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R. sanguineus is one of the most important vectors of worldwide distribution, causing important diseases that affect the nervous system in humans by zoonoses. (1). The singanglion of ticks has not been fully explored (2), in this study we are interested in knowing if there is the presence of the CHH peptide which are documented and have a central role in the homeostatic regulation of energy metabolism, molting, reproduction and stress responses (3), so in future experimental studies it could be a therapeutic target. The objective is identify the CHH / ITP neuropeptide with in silico analysis for future experimental applications in the R. sanguineus tick. The Hypothesis is the neuropeptide CHH / ITP is conserved in the R. sanguineus tick. The study carried out by Andrew , 2008, showing transcripts encoding putative ipodoid neuropeptide precursors was taken as a reference, a phylogenetic tree was performed, a BLAST of the reference sequence was performed, online programs were used to identify the signal peptide, possible glycosylation sites as well as determine the possible secondary structure of the peptide.. Both insects such as crustaceans and ticks belong to the same edge so that this possibility is increased if this peptide is conserved, no glycosylated sites were analyzed and the prediction of its secondary structure showed an alpha helix with a turn and 1 beta sheet. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 ( Betacoronavirus pandemicum ) is responsible for the disease identified by the World Health Organization (WHO) as COVID-19. We designed "CHIVAX 2.1", a multi-epitope vaccine, containing ten immunogenic peptides with conserved B-cell and T-cell epitopes in the receceptor binding domain (RBD) sequences of different SARS-CoV-2 variants of concern (VoCs). We evaluated the immune response of mice immunized with 20 or 60 µg of the chimeric protein with two different alum adjuvants (Alhydrogel ® and Adju-Phos ® ), plus PHAD ® , in a two-immunization regimen (0 and 21 days). Serum samples were collected on days 0, 21, 31, and 72 post first immunization, with antibody titers determined by indirect ELISA, while lymphoproliferation assays and cytokine production were evaluated by flow cytometry. The presence of neutralizing antibodies was assessed by surrogate neutralization assays. Higher titers of total IgG, IgG 1 , and IgG 2a antibodies, as well as increased proliferation rates of specific CD4 + and CD8 + T cells, were observed in mice immunized with 60 μg of protein plus Adju-Phos ® /PHAD ® . This formulation also generated the highest levels of TNF-α and IFN-γ, in addition to the presence of neutralizing antibodies against Delta and Omicron VoC. These findings indicate the potential of this chimeric multi-epitope vaccine with combined adjuvants as a promising platform against viral infections, eliciting a T H1 or T H1 :T H2 balanced cell response.

Pseudomonas aeruginosa is a pathogen of critical priority importance according to the WHO. Due to its multi-resistance and expression of various virulence factors, it is the causal agent of severe healthcare-acquired infections (HAIs). Effective strategies to control infections caused by P. aeruginosa must include early and specific detection of the pathogen for early and timely antibiotic prescription. The need to develop a specific and reproducible diagnostic technique is urgent, which must often be more sensitive and faster than current clinical diagnostic methods. In this study, we implement and standardize the loop-mediated isothermal amplification (LAMP) technique, coupled with the reverse line blot hybridization (RLBH) technique for the detection of P. aeruginosa . A set of primers and probes was designed to amplify a specific region of the P. aeruginosa 16s rRNA gene. The sensitivity of the LAMP-RLBH method was 3 × 10 -4 ng/μL, 1000 times more sensitive than the PCR and LAMP technique (this work), with a sensitivity of 3 × 10 -3 ng/μL. The LAMP-RLBH and LAMP techniques showed specific amplification and no cross-reaction with members of the ESKAPE group and other Pseudomonas species. The present investigation provides a technique that can be easily performed in less time, achieving a faster and more reliable alternative compared to traditional microbial diagnostic methods for the detection of P. aeruginosa .

Tick-borne rickettsioses, caused by Gram-negative bacteria of the Rickettsia genus, pose a growing global threat, with various arthropod vectors contributing to their transmission. Understanding the complex interactions within tick microbiota, including the role of Rickettsia species, is crucial for elucidating the dynamics of rickettsial diseases. Here, we investigate the taxonomic profiles and co-occurrence networks of Rickettsia in Rh. sanguineus sensus lato (s.l.) and Rh. turanicus ticks, revealing significant differences in community composition and local connectivity of Rickettsia species. While the microbiota of both tick species share common taxa, distinct differences in relative abundance and network topology suggest unique ecological niches. Moreover, robustness analysis demonstrates varying resilience to perturbations, indicating different strategies for network organization. Our findings also highlight metabolic differences between tick species, suggesting potential implications for Rickettsia interactions. Overall, this study provides insights into the intricate microbial landscape within ticks, shedding light on the functional redundancy and metabolic pathways associated with Rickettsia, thus advancing our understanding of tick-borne diseases., (© 2024. The Author(s).)

Background: A previous study highlighted the role of antibiotic-induced dysbiosis in the tick microbiota, facilitating the transstadial transmission of Babesia microti from nymph to adult in Haemaphysalis longicornis. This study builds on previous findings by analyzing sequence data from an earlier study to investigate bacterial interactions that could be linked to enhanced transstadial transmission of Babesia in ticks. The study employed antibiotic-treated (AT) and control-treated (CT) Haemaphysalis longicornis ticks to investigate shifts in microbial community assembly. Network analysis techniques were utilized to assess bacterial interactions, comparing network centrality measures between AT and CT groups, alongside studying network robustness and connectivity loss. Additionally, functional profiling was conducted to evaluate metabolic diversity in response to antibiotic treatment., Results: The analysis revealed notable changes in microbial community assembly in response to antibiotic treatment. Antibiotic-treated (AT) ticks displayed a greater number of connected nodes but fewer correlations compared to control-treated (CT) ticks, indicating a less interactive yet more connected microbial community. Network centrality measures such as degree, betweenness, closeness, and eigenvector centrality, differed significantly between AT and CT groups, suggesting alterations in local network dynamics due to antibiotic intervention. Coxiella and Acinetobacter exhibited disrupted connectivity and roles, with the former showing reduced interactions in AT group and the latter displaying a loss of connected nodes, emphasizing their crucial roles in microbial network stability. Robustness tests against node removal showed decreased stability in AT networks, particularly under directed attacks, confirming a susceptibility of the microbial community to disturbances. Functional profile analysis further indicated a higher diversity and richness in metabolic capabilities in the AT group, reflecting potential shifts in microbial metabolism as a consequence of antimicrobial treatment., Conclusions: Our findings support that bacterial interaction traits boosting the transstadial transmission of Babesia could be associated with reduced colonization resistance. The disrupted microbial interactions and decreased network robustness in AT ticks suggest critical vulnerabilities that could be targeted for managing tick-borne diseases., (© 2024. The Author(s).)

Rhipicephalus microplus , the cattle fever tick, is the most important ectoparasite impacting the livestock industry worldwide. Overreliance on chemical treatments for tick control has led to the emergence of acaricide-resistant ticks and environmental contamination. An immunological strategy based on vaccines offers an alternative approach to tick control. To develop novel tick vaccines, it is crucial to identify and evaluate antigens capable of generating protection in cattle. Chitinases are enzymes that degrade older chitin at the time of moulting, therefore allowing interstadial metamorphosis. In this study, 1 R. microplus chitinase was identified and its capacity to reduce fitness in ticks fed on immunized cattle was evaluated. First, the predicted amino acid sequence was determined in 4 isolates and their similarity was analysed by bioinformatics. Four peptides containing predicted B-cell epitopes were designed. The immunogenicity of each peptide was assessed by inoculating 2 cattle, 4 times at 21 days intervals, and the antibody response was verified by indirect ELISA. A challenge experiment was conducted with those peptides that were immunogenic. The chitinase gene was successfully amplified and sequenced, enabling comparison with reference strains. Notably, a 99.32% identity and 99.84% similarity were ascertained among the sequences. Furthermore, native protein recognition was demonstrated through western blot assays. Chitinase peptide 3 reduced the weight and oviposition of engorged ticks, as well as larvae viability, exhibiting a 71% efficacy. Therefore, chitinase 3 emerges as a viable vaccine candidate, holding promise for its integration into a multiantigenic vaccine against R. microplus.

Babesiosis is a growing concern due to the increased prevalence of this infectious disease caused by Babesia protozoan parasites, affecting various animals and humans. With rising worries over medication side effects and emerging drug resistance, there is a notable shift towards researching babesiacidal agents. Antimicrobial peptides, specifically cathelicidins known for their broad-spectrum activity and immunomodulatory functions, have emerged as potential candidates. Aquiluscidin, a cathelicidin from Crotalus aquilus , and its derivative Vcn-23, have been of interest due to their previously observed antibacterial effects and non-hemolytic activity. This work aimed to characterize the effect of these peptides against three Babesia species. Results showed Aquiluscidin's significant antimicrobial effects on Babesia species, reducing the B . bigemina growth rate and exhibiting IC 50 values of 14.48 and 20.70 μM against B . ovata and B . bovis , respectively. However, its efficacy was impacted by serum presence in culture, and it showed no inhibition against a B. bovis strain grown in serum-supplemented medium. Conversely, Vcn-23 did not demonstrate babesiacidal activity. In conclusion, Aquiluscidin shows antibabesia activity in vitro and its efficacy is affected by the presence of serum in the culture medium. Nevertheless, this peptide represents a candidate for further investigation of its antiparasitic properties and provides insights into potential alternatives for the treatment of babesiosis.

Introduction: Rickettsia rickettsii is an obligate, intracellular pathogen and the causative agent of Rocky Mountain spotted fever (RMSF). RMSF is an important zoonotic disease due to its high fatal outcome in humans. The difficulty of clinical diagnosis due to the low sensitivity and specificity of current diagnostic methods are a principal setback. We reported the development of a new method for the detection of R. rickettsii in human and tick DNA samples using loop-mediated isothermal amplification (LAMP), as well as the validation of the LAMP test for R. rickettsii in field samples of infected ticks and humans, determining the diagnostic sensitivity and specificity, as well as the reproducibility of the test., Methods: This technique uses hydroxy naphthol blue (HNB) as an indicator of the formation of magnesium pyrophosphate, a marker for the presence of DNA. Here, we used a putative R. rickettsii gene as a target for three pairs of primers that specifically amplify R. rickettsii DNA by hairpin-based isothermal amplification technique (LAMP)., Results and Discussion: The sensitivity of the assay was ~1.6-3 pg, which is 10 times more sensitive than PCR. To determine the diagnostics specificity and sensitivity, 103 human DNA samples and 30 tick DNA samples were evaluated. For the human samples, a sensitivity for HNB of 93%, a specificity of 70% and a k of 0.53 were obtained. For electrophoresis the sensitivity was 97% with a specificity of 58% and a k of 0.42. For tick samples, a sensitivity of 80% was obtained, a specificity of 93% for HNB and for electrophoresis the sensitivity and specificity were 87%. The k for both was 0.73. The degree of concordance between HNB and electrophoresis was 0.82 for humans and for ticks, it was 0.87. The result is obtained in shorter time, compared to a PCR protocol, and is visually interpreted by the color change. Therefore, this method could be a reliable tool for the early diagnosis of rickettsiosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Carvajal-Gamez, Olguín-Barrera, Tinoco-Gracia, Gordillo-Perez, Dzul-Rosado, Aguilar-Tipacamú, Hidalgo-Ruiz and Mosqueda.)

Introduction: This study evaluated the immune response to a multiepitope recombinant chimeric protein (CHIVAX) containing B- and T-cell epitopes of the SARS-CoV-2 spike's receptor binding domain (RBD) in a translational porcine model for pre-clinical studies., Methods: We generated a multiepitope recombinant protein engineered to include six coding conserved epitopes from the RBD domain of the SARS-CoV-2 S protein. Pigs were divided into groups and immunized with different doses of the protein, with serum samples collected over time to determine antibody responses by indirect ELISA and antibody titration. Peptide recognition was also analyzed by Western blotting. A surrogate neutralization assay with recombinant ACE2 and RBDs was performed. Intranasal doses of the immunogen were also prepared and tested on Vietnamese minipigs., Results: When the immunogen was administered subcutaneously, it induced specific IgG antibodies in pigs, and higher doses correlated with higher antibody levels. Antibodies from immunized pigs recognized individual peptides in the multiepitope vaccine and inhibited RBD-ACE2 binding for five variants of concern (VOC). Comparative antigen delivery methods showed that both, subcutaneous and combined subcutaneous/intranasal approaches, induced specific IgG and IgA antibodies, with the subcutaneous approach having superior neutralizing activity. CHIVAX elicited systemic immunity, evidenced by specific IgG antibodies in the serum, and local mucosal immunity, indicated by IgA antibodies in saliva, nasal, and bronchoalveolar lavage secretions. Importantly, these antibodies demonstrated neutralizing activity against SARS-CoV-2 in vitro ., Discussion: The elicited antibodies recognized individual epitopes on the chimeric protein and demonstrated the capacity to block RBD-ACE2 binding of the ancestral SARS-CoV-2 strain and four VOCs. The findings provide proof of concept for using multiepitope recombinant antigens and a combined immunization protocol to induce a neutralizing immune response against SARS-CoV-2 in the pig translational model for preclinical studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mosqueda, Hernández-Silva, Vega-López, Vega-Rojas, Beltrán, Velasco-Elizondo, Ramírez-Estudillo, Fragoso-Saavedra, Pérez-Almeida, Hernández, Melgoza-González, Hinojosa-Trujillo, Mercado-Uriostegui, Mejía-López, Rivera-Ballesteros and García-Gasca.)

Anti-microbial peptides play a vital role in the defense mechanisms of various organisms performing functions that range from the elimination of microorganisms, through diverse mechanisms, to the modulation of the immune response, providing protection to the host. Among these peptides, cathelicidins, a well-studied family of anti-microbial peptides, are found in various animal species, including reptiles. Due to the rise in anti-microbial resistance, these compounds have been suggested as potential candidates for developing new drugs. In this study, we identified and characterized a cathelicidin-like peptide called Aquiluscidin (Aq-CATH) from transcripts obtained from the skin and oral mucosa of the Querétaro's dark rattlesnake, Crotalus aquilus . The cDNA was cloned, sequenced, and yielded a 566-base-pair sequence. Using bioinformatics, we predicted that the peptide precursor contains a signal peptide, a 101-amino-acid conserved cathelin domain, an anionic region, and a 34-amino-acid mature peptide in the C-terminal region. Aq-CATH and a derived 23-amino-acid peptide (Vcn-23) were synthesized, and their anti-microbial activity was evaluated against various species of bacteria in in vitro assays. The minimal inhibitory concentrations against bacteria ranged from 2 to 8 μg/mL for both peptides. Furthermore, at concentrations of up to 50 μM, they exhibited no significant hemolytic activity (<2.3% and <1.2% for Aquiluscidin and Vcn-23, respectively) against rat erythrocytes and displayed no significant cytotoxic activity at low concentrations (>65% cell viability at 25 µM). Finally, this study represents the first identification of an antimicrobial peptide in Crotalus aquilus , which belongs to the cathelicidin family and exhibits the characteristic features of these peptides. Both Aq-CATH and its derived molecule, Vcn-23, displayed remarkable inhibitory activity against all tested bacteria, highlighting their potential as promising candidates for further antimicrobial research.

This study addresses the variability of the mitochondrial cytochrome oxidase subunit I ( COI ) and 16S rDNA ( 16S ), and nuclear internal transcriber spacer ITS2 ( ITS2 ) genes in a set of field-collected samples of the cattle tick, Rhipicephalus microplus (Canestrini, 1888), and in geo-referenced sequences obtained from GenBank. Since the tick is currently considered to be a complex of cryptic taxa in several regions of the world, the main aims of the study are (i) to provide evidence of the clades of the tick present in the Neotropics, (ii) to explore if there is an effect of climate traits on the divergence rates of the target genes, and (iii) to check for a relationship between geographical and genetic distance among populations (the closest, the most similar, meaning for slow spread). We included published sequences of Rhipicephalus annulatus (Nearctic, Afrotropical, and Mediterranean) and R. microplus (Afrotropical, Indomalayan) to fully characterize the Neotropical populations (total: 74 16S , 44 COI , and 49 ITS2 sequences included in the analysis). Only the clade A of R. microplus spread in the Nearctic-Neotropics. Both the K and Lambda's statistics, two measures of phylogenetic signal, support low divergence rates of the tested genes in populations of R. microplus in the Neotropics. These tests demonstrate that genetic diversity of the continental populations does not correlate either with the geographic distance among samples or with environmental variables. The low variability of these genes may be due to a combination of factors like (i) the recent introduction of the tick in the Neotropics, (ii) a large, effective, and fast exchange of populations, and (iii) a low effect of climate on the evolution rates of the target genes. These results have implications for the ecological studies and control of cattle tick infestations.

Ticks of the Rhipicephalus sanguineus complex are known as the brown dog ticks. This complex groups at least 12 species of ticks that are distributed worldwide. On the American continents, R. sanguineus sensu stricto (s.s.), is distributed in temperate areas, while Rhipicephalus sanguineus sensu lato (s.l.), also called "tropical lineage" is distributed in tropical regions. Previous analyses of brown dog ticks from Mexico have identified the so-called tropical lineage and the country generally has a climate more favorable for these ticks (> 20 o C in average). In addition, some pathogens thought to be transmitted by this lineage (such as Ehrlichia canis, and Rickettsia rickettsii) are prevalent in Mexico. Herein we aim to contribute to the study of brown dog ticks by providing morphological identification and molecular analysis of mt 12S rDNA and 16S rDNA sequences from ticks collected from 12 states in Mexico. Our results indicate that the tropical lineage of R. sanguineus s.l., recently redescribed as R. linnaei is widely distributed in Mexico., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Although vaccines are one of the environmentally friendly means to prevent the spread of ticks, there is currently no commercial vaccine effective against Haemaphysalis longicornis ticks. In this study, we identified, characterized, localized, and evaluated the expression patterns, and tested the immunogenic potential of a homologue of Rhipicephalus microplus ATAQ in H. longicornis (HlATAQ). HlATAQ was identified as a 654 amino acid-long protein present throughout the midgut and in Malpighian tubule cells and containing six full and one partial EGF-like domains. HlATAQ was genetically distant (homology < 50%) from previously reported ATAQ proteins and was expressed throughout tick life stages. Its expression steadily increased ( p < 0.001) during feeding, reached a peak, and then decreased slightly with engorgement. Silencing of HlATAQ did not result in a phenotype that was significantly different from the control ticks. However, H. longicornis female ticks fed on a rabbit immunized with recombinant HlATAQ showed significantly longer blood-feeding periods, higher body weight at engorgement, higher egg mass, and longer pre-oviposition and egg hatching periods than control ticks. These findings indicate that the ATAQ protein plays a role in the blood-feeding-related physiological processes in the midgut and Malpighian tubules and antibodies directed against it may affect these tissues and disrupt tick engorgement and oviposition.

Bovine babesiosis is a tick-transmitted disease caused by intraerythrocytic protozoan parasites of the genus Babesia . Its main causative agents in the Americas are Babesia bigemina and Babesia bovis, while Babesia ovata affects cattle in Asia. All Babesia species secrete proteins stored in organelles of the apical complex, which are involved in all steps of the invasion process of vertebrate host cells. Unlike other apicomplexans, which have dense granules, babesia parasites instead have large, round intracellular organelles called spherical bodies. Evidence suggests that proteins from these organelles are released during the process of invading red blood cells, where spherical body proteins (SBPs) play an important role in cytoskeleton reorganization. In this study, we characterized the gene that encodes SBP4 in B. bigemina . This gene is transcribed and expressed in the erythrocytic stages of B. bigemina . The sbp4 gene consists of 834 nucleotides without introns that encode a protein of 277 amino acids. In silico analysis predicted a signal peptide that is cleaved at residue 20, producing a 28.88-kDa protein. The presence of a signal peptide and the absence of transmembrane domains suggest that this protein is secreted. Importantly, when cattle were immunized with recombinant B. bigemina SBP4, antibodies identified B. bigemina and B. ovata merozoites according to confocal microscopy observations and were able to neutralize parasite multiplication in vitro for both species. Four peptides with predicted B-cell epitopes were identified to be conserved in 17 different isolates from six countries. Compared with the pre-immunization sera, antibodies against these conserved peptides reduced parasite invasion in vitro by 57%, 44%, 42%, and 38% for peptides 1, 2, 3, and 4, respectively ( p < 0.05). Moreover, sera from cattle infected with B. bigemina cattle contained antibodies that recognized the individual peptides. All these results support the concept of spb4 as a new gene in B. bigemina that should be considered a candidate for a vaccine to control bovine babesiosis.

A20 binding inhibitor of nuclear factor kappa B (NF-κB)-1 (ABIN-1), a polyubiquitin-binding protein, is a signal-induced autophagy receptor that attenuates NF-κB-mediated inflammation and cell death. The present study aimed to elucidate the potential role of ABIN-1 in mitophagy, a biological process whose outcome is decisive in diverse physiological and pathological settings. Microtubule-associated proteins 1A/1B light chain 3B-II (LC3B-II) was found to be in complex with ectopically expressed hemagglutinin (HA)-tagged-full length (FL)-ABIN-1. Bacterial expression of ABIN-1 and LC3A and LC3B showed direct binding of ABIN-1 to LC3 proteins, whereas mutations in the LC3-interacting region (LIR) 1 and 2 motifs of ABIN-1 abrogated ABIN-1/LC3B-II complex formation. Importantly, induction of autophagy in HeLa cells resulted in colocalization of ABIN-1 with LC3B-II in autophagosomes and with lysosomal-associated membrane protein 1 (LAMP-1) in autophagolysosomes, leading to degradation of ABIN-1 with p62. Interestingly, ABIN-1 was found to translocate to damaged mitochondria in HeLa-mCherry-Parkin transfected cells. In line with this observation, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated deletion of ABIN-1 significantly inhibited the degradation of the mitochondrial outer membrane proteins voltage-dependent anion-selective channel 1 (VDAC-1), mitofusin-2 (MFN2), and translocase of outer mitochondrial membrane (TOM)20. In addition, short interfering RNA (siRNA)-mediated knockdown of ABIN-1 significantly decreased lysosomal uptake of mitochondria in HeLa cells expressing mCherry-Parkin and the fluorescence reporter mt-mKEIMA. Collectively, our results identify ABIN-1 as a novel and selective mitochondrial autophagy regulator that promotes mitophagy, thereby adding a new player to the complex cellular machinery regulating mitochondrial homeostasis.

Background: In recent years, promising vaccination strategies against rickettsiosis have been described in experimental animal models and human cells. OmpB is considered an immunodominant antigen that is recognized by T and B cells. The aim of this study was to identify TCD4+INF-γ+ and TCD8+INF-γ+ lymphocytes in an autologous system with macrophages transfected with the vaccine candidate pVAX1-OmpB24. Lymphocytes and monocytes from 14 patients with Rickettsia were isolated from whole blood. Monocytes were differentiated into macrophages and transfected with the plasmid pVAX1-OmpB24 pVax1. Isolated lymphocytes were cultured with transfected macrophages. IFN-γ-producing TCD4+ and TCD8+ lymphocyte subpopulations were identified by flow cytometry, as was the percentage of macrophages expressing CD40+, CD80+, HLA-I and HLA-II. Also, we analyzed the exhausted condition of the T lymphocyte subpopulation by PD1 expression. Macrophages transfected with pVAX1-OmpB24 stimulated TCD4+INF-γ+ cells in healthy subjects and patients infected with R. typhi . Macrophages stimulated TCD8+INF-γ+ cells in healthy subjects and patients infected with R. rickettsii and R. felis . Cells from healthy donors stimulated with OmpB-24 showed a higher percentage of TCD4+PD1+. Cells from patients infected with R. rickettsii had a higher percentage of TCD8+PD-1+, and for those infected with R. typhi the larger number of cells corresponded to TCD4+PD1+. Human macrophages transfected with pVAX1-OmpB24 activated TCD4+IFN-γ+ and CD8+IFN-γ+ in patients infected with different Rickettsia species. However, PD1 expression played an important role in the inhibition of T lymphocytes with R. felis.

Bovine babesiosis is a tick-borne disease caused by protozoan parasites of the genus Babesia . Babesia bigemina is one of the most prevalent and economically important parasite species that infects cattle because of its impact on the meat and milk production industry. Effective disease control strategies should include detection of reservoir animals and early and specific pathogen detection using rapid, economical, sensitive, and specific detection techniques. The loop-mediated isothermal amplification technique (LAMP) is a one-step molecular reaction that amplifies DNA sequences with high sensitivity and specificity under isothermal conditions and requires no special equipment. The results can be observed by the naked eye as color changes. The aim of this work was to develop and standardize the LAMP technique for B. bigemina detection and its visualization using hydroxynaphtol blue. For this situation, primers were designed from the conserved sequences of the B. bigemina ama-1 gene. The results showed that at 63 °C in 1 h and under standardized conditions, this technique could amplify B. bigemina DNA as indicated by the characteristic colorimetric change. Sensitivity evaluation indicated that DNA was amplified at a 0.00000001% parasitemia, and it was demonstrated that this technique specifically amplified the DNA of B. bigemina . Additionally, this technique could amplify DNA from 10 strains of B. bigemina from three different countries. It is concluded that the LAMP technique as modified in our case could specifically amplify B. bigemina DNA and shows high sensitivity, does not cross-react with related organisms, and the product is observed by 60 min of reaction time based on color changes. This report is the first LAMP report that uses sequences that are conserved between strains of the ama-1 gene, demonstrates the results by color changes using hydroxynaphtol blue. We propose LAMP as a rapid and economical alternative method for the molecular detection of B. bigemina ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lizarazo-Zuluaga, Carvajal-Gamez, Wilkowsky, Cravero, Trangoni and Mosqueda.)