Efficacy of Entomopathogenic Staphylococcus Bacteria as a Biocontrol Agent against Rhipicephalus microplus Ticks: Assessing Reproductive Inhibition and Mortality Rates.

Rhipicephalus microplus is a persistent ectoparasite of cattle that causes bovine anaplasmosis and babesiosis, causing economic losses worldwide. Chemical treatment is the primary method for tick control, but the emergence of pesticide-resistant ticks is a major challenge. Alternative biocontrol strategies utilizing entomopathogenic microorganisms are being explored. This study aimed to validate the species identification and assess the efficacy of four strains of Staphylococcus bacteria (S. shinii S1 and S-2, S. succinus, and S. xylosus) previously reported as being entomopathogenic to R. microplus ticks. According to the bioassays, S. shinii S-1 exhibited the greatest degree of reproductive inhibition (47%), followed by S. succinus (44.3%) at a concentration of 1 × 108 cfu/mL. S. xylosus displayed decreased reproductive inhibition (6.3%). In an additional bioassay, S. shinii S-1 exhibited a significant larval mortality of 67.63%, followed by S. succinus with 66.75%, S. shinni S-2 with 64.61%, and S. xylosus with 28.18% mortality. The common signs of infection observed on these ticks included swelling, yellowish exudate on the hypostome, and reduced limb mobility and color change, except for S. succinus, which did not cause color changes. These bacteria were naturally found on bovine skin. However, further studies are needed to confirm their potential as promising alternatives or complementary agents to existing acaricidal compounds.

Relationship between acaricide resistance and acetylcholinesterase gene polymorphisms in the cattle tick Rhipicephalus microplus.

In this study, we aimed to develop a comprehensive methodology for identifying amino acid polymorphisms in acetylcholinesterase transcript 2 (AChE2) in acaricide-resistant Rhipicephalus microplus ticks. This included assessing AChE2 expression levels through qPCR and conducting 3D modeling to evaluate the interaction between acaricides and AChE2 using docking techniques. The study produced significant results, demonstrating that acaricide-resistant R. microplus ticks exhibit significantly higher levels of AChE expression than susceptible reference ticks. In terms of amino acid sequence, we identified 9 radical amino acid substitutions in AChE2 from acaricide-resistant ticks, when compared to the gene sequence of the susceptible reference strain. To further understand the implications of these substitutions, we utilized 3D acaricide-AChE2 docking modeling to examine the interaction between the acaricide and the AChE2 catalytic site. Our models suggest that these amino acid polymorphisms alter the configuration of the binding pocket, thereby contributing to differences in acaricide interactions and ultimately providing insights into the acaricide-resistance phenomenon in R. microplus.

Title: Relations entre la résistance aux acaricides et les polymorphismes du gène de l'acétylcholinestérase chez la tique du bétail Rhipicephalus microplus. Abstract: Notre étude vise à développer une méthodologie complète pour identifier les polymorphismes d'acides aminés dans le transcrit 2 de l'acétylcholinestérase (AChE2) chez les tiques Rhipicephalus microplus résistantes aux acaricides. Cela comprend l'évaluation des niveaux d'expression d'AChE2 via qPCR et la réalisation d'une modélisation 3D pour évaluer l'interaction entre les acaricides et l'AChE2 à l'aide de techniques d'amarrage moléculaire. L'étude a produit des résultats significatifs, démontrant que les tiques R. microplus résistantes aux acaricides présentent des niveaux d'expression d'AChE significativement plus élevés que les tiques sensibles de référence. En termes de séquence d'acides aminés, nous avons identifié 9 substitutions d'acides aminés dans AChE2 provenant de tiques résistantes aux acaricides par rapport à la séquence génétique de la souche sensible de référence. Pour mieux comprendre les implications de ces substitutions, nous avons utilisé la modélisation de l'amarrage acaricide-AChE2 pour examiner l'interaction entre l'acaricide et le site catalytique AChE2. Nos modèles suggèrent que ces polymorphismes d'acides aminés modifient la configuration de la poche de liaison, contribuant ainsi aux différences dans les interactions acaricides et fournissant finalement un aperçu du phénomène de résistance aux acaricides chez R. microplus.

Single Amino Acid Polymorphisms in the Fasciola hepatica Carboxylesterase Type B Gene and Their Potential Role in Anthelmintic Resistance.

The expression of the Fasciola hepatica carboxylesterase type B (CestB) gene is known to be induced upon exposure to the anthelmintic triclabendazole (TCBZ), leading to a substantial rise in enzyme-specific activity. Furthermore, the nucleotide sequence of the CestB gene displays variations that can potentially result in radical amino acid substitutions at the ligand binding site. These substitutions hold the potential to impact both the ligand-protein interaction and the catalytic properties of the enzyme. Thus, the objective of our study was to identify novel CestB polymorphisms in TCBZ-resistant parasites and field isolates obtained from a highly endemic region in Central Mexico. Additionally, we aimed to assess these amino acid polymorphisms using 3D modeling against the metabolically oxidized form of the anthelmintic TCBZSOX. Our goal was to observe the formation of TCBZSOX-specific binding pockets that might provide insights into the role of CestB in the mechanism of anthelmintic resistance. We identified polymorphisms in TCBZ-resistant parasites that exhibited three radical amino acid substitutions at positions 147, 215, and 263. These substitutions resulted in the formation of a TCBZSOX-affinity pocket with the potential to bind the anthelmintic drug. Furthermore, our 3D modeling analysis revealed that these amino acid substitutions also influenced the configuration of the CestB catalytic site, leading to alterations in the enzyme's interaction with chromogenic carboxylic ester substrates and potentially affecting its catalytic properties. However, it is important to note that the TCBZSOX-binding pocket, while significant for drug binding, was located separate from the enzyme's catalytic site, rendering enzymatic hydrolysis of TCBZSOX impossible. Nonetheless, the observed increased affinity for the anthelmintic may provide an explanation for a drug sequestration type of anthelmintic resistance. These findings lay the groundwork for the future development of a molecular diagnostic tool to identify anthelmintic resistance in F. hepatica.

An alternative vaccine target for bovine Anaplasmosis based on enolase, a moonlighting protein.

The discovery of new targets for preventing bovine anaplasmosis has moved away from focusing on proteins that have already been extensively studied in Anaplasma marginale, including the Major Surface Proteins, Outer Membrane Proteins, and Type IV Secretion System proteins. An alternative is moonlighting or multifunctional proteins, capable of performing various biological functions within various cellular compartments. There are several reports on the role of moonlighting proteins as virulence factors in various microorganisms. Moreover, it is known that about 25% of all moonlighting is involved in the virulence of pathogens. In this work, for the first time, we present the identification of three enolase proteins (AmEno01, AmEno15, and AmEno31) in the genome of Mexican strains of A. marginale. Using bioinformatics tools, we predicted the catalytic domains, enolase signature, and amino acids binding magnesium ion of the catalytic domain and performed a phylogenetic reconstruction. In addition, by molecular docking analysis, we found that AmEno01 would bind to erythrocyte proteins spectrin, ankyrin, and stomatin. This adhesion function has been reported for enolases from other pathogens. It is considered a promising target since blocking this function would impede the fundamental adhesion process that facilitates the infection of erythrocytes. Additionally, molecular docking predicts that AmEno01 could bind to extracellular matrix protein fibronectin, which would be significant if we consider that some proteins with fibronectin domains are localized in tick gut cells and used as an adhesion strategy to gather bacteria before traveling to salivary glands. Derived from the molecular docking analysis of AmEno01, we hypothesized that enolases could be proteins driven by the pathogen and redirected at the expense of the pathogen's needs.

A Novel Rhipicephalus microplus Estrogen Related Receptor (RmERR), a Molecular and In Silico Characterization of a Potential Protein Binding Estrogen.

The search for targets to control ticks and tick-borne diseases has been an ongoing problem, and so far, we still need efficient, non-chemical alternatives for this purpose. This search must consider new alternatives. For example genomics analysis is a widely applied tool in veterinary health studies to control pathogens. On the other hand, we propose that regulation of endocrine mechanisms represents a feasible alternative to biologically controlling tick infestations. Thus, we performed the molecular identification of an estrogen-related receptor gene of Rhipicephalus microplus called RmERR by RT-PCR in tick ovaries, embryonic cells, and hemolymph, which allowed us to analyze its expression and propose potential functions in endocrine mechanisms and developmental stages. In addition, we performed an in silico characterization to explore the molecular interactions of RmERR with different estrogens, estrogenic antagonists, and endocrine disruptor Bisphenol A (BPA), finding potential interactions predicted by docking analysis and supported by negative values of ΔG (which suggests the potential interaction of RmERR with the molecules evaluated). Additionally, phylogenetic reconstruction revealed that RmERR is grouped with other tick species but is phylogenetically distant from host vertebrates' ERRs. In summary, this study allowed for the identification of an ERR in cattle tick R. microplus for the first time and suggested its interaction with different estrogens, supporting the idea of a probable transregulation process in ticks. The elucidation of this interaction and its mechanisms unveiled its potential as a target to develop tick control strategies.

An Update of Bovine Hemoplasmas Based on Phylogenetic and Genomics Analysis.

Mycoplasma wenyonii and 'Candidatus Mycoplasma haemobos' are bacteria that have been described as significant hemoplasmas that infect cattle worldwide. Currently, three bovine hemoplasma genomes are known. This work aimed to describe the main genomic characteristics and the evolutionary relationships between hemoplasmas, and provide a list of epitopes predicted by immunoinformatics as diagnostic candidates for bovine hemoplasmosis. Thus far, there is no vaccine to prevent this disease that economically impacts cattle production worldwide. Additionally, there is a lack of vaccines against bovine hemoplasmosis. In this work, we performed a genomic characterization of hemoplasmas, including two Mexican strains reported in bovines in the last few years. The generated information is a new scenario about the phylogeny of hemoplasmas. Also, we show genomic features among hemoplasmas that strengthen their characteristic genome plasticity of intracellular lifestyles. Finally, the elucidation of antigenic proteins in Mexican strains represents an opportunity to develop molecular detection methods and diagnoses.

A Single Nucleotide Polymorphism Translates into a Radical Amino Acid Substitution at the Ligand-Binding Site in Fasciola hepatica Carboxylesterase B.

Fasciola hepatica anthelmintic resistance may be associated with the catalytic activity of xenobiotic metabolizing enzymes. The gene expression of one of these enzymes, identified as carboxylesterase B (CestB), was previously described as inducible in adult parasites under anthelmintic treatment and exhibited a single nucleotide polymorphism at position 643 that translates into a radical amino acid substitution at position 215 from Glutamic acid to Lysine. Alphafold 3D models of both allelic sequences exhibited a significant affinity pocket rearrangement and different ligand-docking modeling results. Further bioinformatics analysis confirmed that the radical amino acid substitution is located at the ligand affinity site of the enzyme, affecting its affinity to serine hydrolase inhibitors and preferences for ester ligands. A field genotyping survey from parasite samples obtained from two developmental stages isolated from different host species from Argentina and Mexico exhibited a 37% allele distribution for 215E and a 29% allele distribution for 215K as well as a 34% E/K heterozygous distribution. No linkage to host species or geographic origin was found in any of the allele variants.

Mexican Strains of Anaplasma marginale: A First Comparative Genomics and Phylogeographic Analysis.

The One Health approach looks after animal welfare and demands constant monitoring of the strains that circulate globally to prevent outbreaks. Anaplasma marginale is the etiologic agent of bovine anaplasmosis and is endemic worldwide. This study aimed to analyze, for the first time, the genetic diversity of seven Mexican strains of A. marginale and their relationship with other strains reported. The main features of A. marginale were obtained by characterizing all 24 genomes reported so far. Genetic diversity and phylogeography were analyzed by characterizing the msp1a gene and 5'-UTR microsatellite sequences and constructing a phylogenetic tree with 540 concatenated genes of the core genome. The Mexican strains show 15 different repeat sequences in six MSP1a structures and have phylogeographic relationships with strains from North America, South America, and Asia, which confirms they are highly variable. Based on our results, we encourage the performance of genome sequencing of A. marginale strains to obtain a high assembly level of molecular markers and the performance of extensive phylogeographic analysis. Undoubtedly, genomic surveillance helps build a picture of how a pathogen changes and evolves in geographical regions. However, we cannot discard the study of relationships pathogens establish with ticks and how they have co-evolved to establish themselves as a successful transmission system.

Sexual Dimorphism of the Neuroimmunoendocrine Response in the Spleen during a Helminth Infection: A New Role for an Old Player?

The interaction of the nervous, immune, and endocrine systems is crucial in maintaining homeostasis in vertebrates, and vital in mammals. The spleen is a key organ that regulates the neuroimmunoendocrine system. The Taenia crassiceps mouse system is an excellent experimental model to study the complex host-parasite relationship, particularly sex-associated susceptibility to infection. The present study aimed to determine the changes in neurotransmitters, cytokines, sex steroids, and sex-steroid receptors in the spleen of cysticercus-infected male and female mice and whole parasite counts. We found that parasite load was higher in females in comparison to male mice. The levels of the neurotransmitter epinephrine were significantly decreased in infected male animals. The expression of IL-2 and IL-4 in the spleen was markedly increased in infected mice; however, the expression of Interleukin (IL)-10 and interferon (IFN)-γ decreased. We also observed sex-associated differences between non-infected and infected mice. Interestingly, the data show that estradiol levels increased in infected males but decreased in females. Our studies provide evidence that infection leads to changes in neuroimmunoendocrine molecules in the spleen, and these changes are dimorphic and impact the establishment, growth, and reproduction of T. crassiceps. Our findings support the critical role of the neuroimmunoendocrine network in determining sex-associated susceptibility to the helminth parasite.

A newly optimized protocol to extract high-quality hemolymph from the cattle tick Rhipicephalus microplus: Improving the old conditions.

Ticks are hematophagous ectoparasites with importance to animal and human health. In recent years, the study of ticks has had significant development, including immune response, vector-host interactions, physiological and multi-omics approaches. However, one of the main impediments is obtaining a significant amount of high-quality hemolymph. For this reason, we developed a protocol that allows obtaining up to 100 µl of hemolymph free of host blood per engorged tick. The technique consists of continuous hipocuticular punctures of the tick dorsum and an anticoagulant buffer that impedes hemolymph coagulation, allowing constant extravasation and ensuring high yields. Additionally, the hemocytes recovered with this protocol are intact and can be used for further analysis. The high-quality hemolymph obtained using this protocol and its applications will help to better understand the processes involving the hemolymph and its components. Although there are other hemolymph extraction protocols, the method developed here is very well suited for Rhipicephalus microplus, and in our experience, results in better yields and high-quality samples.

Transcriptome-Based Identification of a Functional Fasciola hepatica Carboxylesterase B.

Bioinformatics analysis of the complete transcriptome of Fasciola hepatica, identified a total of ten putative carboxylesterase transcripts, including a 3146 bp mRNA transcript coding a 2205 bp open reading frame that translates into a protein of 735 amino acids, resulting in a predicted protein mass of 83.5 kDa and a putative carboxylesterase B enzyme. The gene coding for this enzyme was found in two reported F. hepatica complete genomes stretching 23,230 bp, containing two exons of 1282 and 1864 bp, respectively, as well as a 20,084 bp intron between the exons. The enzymatic activity was experimentally assayed on F. hepatica protein extracts by SDS-PAGE zymograms using synthetic chromogenic substrates, confirming both the theoretical molecular weight and carboxylesterase enzymatic activity. Further bioinformatics predicted that this enzyme is an integral component of the cellular membrane that should be active as a 167 kDa homodimer complex and polyacrylamide gel electrophoresis (PAGE) zymograms experiments confirmed the analysis. Additional bioinformatics analysis showed that DNA sequences that code for this particular enzyme are highly conserved in other parasitic trematodes, although they are labeled hypothetical proteins.

Tick Immunobiology and Extracellular Traps: An Integrative Vision to Control of Vectors.

Ticks are hematophagous ectoparasites that infest a diverse number of vertebrate hosts. The tick immunobiology plays a significant role in establishing and transmitting many pathogens to their hosts. To control tick infestations, the acaricide application is a commonly used method with severe environmental consequences and the selection of tick-resistant populations. With these drawbacks, new tick control methods need to be developed, and the immune system of ticks contains a plethora of potential candidates for vaccine design. Additionally, tick immunity is based on an orchestrated action of humoral and cellular immune responses. Therefore, the actors of these responses are the object of our study in this review since they are new targets in anti-tick vaccine design. We present their role in the immune response that positions them as feasible targets that can be blocked, inhibited, interfered with, and overexpressed, and then elucidate a new method to control tick infestations through the development of vaccines. We also propose Extracellular Traps Formation (ETosis) in ticks as a process to eliminate their natural enemies and those pathogens they transmit (vectorial capacity), which results attractive since they are a source of acting molecules with potential use as vaccines.

Advances in the Study of the Tick Cattle Microbiota and the Influence on Vectorial Capacity.

The information from the tick cattle microbiota suggests that the microbial populations may modulate a successful infection process of the tick-borne pathogens. Therefore, there is a need to know the microbial population and their interactions. In this mini-review, we present several examples of how microbiota regulates the survival of pathogens inside the tick and contributes to fitness, adaptation, and tick immunity, among others. The communication between the tick microbiota and the host microbiota is vital to understanding the pathogen transmission process. As part of the tick microbiota, the pathogen interacts with different microbial populations, including the microorganisms of the host microbiota. These interactions comprise a microsystem that regulates the vectorial capacity involved in tick-borne diseases. The knowledge we have about the vectorial capacity contributes to a better understanding of tick-borne pathogens. Additionally, using approaches based on multi-omics strategies applied to studying the microbiota and its microbiome allows the development of strategies to control ticks. The results derived from those studies reveal the dynamics of the microbiota and potential targets for anti-tick vaccine development. In this context, the anti-microbiota vaccines have emerged as an alternative with a good prognosis. Some strategies developed to control other arthropods vectors, such as paratransgenesis, could control ticks and tick-borne diseases.

Transcriptome assembly dataset of anthelmintic response in Fasciola hepatica.

Fasciola hepatica is a worldwide distributed zoonotic parasitic trematode, which causes a severe liver disease clinically known as fasciolasis in a large number of wild animals, several livestock species as well as humans, prevention and control of fasciolasis is made by massive use of anthelmintic compounds on livestock and inevitably this practice has led to the emergence of anthelmintic resistant Fasciola hepatica and there is a great scientific effort to elucidate the molecular basis of anthelmintic resistance of parasitic helminths in general and of Fasciola hepatica in particular that may lead to improved anthelmintic compounds. In our project, we sequenced the transcriptomes obtained from the anthelmintic response to Triclabendazole and Albendazole on four samples from sensitive and resistant strains of Fasciola hepatica on Illumina HiSeq 4000 Platform and generated about 10.03 Gb per sample. The average genome-mapping rate is 81.29% and the average gene-mapping rate is 62.81%. 30,105 genes were identified in which 28,669 of them are known genes and 1,237 of them are novel genes from novel coding transcripts without any known features, 20,743 novel RNA transcripts were identified of which 14,293 of them are previously unknown splicing event for known genes but no alternative splicing was detected, the remaining 5,213 transcripts were found to be long noncoding RNA.

Immunoinformatic Analysis to Identify Proteins to Be Used as Potential Targets to Control Bovine Anaplasmosis.

Omics sciences and new technologies to sequence full genomes provide valuable data that are revealed only after detailed bioinformatic analysis is performed. In this work, we analyzed the genomes of seven Mexican Anaplasma marginale strains and the data from a transcriptome analysis of the tick Rhipicephalus microplus. The aim of this analysis was to identify protein sequences with predicted features to be used as potential targets to control the bacteria or tick-vector transmission. We chose three amino acid sequences different to all proteins previously reported in A. marginale that have been used as potential vaccine candidates, and also, we report, for the first time, the presence of a peroxinectin protein sequence in the transcriptome of R. microplus, a protein associated with the immune response of ticks. The bioinformatics analyses revealed the presence of B-cell epitopes in all the amino acid sequences chosen, which opens the way for their likely use as single or arranged peptides to develop new strategies for the control and prevention of bovine anaplasmosis transmitted by ticks.

Pyrethroid Acaricide Resistance Is Proportional to P-450 Cytochrome Oxidase Expression in the Cattle Tick Rhipicephalus microplus.

The goal of the present study was to assess the gene expression of xenobiotic metabolizing enzymes (XMEs) Cytochrome P-450 (CYP) and carboxylesterase (CE) related to detoxification of synthetic pyrethroids, plus acetylcholinesterase (AChE), in field isolates of acaricide-resistant Rhipicephalus microplus. The XMEs expression levels were assessed by mRNA measurement using quantitative reverse transcription PCR. The XME expression levels of field-isolated acaricide-resistant ticks were compared against acaricide-susceptible reference ticks used in this study as a gene expression baseline and represented as relative expression units (REU). Field isolates were subjected to toxicological bioassays and determined resistant to all the Pyr acaricides (Pyr), whereas most of them were found susceptible to organophosphorous acaricides (OP), with the exception of three isolates, which exhibited moderate resistance to Diazinon. Significantly higher levels of CYP were detected in pyrethroid-resistance ticks when compared to Su ticks (P<0.01). A linear regression analysis showed that pyrethroid acaricide resistance levels of R. microplus were proportional to the CYP expression levels (correlation coefficient (R):0.85; P<0.05). Analysis on CE expression levels showed only one isolate resistant to Pyr and OP with a statistically significant increase (P<0.01). AChE expression levels showed statistically significant (P<0.01) subexpression in all tick isolates when compared to the susceptible reference. Our results suggest that pyrethroid acaricide resistance in the cattle tick may be diagnosed by measuring the CYP expression levels using quantitative PCR.

Prolactin as immune cell regulator in Toxocara canis somatic larvae chronic infection.

Toxocariasis is a zoonotic disease produced by ingestion of larval Toxocara spp. eggs. Prolactin (PRL) has been considered to have an important role in Toxocara canis infection. Recent evidence has found that PRL directly can increase parasite growth and differentiation of T. canis The present study, evaluated the effect of high PRL levels on the immune system's response and parasites clearance in chronic infection. Our results showed that hyperprolactinemia did not affect the number of larvae recovered from several tissues in rats. Parasite-specific antibody production, showed no difference between the groups. Lung tissue presented eosinophilic granulomas typical of a chronic infection in all the experimental groups. Flow cytometry analysis was made in order to determine changes in the percentage of innate and adaptive immune cell subpopulations in the spleen, peripheric (PLN) and mesenteric (MLN) lymphatic nodes. The results showed a differential effect of PRL and infection on different immune compartments in the percent of total T cells, T helper cells, T cytotoxic cells, B cells, NK cells, and Tγδ cells. To our knowledge, for the first time it is demonstrated that PRL can have an immunomodulatory role during T. canis chronic infection in the murine host.

A novel progesterone receptor membrane component (PGRMC) in the human and swine parasite Taenia solium: implications to the host-parasite relationship.

BACKGROUND: We have previously reported that progesterone (P4) has a direct in vitro effect on the scolex evagination and growth of Taenia solium cysticerci. Here, we explored the hypothesis that the P4 direct effect on T. solium might be mediated by a novel steroid-binding parasite protein. METHODS: By way of using immunofluorescent confocal microscopy, flow cytometry analysis, double-dimension electrophoresis analysis, and sequencing the corresponding protein spot, we detected a novel PGRMC in T. solium. Molecular modeling studies accompanied by computer docking using the sequenced protein, together with phylogenetic analysis and sequence alignment clearly demonstrated that T. solium PGRMC is from parasite origin. RESULTS: Our results show that P4 in vitro increases parasite evagination and scolex size. Using immunofluorescent confocal microscopy, we detected that parasite cells showed expression of a P4-binding like protein exclusively located at the cysticercus subtegumental tissue. Presence of the P4-binding protein in cyst cells was also confirmed by flow cytometry. Double-dimension electrophoresis analysis, followed by sequencing the corresponding protein spot, revealed a protein that was previously reported in the T. solium genome belonging to a membrane-associated progesterone receptor component (PGRMC). Molecular modeling studies accompanied by computer docking using the sequenced protein showed that PGRMC is potentially able to bind steroid hormones such as progesterone, estradiol, testosterone and dihydrodrotestosterone with different affinities. Phylogenetic analysis and sequence alignment clearly demonstrated that T. solium PGRMC is related to a steroid-binding protein of Echinoccocus granulosus, both of them being nested within a cluster including similar proteins present in platyhelminths such as Schistocephalus solidus and Schistosoma haematobium. CONCLUSION: Progesterone may directly act upon T. solium cysticerci probably by binding to PGRMC. This research has implications in the field of host-parasite co-evolution as well as the sex-associated susceptibility to this infection. In a more practical matter, present results may contribute to the molecular design of new drugs with anti-parasite actions.

Parasiticidal effect of synthetic bovine lactoferrin peptides on the enteric parasite Giardia intestinalis.

Giardia intestinalis is the most common infectious protozoan parasite in children. Despite the effectiveness of some drugs, the disease remains a major worldwide problem. Consequently, the search for new treatments is important for disease eradication. Biological molecules with antimicrobial properties represent a promising alternative to combat pathogens. Bovine lactoferrin (bLF) is a key component of the innate host defense system, and its peptides have exhibited strong antimicrobial activity. Based on these properties, we evaluated the parasiticidal activity of these peptides on G. intestinalis. Trophozoites were incubated with different peptide concentrations for different periods of time, and the growth or viability was determined by carboxyfluorescein-succinimidyl-diacetate-ester (CFDA) and propidium iodide (PI) staining. Endocytosis of peptides was investigated by confocal microscopy, damage was analyzed by transmission and scanning electron microscopy, and the type of programmed cell death was analyzed by flow cytometry. Our results showed that the LF peptides had giardicidal activity. The LF peptides interacted with G. intestinalis and exposure to LF peptides correlated with an increase in the granularity and vacuolization of the cytoplasm. Additionally, the formation of pores, extensive membrane disruption, and programmed cell death was observed in trophozoites treated with LF peptides. Our results demonstrate that LF peptides exhibit potent in vitro antigiardial activity.

Protection against Amoebic Liver Abscess in Hamster by Intramuscular Immunization with an Autographa californica Baculovirus Driving the Expression of the Gal-Lectin LC3 Fragment.

In a previous study, we demonstrated that oral immunization using Autographa californica baculovirus driving the expression of the Gal-lectin LC3 fragment (AcNPV-LC3) of Entamoeba histolytica conferred protection against ALA development in hamsters. In this study, we determined the ability of AcNPV-LC3 to protect against ALA by the intramuscular route as well as the liver immune response associated with protection. Results showed that 55% of hamsters IM immunized with AcNPV-LC3 showed sterile protection against ALA, whereas other 20% showed reduction in the size and extent of abscesses, resulting in some protection in 75% of animals compared to the sham control group. Levels of protection showed a linear correlation with the development and intensity of specific antiamoeba cellular and humoral responses, evaluated in serum and spleen of hamsters, respectively. Evaluation of the Th1/Th2 cytokine patterns expressed in the liver of hamsters showed that sterile protection was associated with the production of high levels of IFNγ and IL-4. These results suggest that the baculovirus system is equally efficient by the intramuscular as well as the oral routes for ALA protection and that the Gal-lectin LC3 fragment is a highly protective antigen against hepatic amoebiasis through the local induction of IFNγ and IL-4.