise to the matrix, capsid (CA, p24), and nucleocapsid (NC, p12) proteins, while the env gene encodes the surface (SU, gp51) and transmembrane (TM, gp30) glycoproteins (Llames 2001). The immunogenic capacity of these structural proteins varies in infected cattle. Antibodies against the SU protein (gp51) are durable, whereas the TM protein (gp30) induces a weak immune response (Bruck 1982). Conversely, the non-glycosylated capsid protein (p24) can elicit antibodies in early infection stages, even before gp51 antibodies appear (Barez 2015). Consequently, IgG antibodies against p24 and gp51 are the most prevalent during infection and are the primary targets for serological diagnosis (Johston 2002). BLV has been classified into 12 genotypes based on the env gene, with genotype 1 being the most widely distributed globally (Pereira 2023). This genetic variability can impact the diagnostic efficiency of serological tests. In Mexico, various seroprevalence studies have confirmed BLV infection in numerous herds and geographic regions (Cerón-Téllez 2020). Genotyping studies in Mexican dairy cattle have identified BLV genotypes 1 and 3 (Heinecken 2017; Cerón-Téllez 2020). Serological testing is the most cost-effective method for detecting BLV-infected animals. Enzyme-linked immunosorbent assay (ELISA) is the most widely used and recommended commercial test in many countries for infection control. (Barajas-Rojas 1993; Milićević 2025). The BLV nucleocapsid protein (p12) has been studied for its role in the viral replication cycle (Bicka 2001; Gutiérrez 2009) and in mapping T-cell recognition epitopes (Knapen 1944; Larsen 2013; Bai 2015; Andreolla 2018). Despite this, its potential as a diagnostic antigen remains unexplored. While numerous studies have expressed BLV p24 and gp51 using baculovirus and bacterial systems due to their high antigenicity, bacterial expression systems like E. coli are generally more economical and productive than eukaryotic cell culture systems (Bai 2019; Bai 2023). The objective of this study was to generate a recombinant chimeric capsid-nucleocapsid (CA-NC, p24-p12) protein from BLV genotype 1 and determine its diagnostic efficiency for detecting antibodies in cattle naturally infected with BLV.

Materials and methods

Study animals

A total of 680 plasma samples were selected from a sample bank stored at the Virology, Genetics, and Molecular Biology Laboratory of FES-Cuautitlán. The samples were obtained from Holstein Friesian dairy cattle (females older than 3 years) from different states of Mexico (Aguascalientes, Coahuila, Guanajuato, Jalisco, Puebla, Querétaro, Hidalgo, and Tlaxcala). These samples had been previously tested with a commercial ELISA (VMRD^®, USA) detecting antibodies against BLV gp51. Furthermore, some samples had been genotyped, identifying BLV genotype 1 in most animals and genotype 3 in only one animal from Jalisco (Cerón-Téllez 2020; Heinecke 2017). The review of this experimental project was conducted by the Internal Committee for the Care and Use of Experimental Animals (CICUAE-FESC), which established that no experiments would be performed on animals for this specific study; therefore, the approval of such procedures did not fall under its purview for this project.

B-cell epitope prediction

An analysis to predict antigenic determinants was performed on a set of gp51, p24, and p12 protein sequences of BLV available in GenBank and on a complete BLV genotype 1 genome (accession number AP019592.1) using the online tool http://tools.iedb.org/bcell/ (Kolaskar y Tongaonkar 1990). The available methods use the physicochemical properties of amino acid residues and their frequencies of occurrence to predict antigenic determinants in protein sequences. The Kolaskar and Tongaonkar algorithm (Kolaskar y Tongaonkar 1990) has demonstrated an accuracy of approximately 75% for predicting antigenic determinants. Parker’s algorithm evaluates hydrophilic, accessible, or mobile regions that correlates to the known antigenic sites from immunological studies (Parker 1986). Based on this analysis, the decision was made to clone a chimeric protein comprising the capsid and nucleocapsid proteins of BLV genotype 1.

Cloning, expression, and purification of BLV RCP24-12

The synthesis and expression of the recombinant chimeric protein consisting of the p24 (CA) and p12 (NC) proteins of BLV (RCP24-12), were carried out through a cloning process. Specific primers flanking the sequences corresponding to the p24 and p12 proteins of the BLV genotype 1 genome (GenBank accession AP019592.1) were designed using the IDT DNA OligoAnalyzer™ tool. The sequences were as follows: Fw-p24-p12: 5’-CCA ATC ATA TCT GAA GGG AAT CGC-3’ and Rv-p24-p12: 5’-CTA CTA TGA TTT GAG GGT TGG ACA GTC-3’. The reverse primer (Rv-p24-p12) included an additional stop codon to ensure the synthesis of the full ORF protein. DNA from a naturally BLV-infected bovine was used as a template to obtain the insert. PCR amplification with the Fw-p24-p12 and Rv-p24-p12 primers generated an 851 bp amplicon corresponding to the fused p24 and p12 sequences of BLV.

This PCR product was cloned into the pJET1.2/blunt cloning vector (Thermo Fisher Scientific™, USA), designed for blunt-end cloning. This vector also employs a positive selection system, facilitating the identification of cells containing the desired insert. Subsequently, competent Escherichia coli TOP10 cells (Invitrogen Life Technologies, Carlsbad, CA, USA) were transformed. These cells, capable of incorporating exogenous DNA with high efficiency, were incubated with the recombinant vector obtained from pJET1.2/blunt, allowing for the incorporation of the DNA into the pETSUMO expression vector (Invitrogen Life Technologies, Carlsbad, CA, USA). The transformed cells were then selected on agar medium containing ampicillin, facilitating the identification of cells that successfully incorporated the recombinant plasmid, thanks to the antibiotic resistance marker present in the pJET1.2/blunt vector. Once the insert cloning was confirmed, ligation into the pETSUMO expression vector was performed following the methods described in previous studies (Lara-Romero 2022; Castañeda-Montes 2023). BLV RCP24–12 was purified using HisTrap HP 5 mL Ni-NTA agarose columns (GE Healthcare, USA) according to the manufacturer’s instructions. The presence of purified chimeric protein was confirmed using SDS-PAGE, Western blot, and quantified by Bradford method (Bradford 1976) using bovine serum albumin as the standard. The recombinant chimeric protein was dialyzed, lyophilized, and stored at − 20 °C until use.

BLV RCP24-12 ELISA

An indirect ELISA (iELISA) was standardized using the BLV RCP24-12 in 96-well plates (Thermo Fisher Scientific™, USA). Different antigen concentrations (BLV RCP24-12) were evaluated, ranging from 23 ng/well to 300 ng/well. Plasma was tested at dilutions from 1/10 to 1/400. Similarly, the concentration of horseradish peroxidase-conjugated Protein G (Thermo Scientific™, USA) was evaluated at dilutions from 1/1050 to 1/5000. The iELISA technique was standardized by adding different concentrations of BLV RCP24-12 to the wells in a final volume of 50 µl of carbonate buffer pH 9.6 (Sigma-Aldrich, USA). Plates were incubated for 16 h at 4 °C. After incubation, three washes were performed with PBS (Quick Silver™, Accuris, USA) and 0.1% Tween 20 (Sigma-Aldrich, USA). The plate was then blocked with 5% casein (Sigma-Aldrich, USA), adding 230 µl per well, and incubated for 2 h at 37 °C. After blocking, three washes with PBS-Tween 20 were performed again, and plasma was added at the dilutions (100 µl of plasma diluted with 2.5% casein in sterile PBS - dilution buffer) and incubated for 1 h at 37 °C. The washing process was repeated, and 100 µl of horseradish peroxidase-conjugated Protein G (Thermo Scientific™, USA) was added to each well, followed by incubation for 1 h at 37 °C. After this incubation, three washes with PBS-Tween 20 and three washes with sterile PBS were performed. Finally, 100 µl of ABTS substrate (Chemicon Millipore, USA) was added to each well, and plates were incubated for 30 min at room temperature in the dark. Absorbance was read spectrophotometrically at a wavelength of 405 nm. Positive and negative control samples, previously confirmed as seropositive and seronegative by both commercial ELISA and Western Blot (WB), were used. To determine sample reactivity on each plate, the cut-off value was determined using sample reaction index=). In addition, a threshold was established above the negative control index to ensure adequate discrimination between positive and negative samples. Negative control index= ).

BLV RCP24-12 western blot

Purified BLV RCP24-12 was placed in reducing buffer (20% glycerol, 2% SDS, 5% β-mercaptoethanol; 2-mercaptoethanol, 0.0623 M Tris-HCl, pH 6.8) for 10 min in a boiling water bath (100 °C). The recombinant protein concentration per lane was 100 ng in a final volume of 15 µl, separated on a 13% SDS-PAGE gel. After electrophoresis, transfer was performed onto PVDF (polyvinylidene fluoride) membranes (Thermo Scientific™, USA), activated with methanol. Transfer was conducted at 90 volts for 55 min. After transfer, the membrane was dried, then placed in methanol for 30 s, in distilled water for 5 min, and in PBS-Tween 20 three times for 10 min each, with agitation at each step. Transferred membranes were incubated with 25 ml of blocking solution (2.5% casein) at room temperature with agitation for 1 h and 20 min. After this time, the membrane was washed three times with PBS-Tween 20 for 5 min each. Subsequently, the membrane was incubated with plasma diluted 1/10 for 1 h at room temperature with agitation. The washing process with PBS-Tween 20 (5 min, three times, with agitation) was repeated. Horseradish peroxidase-conjugated Protein G was then added at a dilution of 1/2500 and incubated for 1 h at room temperature with agitation. The washing process was repeated as described. Finally, DAB (diaminobenzidine, Thermo Scientific™, USA) with H₂O₂ was added for approximately 30 s, and the membrane was immersed in distilled water to stop the development reaction.

Statistical analysis

The diagnostic efficient of the BLV RCP 24 − 12 ELISA was analyzed by comparing it with the commercial ELISA (gold standard), correlating predictive values between tests (Cohen´s kappa statistic ± standard error and 95% confidence intervals for this statistic, calculated at https://www.graphpad.com/quickcalcs/kappa1/) according to the criteria established by Landis and Koch (1977). Sensitivity and specificity were determined using Win Episcope 2.0 (http://www.winepi.net/uk/index.htm) when making comparisons with the commercial ELISA test. Samples with discordant results between both ELISA tests were evaluated by WB.

Results

BLV RCP24-12

The primer sequences designed to amplify the capsid and nucleocapsid region of BLV genotype 1 were: Fw-p24-p12: 5’-CCA ATC ATA TCT GAA GGG AAT CGC-3’ and Rv-p24-p12: 5’-CTA CTA TGA TTT GAG GGT TGG ACA GTC-3’. An 851 bp fragment of the gag gene from BLV genotype 1, encoding the capsid and nucleocapsid proteins (p24-p12), was expressed in the Champion™ pET-Sumo vector (Fig. 1a, b and c). The recombinant chimeric protein (RCP24-12) consistently migrated as a band with a molecular weight of 71 kDa (Fig. 1d and e). This weight was the expected size for the recombinant protein and was confirmed using bioinformatic tools (https://www.bioinformatics.org/sms/prot_mw.html) (22). Antigenic epitopes for B lymphocytes were identified in the gp51, p24, and p12 protein sequences of BLV using the Kolaskar & Tongaonkar and Parker algorithms (Fig. 2).

Fig. 1.

(a) Ethidium bromide-stained agarose gel showing the PCR amplification product using the primers Fw-p24-p12 and Rv-p24-p12. The DNA was obtained from naturally infected cattle; the expected amplicon is shown in bold in base pairs (bp). (b) Diagram of the pET SUMO expression system vector. The black arrow indicates the cloned recombinant chimeric protein (c) Bacterial culture in a Petri dish showing the growth of bacterial colonies containing a recombinant plasmid (with the recombinant DNA insert). (d) Analysis of recombinant chimeric protein (RCP24-12) expression in the BL21/DE3 expression system by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. (e) RCP24-12 specificity was determined by Western blot using 6X His-tag antibody. Different batches of clone RCP24-12 were loaded to visualize its integrity

Fig. 2.

Methods for predicting continuous antibody epitopes from protein sequences. Epitope prediction graphs for the BLV proteins gp51, p24, and p12 are shown using the Parker and Kolaskar & Tongaonkar algorithms. The regions with the highest scores are shown as yellow peaks, and the score scale is indicated on the y-axis

BLV RCP24-12 elisa results

The immunogenicity of BLV RCP24-12 was analyzed in an iELISA using 680 bovine samples previously tested with the commercial VMRD^® ELISA based on the gp51 protein. The absorbance values for each ELISA are shown in Fig. 3, and the infectious status (seropositive or seronegative) for BLV by region and technique is listed in Table 1. Higher absorbance values were obtained with the RCP24-12 BLV ELISA for several samples compared with the commercial VMRD^® ELISA. For the RCP24-12 ELISA, the average cut-off value of all plates was 0.292, while the average cut-off for the commercial ELISA was 0.356.

Fig. 3.

Absorbances obtained from the VMRD^® ELISA test that detects antibodies against gp51 (blue circle) and the homemade RCP24-12 ELISA test that detects antibodies against p24-p12 (orange circle). The cutoff calculated for ELISA RCP24-12 BLV and VMRD^® or cutoff line continuous line and dotted line, respectively

Table 1.

Results obtained using a commercial ELISA test (VMRD^® gp51) and a homemade ELISA (RCP24-12 BLV) for the detection of antibodies against BLV. In addition, the result obtained by WB of the samples that were discordant between the ELISA tests used is shown

State	Samples	VMRD+/RCP24-12+	VMRD-/RCP24-12-	Discordants ELISA test	WB+	WB-
Aguascalientes	105	76	1	28	21	7
Coahuila	104	91	1	12	8	4
Guanajuato	87	43	6	38	19	19
Jalisco	207	154	49	4	3	1
Puebla	54	28	12	14	8	6
Querétaro	9	9	0	0	0	0
Hidalgo	14	14	0	0	0	0
Tlaxcala	100	52	5	43	34	9
Total	680	467	74	139	93	46

Of the total 680 bovine plasma samples used in this study, 467 were concordant seropositive in both ELISA tests (VMRD^® gp51 BLV ELISA and RCP24-12 BLV ELISA), while 74 samples were concordant seronegative in both tests (Table 1). Regarding discordant samples (20.44%, n = 139), 84 were seropositive by VMRD^® ELISA but seronegative by RCP24-12 ELISA, whereas 55 were seronegative by VMRD^® ELISA but seropositive by RCP24-12 ELISA. Of these 139 discordant samples, 93 tested positives by Western Blot (WB) using the BLV RCP24-12 protein as antigen. In total, using BLV RCP24-12 as antigen (ELISA and WB), antibodies against BLV were identified in 560 Holstein Friesian bovine plasma samples, corresponding to 82.35% of the analyzed samples.

When comparing the initial results from the commercial ELISA test (used as the gold standard) with a 95% significance level, the RCP24-12 BLV ELISA showed a sensitivity of 89.5% and a specificity of 46.8%, with a kappa agreement index of 0.388 (fair). In contrast, when discordant samples were evaluated by WB and these results were used to establish the gold standard and determine the diagnostic accuracy of the RCP24-12 BLV ELISA, a sensitivity of 88.8% and a specificity of 79.2% were obtained, with a kappa value of 0.604, maintaining the agreement level as moderate. On the other hand, in samples from cattle from two different states (Querétaro and Hidalgo), no discordant samples were identified, and the entire population was seropositive, although it should be noted that these were also the regions with the lowest number of samples. In contrast, samples from cattle from three other states (Aguascalientes, Guanajuato, and Tlaxcala) had a high number of samples discordant between the ELISA tests used, approximately 78% (Fig. 4).

Fig. 4.

Map of Mexico showing the states from which blood samples were obtained from Holstein cattle. The bars show the number of animals that tested positive (orange), negative (blue), and discordant (green). Discordant animals were evaluated using the WB technique, and positive (orange) and negative (blue) results are shown in color

Discussion

This study successfully expressed a recombinant chimeric capsid/p24-nucleocapsid/p12 protein of BLV genotype 1. This protein proved to be a suitable antigen recognized by antibodies from cattle naturally infected with BLV, yielding satisfactory diagnostic results comparable to those obtained with a commercial ELISA detecting antibodies against the bovine leukemia virus gp51 protein. While there is no vaccine or treatment for enzootic bovine leukosis, all infected animals mount an immune response through antibody production, making serological tests the best option for identifying BLV infection. The seroprevalence of Bovine Leukemia Virus (BLV) infection in Mexico has shown an increasing trend in recent decades, reflecting high viral circulation, particularly in intensive dairy production systems. Early reports in the country from 1987 in the state of Veracruz indicated a seroprevalence of 15.78% in dual-purpose cattle detected by agar gel immunodiffusion (Monroy 1987). Other studies have reported an average seroprevalence of 9.83% in five southeastern states, although data were generated from sampling a single herd per state, limiting representativeness (Cordero-Pulido 2023). In more recent years, studies with broader coverage and using more sensitive diagnostic methods, such as indirect ELISA, have revealed considerably higher seroprevalence levels, ranging from 55.9% to 58.7% in states like Puebla, Aguascalientes, Coahuila, and Tlaxcala. These seroprevalences demonstrate the significant expansion of BLV infection in dairy herds nationwide (Heinecken 2017; Cerón-Téllez 2020; Montero-Machuca 2022; González-Méndez 2023). On an international scale, Mexico would rank in a high seroprevalence range (82.35% obtained in this study), similar to that described in other countries in the Americas. Countries such as the United States and Argentina have published studies with seroprevalence exceeding 80% or 85%. Canada also reports high figures (78%–89.3%), while Brazil documents variable ranges from 17.1% to 60.8% depending on the geographic region. Conversely, Japan reports a seroprevalence of 49.1% in dairy cattle. However, despite the high seroprevalences described, countries like the United Kingdom, Denmark, Sweden, Finland, New Zealand, and Australia have successfully eradicated BLV through rigorous sanitary policies, including early diagnosis, movement control, and culling of seropositive cattle (Lv 2024). Serological tests are highly useful and easy to implement for detecting infection-induced antibodies and have been extensively used to identify BLV-infected cattle due to their speed, cost-effectiveness, and ease of interpretation (Gutiérrez 2009; Andreolla 2018). For this reason, various commercial ELISA formats for identifying antibodies against BLV have been available on the market for decades (González et al. 2001; Gutiérrez 2009). These tests use the viral envelope glycoprotein gp51 and the capsid protein p24 as antigens, as these are the primary targets of antibodies in a natural infection (Cowley 1992; Larsen 2013; Troiano 2013; Kuezewski 2018). Some studies have described those antibodies against gp51 appear early and in high titers compared to antibodies binding the p24 protein (González et al. 2001; Bartlett 2013; Bai 2023). The present study analyzed the utility of a recombinant chimeric protein including p24 and the p12 protein in an indirect ELISA format. The results obtained from a large number of samples showed high optical density (OD) values (Fig. 3), exceeding those obtained using gp51 in a commercial ELISA. This difference in OD values could be related to antibody titers and could be associated with the animals in this study not being in an early stage of infection, where antibodies against gp51 might be low or absent. Another possible explanation for this low antibody concentration could be related to gp51 variability (Olech 2023), i.e., the possibility of an infecting genotype different from the one used in the commercial diagnostic test. This would also explain the high number of discordant samples between ELISA techniques identified in the study, particularly in the geographic regions of Guanajuato, Tlaxcala, and Aguascalientes. It is important to note that BLV genotype 3 was previously identified in the state of Aguascalientes, differing from other regions of the country where genotype 1 prevailed (Cerón-Téllez 2020). Alternatively, the p12 protein might stimulate the production of high antibody titers in infected animals, as suggested by the B-cell epitope analysis, which revealed at least two immunodominant epitopes (Fig. 2). However, the capsid and nucleocapsid proteins were not evaluated independently, precluding definitive conclusions on this hypothesis.

The RCP24-12 BLV ELISA detected antibodies against BLV in 467 of 680 cattle, representing 68.67% of samples. In both tests, 74 samples were seronegative, equivalent to 10.88%. Conversely, 55 samples negative by VMRD^® ELISA but positive by RCP24-12 BLV iELISA represent 8.08% of the total, while 84 samples seropositive by VMRD^® ELISA but negative by RCP24-12 BLV ELISA correspond to 12.35%. Using RCP24-12 as the antigen to recognize BLV antibodies by ELISA and WB, 497 seropositive animals were detected, corresponding to 73.08% seropositivity, and 95 seronegative animals (13.97%). This suggests that to achieve results similar to the commercial ELISA, samples seronegative by the RCP24-12 ELISA should be evaluated by WB using the same recombinant protein. The analysis for identifying B-cell epitopes based on antigenicity and hydrophilicity for gp51, p24, and p12 proteins generally showed better scores for the surface protein (Fig. 2). Nevertheless, a similar number of peaks was observed with both algorithms for all proteins, demonstrating that p12 contributes 2–3 immunodominant epitopes, which undoubtedly favored the reactivity of the RCP24-12 BLV ELISA in conjunction with p24. The BLV nucleocapsid protein has not been studied as a potential diagnostic antigen; research has only focused on its role in the viral replication cycle (Bicka 2001; Gutiérrez 2009) and in mapping T-cell recognition epitopes (Knapen 1994; Larsen 2013; Bai 2015; Andreolla 2018). It would be important to analyze the antigenic capacity of p12 individually to precisely determine its value as a diagnostic antigen and to analyze its reactivity across different BLV genotypes. However, it is necessary for full operational validation (intra-/inter-assay variability, reproducibility) before field deployment as a screening tool. The use of antigens prevalent in different geographical areas appears crucial for improving diagnostic efficiency, not only because they are immunodominant but also because they relate to the prevalent infecting genotype in the study region. This was shown in the present study, where the recombinant protein from BLV genotype 1 identified a slightly higher number of seropositive animals, as this genotype appears to be prevalent in Mexican dairy herds (Heinecken 2017; Cerón-Téllez 2020; Montero-Machuca 2022). In conclusion in this study, a recombinant chimeric capsid-nucleocapsid (p24CA-p12NC) protein from BLV genotype 1 was successfully expressed. The recombinant RCP24-12 protein evaluated in the ELISA showed a sensitivity of 88.8% and a specificity of 79.2%, with a Kappa value of 0.604. Using the RCP24-12 protein as an antigen, high reactivity in recognizing antibodies against BLV was identified in a large number of samples.

Author contributions

All authors contributed to the conception of this study. José Hiram Sánchez Gasca and Hugo Ramírez Álvarez designed the research; José Hiram Sánchez Gasca, José Luis Cerriteño Sánchez and Cecilia Rodriguez-Murillo performed laboratory analyses. Data analysis and statistical evaluation were conducted by Julieta Sandra Cuevas Romero and Gabriel Eduardo Acevedo-Jimenez. The first draft of the manuscript was written by José Hiram Sánchez Gasca and Hugo Ramírez Álvarez, and all authors provided comments on subsequent versions. José Luis Cerriteño Sánchez, Julieta Sandra Cuevas Romero and Gabriel Eduardo Acevedo-Jimenez edited the final draft. Funding acquisition was led by Hugo Ramírez Álvarez. All authors have read and approved the published version of the manuscript.

Funding

This research was supported by Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT), Dirección General de Asuntos del Personal Académico (DGAPA), Universidad Nacional Autónoma de México (UNAM) (Project Code: IN206924), and by the Research Chair CI2478 of Facultad de Estudios Superiores Cuautitlán, UNAM.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

This study was approved by the Institutional Committee for the Care and Use of Experimental Animals from the Facultad de Estudios Superiores Cuautitlán (CICUAE-FESC).

Competing interests

The authors declare no competing interests.