Instituto de Investigación en Recursos Cinegéticos

Is Zebrafish a Good Model for the Alpha-Gal Syndrome?

The alpha-Gal syndrome (AGS) is an underdiagnosed tick-borne allergy characterized by both immediate and delayed IgE-mediated anaphylactic reactions to the galactose-alpha-1,3-galactose (alpha-Gal) epitope. Common manifestations include gastrointestinal, cutaneous, and respiratory symptoms appearing 2-6 h after the consumption of mammalian meat or derived products. Zebrafish (Danio rerio) are emerging as essential animal models in biomedical studies, due to their anatomical, genetic, and physiological similarities to humans, with significant applications in toxicology, behavioral research, oncology, and inflammation studies. The mechanisms associated with AGS are sustained by studies in the humanized α1,3GalT-KO C57BL/6 mouse (Mus musculus) and zebrafish animal models for the production of anti-alpha-Gal antibodies in response to tick saliva, the development of allergic reactions in animals sensitized with tick protein extracts following mammalian meat consumption, and the identification of immune mechanisms. The immune mechanisms characterized in both models are associated with a skewed type 2 immune response, triggering Toll-Like receptor (TLR) signaling pathways, IL-4 production, and humoral activity. These results support the use of both models rather than a single one for a more comprehensive characterization of AGS-associated immune mechanisms. In this study, we focused on the use of zebrafish as a model for biomedicine research in immunity, infectious, and allergic diseases, with a particular emphasis on the AGS and the identification of candidate therapeutic interventions. Based on insights from multiple studies, we concluded that zebrafish is a suitable model for studying the AGS, considering the addressed limitations and in combination with the α1,3GalT-KO mouse model.

Tick salivary proteins metalloprotease and allergen-like p23 are associated with response to glycan α-Gal and mycobacterium infection.

The alpha-Gal syndrome (AGS) evolved as a catastrophic selection associated with anti-α-Gal IgM/IgG protective response against pathogen infection and tick-borne food allergy caused by IgE-type antibodies against this glycan present in glycoproteins and glycolipids from mammalian meat and derived products. The immune response to α-Gal is modulated by tick salivary proteins with and without α-Gal modifications in combination with tick saliva non-protein fraction. Herein, we characterized the role of tick salivary proteins, metalloprotease and allergen-like p23 in AGS and protection against tuberculosis in the AGS zebrafish animal model. Metalloprotease and p23 are involved in allergic reactions after mammalian meat consumption through upregulation of pro-inflammatory protein-coding genes prkdc, tlr2, tnfα and il1b. Challenge with Mycobacterium marinum activated Th1-mediated immune protective response with reduced pathogen infection, ameliorating Th2-associated allergic reactions associated with AGS. These results highlight molecular mechanisms modulated by tick proteins in response to α-Gal and provide insights to reduce AGS impact on human health.

Publisher Correction: Tick salivary proteins metalloprotease and allergen-like p23 are associated with response to glycan α-Gal and mycobacterium infection.

Allergic reactions associated with tsetse fly bites.

Allergic reactions in response to arthropod bites are a growing concern worldwide. Alpha-Gal syndrome (AGS) is a red meat allergy associated with IgE antibodies against galactose-alpha-1,3-galactose (alpha-Gal), typically linked to tick bites but other arthropods may also play a role. The objective of this study was to investigate the potential association between tsetse fly ( spp.) bites and AGS by analyzing clinical cases, citizen science data, and the possible production of alpha-Gal by tsetse flies. Data from five case series involving tsetse fly bites included demographic information such as age, gender and geographical location, as well as clinical details like symptoms and diagnosis. The production of alpha-Gal by tsetse flies focused on epitope content analysis by ELISA and the expression of several galactosyltransferases using RT-qPCR in the whole body of Ugandan tsetse flies and midgut tissues. The cases included patients bitten by tsetse flies in Uganda-Rwanda and, with different symptoms. Individuals resided in Spain, the USA or Uganda, with no record of or reaction to bites from ticks or other arthropods. Cases 1 and 2 were diagnosed with AGS, whereas Ugandan Case 3 presented symptoms consistent with AGS. Ugandan participative or citizen science survey identified one individual (Case 4) who experienced allergic reactions to red meat following multiple tsetse fly bites, while eight individuals (summarized as grouped Case 5) exhibited only local skin reactions to several bites. The expression of galactosyltransferases suggested the possible production of alpha-Gal modification in these arthropods. Allergic anaphylactic reactions to tsetse fly bites may occur in some individuals, potentially linking tsetse fly bites to AGS. Diagnosis and monitoring should consider factors beyond anti-alpha-Gal IgE antibody levels, such as arthropod salivary compounds and human immune biomarkers.

Public perspectives on tick bite exposure, healthcare visits and associated allergies in iberia.

Tick bites and tick-borne diseases (TBDs) are a worldwide concern, with growing evidence of an ongoing spread and emergence of new cases. This study applied a participatory citizen science across Spain and Portugal to gather public perspectives on the impact of tick bites, the presence of TBDs and a potential connection with the α-Gal syndrome (AGS). Data collected from the questionnaire (380 participants) was geographically represented using QGIS Geographic Information System and fitted into multiple generalized linear models (GLMs). Statistical analysis identified factors influencing the occurrence of local skin reactions post-tick bite, AGS-compatible symptoms and visits to health centers following tick exposure. Results showed that the probability of developing localized skin reactions to tick bites rises with increasing age ( = 0.006,  < 0.05), the occurrence of multiple bites ( = 0.006,  = 0.063) and among individuals bitten in the center-north part of the peninsula ( = 0.006,  = 0.058). Seeking medical care following tick bite was reported by 21.6% of respondents, being more common in first-time bite cases ( = 0.002, p < 0.01), men ( = 0.002,  < 0.01) or individuals presenting localized skin symptoms ( = 0.002,  < 0.05). Although 38 inquiries (10.0%) showed signs of AGS, only 3 had received a formal diagnosis, with the odds of developing this disease marginally rising with advancing age ( = 0.215,  = 0.066). This study provides valuable insights that should be considered to improve TBDs surveillance and diagnostic strategies, as well as developing preventive measures to reduce tick bite exposure and TBD cases.Developing local skin reactions to tick bites increases with age, multiple bites and in individuals bitten in central-northern Iberia.Seeking medical care following a tick bite is more common in first-time bite cases, men or individuals with local skin symptoms.Although 38 inquiries (10.0%) showed signs of AGS, only 3 were formally diagnosed, with risk increasing with age.

Transcriptomics insights into the functional role of tick Ixodes ricinus proteins metalloprotease and antigen p23.

Ixodes ricinus ticks (Acari: Ixodidae) are hematophagous ectoparasites and a major European vector of zoonotic diseases affecting global health. Tick salivary and midgut proteins antigen p23 (A0A0K8RKR7) and metalloprotease (A0A0K8RCY8) were previously implicated in the pathophysiology of the tick-borne allergy alpha-Gal syndrome (AGS). This study aimed to functionally characterize these two biomolecules, focusing on their role in I. ricinus tick feeding and reproduction through gene knockdown by RNA interference and midgut transcriptomic analysis. Validation of RNA-seq data was conducted using RT-qPCR analysis on tick midgut and salivary gland tissues. Silencing the expression of p23 and metalloprotease did not result in any significant differences in tick engorgement and egg batch weights compared to the control group. Gene set enrichment analysis following antigen p23 gene knockdown identified significantly upregulated pathways associated with protein production and suppressed routes mostly correlated with ion transport, lipid metabolism, catalytic activity, protein modification, and G-protein activity. Partial knockdown of the metalloprotease led to the upregulation of several biological and functional pathways associated with RNA splicing and significantly suppressed routes connected with detoxification, protein modification, catalytic activity and molecule binding. Antigen p23 appears to play a functional role in tick midgut cell homeostasis, primarily by participating in regulatory and signaling processes essential for cell viability. Metalloprotease is potentially involved in regulating midgut response to oxidative stress, thereby reducing tissue damage and promoting regular cell proliferation, growth and behavior. These results provide insights into tick physiology and bases for further research on tick-host interactions and AGS pathogenesis.

Tick salivary proteome and lipidome with low glycan content correlate with allergic type reactions in the zebrafish model.

Ticks, as hematophagous ectoparasites, can manipulate host immune and metabolic processes, causing tick-borne allergies such as α-Gal syndrome (AGS). Glycolipids with bound galactose-alpha-1-3-galactose (α-Gal) are potential allergenic molecules associated with AGS. Nevertheless, proteins and lipids lacking α-Gal modifications may contribute to tick salivary allergies and be linked to AGS. In this study, we characterized the effect of deglycosylated tick salivary proteins without lipids on treated zebrafish fed with dog food formulated with mammalian (beef, lamb, pork) meat by quantitative proteomics analysis of intestinal samples. The characterization and functional annotations of tick salivary lipids with low representation of glycolipids was conducted using a lipidomics approach. Results showed a significant effect of treatment with saliva and saliva deglycosylated protein fraction on zebrafish abnormal or no feeding (p < 0.005). Treatment with this fraction affected multiple metabolic pathways, defense responses to pathogens and protein metabolism, which correlated with abnormal or no feeding. Lipidomics analysis identified 23 lipid classes with low representation of glycolipids (0.70% of identified lipids). The lipid class with highest representation was phosphatidylcholine (PC; 26.66%) and for glycolipids it corresponded to diacylglycerol (DG; 0.48%). Qualitative analysis of PC antibodies revealed that individuals bitten by ticks were more likely to produce PC-IgG antibodies (p < 0.001). DG levels were significantly higher in tick salivary glands (p < 0.05) compared with tick saliva and salivary fractions. The α-Gal content was higher in tick saliva than in deglycosylated saliva and lipid fractions. These results support a possible role for tick salivary proteins and lipids without α-Gal modifications in AGS.

The alpha-Gal Syndrome is Underdiagnosed.

Zebrafish gut microbiota composition in response to tick saliva biomolecules correlates with allergic reactions to mammalian meat consumption.

The α-Gal syndrome (AGS) is an IgE-mediated tick borne-allergy that results in delayed anaphylaxis to the consumption of mammalian meat and products containing α-Gal. Considering that α-Gal-containing microbiota modulates natural antibody production to this glycan, this study aimed to evaluate the influence on tick salivary compounds on the gut microbiota composition in the zebrafish (Danio rerio) animal model. Sequencing of 16 S rDNA was performed in a total of 75 zebrafish intestine samples, representing different treatment groups: PBS control, Ixodes ricinus tick saliva, tick saliva non-protein fraction (NPF), tick saliva protein fraction (PF), and tick saliva protein fractions 1-5 with NPF (F1-5). The results revealed that treatment with tick saliva and different tick salivary fractions, combined with α-Gal-positive dog food feeding, resulted in specific variations in zebrafish gut microbiota composition at various taxonomic levels and affected commensal microbial alpha and beta diversities. Metagenomics results were corroborated by qPCR, supporting the overrepresentation of phylum Firmicutes in the tick saliva group, phylum Fusobacteriota in group F1, and phylum Cyanobacteria in F2 and F5 compared to the PBS-control. qPCRs results at genus level sustained significant enrichment of Plesiomonas spp. in groups F3 and F5, Rhizobium spp. in NPF and F4, and Cloacibacterium spp. dominance in the PBS control group. This study provides new results on the role of gut microbiota in allergic reactions to tick saliva components using a zebrafish model of AGS. Overall, gut microbiota composition in response to tick saliva biomolecules may be associated with allergic reactions to mammalian meat consumption in AGS.

Netosis and trained immunity in tick-borne diseases: a possible pathogenetic role.

Various types of pathogens transmitted by ticks elicit distinct immune responses just like the emerging α-Gal syndrome that is associated with allergic reactions to tick bites. The mechanisms of Neutrophil Extracellular Traps release (called NETosis) and trained immunity in response to tick-borne microbes have not been extensively investigated. In our paper, we explored the intricate interplay of NETosis and trained immunity within the realm of infectious diseases triggered by tick bites and their possible pathogenetic role in autoimmunity. We conducted an extensive literature search to identify studies for this review, considering articles and reviews published in English within the last years. Additionally, we scrutinized the references of all included papers and relevant review articles to ensure comprehensive coverage. We shed light on a plausible correlation between these innate immune responses and their potential implication in certain pathological conditions, with a specific focus on some autoimmune diseases. These findings offer new perspectives for a more profound comprehension of the immunopathogenesis of certain autoimmune-like signs where clinicians should include Tick-Borne Diseases (TBDs) in their differential diagnoses, in those geographical areas of tick infestation.

Citizen Science on Lyme Borreliosis in Spain Reveals Disease-Associated Risk Factors and Control Interventions.

Lyme borreliosis (LB) caused by lato complex spirochetes is one of the tick-borne diseases with high prevalence and social/economic burden in the United States, Spain, and other European countries. The objective is to address limited information available about the incidence, prevalence, and symptoms of LB, current prevention, and treatment interventions that are not adequately focused and thus not very effective against this disease. To address these limitations, in this study, we used a citizen science approach to evaluate the LB-associated risks and implementation of control interventions in Spain. A total of 405 participants in the survey were included in the analysis. Responses to the questionnaire were received during January-July 2022. The questionnaire contained qualitative and quantitative questions. Homogeneity among binary variables was analyzed using a Fisher's exact test. Despite limitations of the study associated with response to the questionnaire and information on tick species, the results evidenced the effect of factors such as age, gender, tick bites, disease clinical signs, comorbidities such as alpha-gal syndrome, health care services, and treatment effectiveness affecting LB. The main conclusions of the study highlight the need for better surveillance of tick infestations, pathogen infection, and diagnosis of LB and related comorbidities. To advance in disease prevention, diagnosis, and treatment, new interventions need to be developed and implemented in both public and private health care services. Providing access to these results to the society, health care system, and scientists is important to further advance in disease surveillance, diagnosis, control, and prevention.

Allergic reactions to tick saliva components in zebrafish model.

Alpha-Gal syndrome (AGS) is a tick-borne food allergy caused by IgE antibodies against the glycan galactose-alpha-1,3-galactose (α-Gal) present in glycoproteins and glycolipids from mammalian meat. To advance in the diagnosis and treatment of AGS, further research is needed to unravel the molecular and immune mechanisms underlying this syndrome. The objective of this study is the characterization of tick salivary components and proteins with and without α-Gal modifications involved in modulating human immune response against this carbohydrate. Protein and α-Gal content were determined in tick saliva components, and proteins were identified by proteomics analysis of tick saliva fractions. Pathophysiological changes were recorded in the zebrafish (Danio rerio) model after exposure to distinct Ixodes ricinus tick salivary components. Serum samples were collected from zebrafish at day 8 of exposure to determine anti-α-Gal, anti-glycan, and anti-tick saliva protein IgM antibody titers by enzyme-linked immunosorbent assay (ELISA). Zebrafish treated with tick saliva and saliva protein fractions combined with non-protein fractions demonstrated significantly higher incidence of hemorrhagic type allergic reactions, abnormal behavioral patterns, or mortality when compared to the phosphate-buffered saline (PBS)-treated control group. The main tick salivary proteins identified in these fractions with possible functional implication in AGS were the secreted protein B7P208-salivary antigen p23 and metalloproteases. Anti-α-Gal and anti-tick salivary gland IgM antibody titers were significantly higher in distinct saliva protein fractions and deglycosylated saliva group when compared with PBS-treated controls. Anti-glycan antibodies showed group-related profiles. Results support the hypothesis that tick salivary biomolecules with and without α-Gal modifications are involved in modulating immune response against this carbohydrate.

Serum biomarkers for nutritional status as predictors in COVID-19 patients before and after vaccination.

The aim of this study was to characterize serum protein biomarkers for nutritional status that may be used as predictors for disease symptomatology in COVID-19 patients before and after vaccination. In pre-vaccine cohorts, proteomics analysis revealed significant differences between groups, with serum proteins alpha-1-acid glycoproteins (AGPs) 1 and 2, C-reactive protein (CRP) and retinol binding protein (RBP) increasing with COVID-19 severity, in contrast with serum albumin, transthyretin (TTR) and serotransferrin (TF) reduction as the symptomatology increased. Immunoassay reproduced and validated proteomics results of serum proteins albumin and RBP. In post-vaccine cohorts, the results showed the same pattern as in pre-vaccine cohorts for serum proteins AGPs, CRP, albumin and TTR. However, TF levels were similar between groups and RBP presented a slight reduction as COVID-19 symptomatology increased. In these cohorts, immunoassay validated proteomics results of serum proteins albumin, TTR and TF. Additionally, immune response to α-Gal in pre-vaccine cohorts varied in predominant immunoglobulin type profile, while post-vaccine groups presented mainly anti-α-Gal protective IgG antibodies. The study identified serum nutritional biomarkers that could potentially predict an accurate prognostic of COVID-19 disease to provide an appropriate nutritional care and guidance in non-vaccinated and vaccinated individuals against SARS-CoV-2. These results highlight the importance of designing personalized nutrition protocols to improve diet along with the application of prebiotics or probiotics for the control of COVID-19 and other infectious diseases.

Multi-omics analysis of zebrafish response to tick saliva reveals biological processes associated with alpha-Gal syndrome.

The alpha-Gal syndrome (AGS) is a tick-borne allergy. A multi-omics approach was used to determine the effect of tick saliva and mammalian meat consumption on zebrafish gut transcriptome and proteome. Bioinformatics analysis using R software was focused on significant biological and metabolic pathway changes associated with AGS. Ortholog mapping identified highly concordant human ortholog genes for the detection of disease-enriched pathways. Tick saliva treatment increased zebrafish mortality, incidence of hemorrhagic type allergic reactions and changes in behavior and feeding patterns. Transcriptomics analysis showed downregulation of biological and metabolic pathways correlated with anti-alpha-Gal IgE and allergic reactions to tick saliva affecting blood circulation, cardiac and vascular smooth muscle contraction, behavior and sensory perception. Disease enrichment analysis revealed downregulated orthologous genes associated with human disorders affecting nervous, musculoskeletal, and cardiovascular systems. Proteomics analysis revealed suppression of pathways associated with immune system production of reactive oxygen species and cardiac muscle contraction. Underrepresented proteins were mainly linked to nervous and metabolic human disorders. Multi-omics data revealed inhibition of pathways associated with adrenergic signaling in cardiomyocytes, and heart and muscle contraction. Results identify tick saliva-related biological pathways supporting multisystemic organ involvement and linking α-Gal sensitization with other illnesses for the identification of potential disease biomarkers.

Allergic reactions associated with medically relevant arthropods.

Vector-borne diseases are a growing burden worldwide. In particular, the risks of allergic reactions to bites are associated with growing arthropod populations in contact with the public. The diversity of allergic reactions associated with host and arthropod factors difficult disease diagnosis, prognosis and prevention. Therefore, arthropod-associated allergies are underdiagnosed and require better surveillance of arthropod populations and disease diagnosis and management. To face these challenges, in this study, we describe five cases to illustrate arthropod-associated allergies with different symptomatology, including alpha-gal syndrome (AGS) associated with anti-alpha-gal IgE antibody titres. Information on symptoms in response to arthropod bites was collected from patients and medical doctors. The five cases included patients bitten by a robber fly and different tick species. Cases were in Spain or U.S.A. Two cases were diagnosed with AGS and one case was diagnosed with anaphylaxis in response to tick bite with high anti-alpha-gal IgE levels. The symptoms in response to arthropod bites vary between different cases. Allergic reactions and symptoms in response to arthropod bites vary in association with host and arthropod factors. Herein we propose recommendations to control allergic symptoms, associated disease risk factors and the way forward to advance in the prevention and control of arthropod-associated allergies.

Zebrafish Animal Model for the Study of Allergic Reactions in Response to Tick Saliva Biomolecules.

Ticks are arthropod vectors that cause disease by pathogen transmission and whose bites could be related to allergic reactions impacting human health worldwide. In some individuals, high levels of immunoglobulin E antibodies against the glycan Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) have been induced by tick bites. Anaphylactic reactions mediated by glycoproteins and glycolipids containing the glycan α-Gal, present in tick saliva, are related to alpha-Gal syndrome (AGS) or mammalian meat allergy. Zebrafish (Danio rerio) has become a widely used vertebrate model for the study of different pathologies. In this study, zebrafish was used as a model for the study of allergic reactions in response to α-Gal and mammalian meat consumption because, like humans, they do not synthesize this glycan. For this purpose, behavioral patterns and hemorrhagic anaphylactic-type allergic reactions in response to Ixodes ricinus tick saliva and mammalian meat consumption was evaluated. This experimental approach allows the obtention of valid data that support the zebrafish animal model for the study of tick-borne allergies including AGS.

Heat inactivated mycobacteria, alpha-Gal and zebrafish: Insights gained from experiences with two promising trained immunity inductors and a validated animal model.

Trained immunity (TRAIM) may be defined as a form of memory where innate immune cells such as monocytes, macrophages, dendritic and natural killer (NK) cells undergo an epigenetic reprogramming that enhances their primary defensive capabilities. Cross-pathogen protective TRAIM can be triggered in different hosts by exposure to live microbes or microbe-derived products such as heat-inactivated Mycobacterium bovis or with the glycan α-Gal to elicit protective responses against several pathogens. We review the TRAIM paradigm using two models representing distinct scales of immune sensitization: the whole bacterial cell and one of its building blocks, the polysaccharides or glycans. Observations point out to macrophage lytic capabilities and cytokine regulation as two key components in non-specific innate immune responses against infections. The study of the TRAIM response deserves attention to better characterize the evolution of host-pathogen cooperation both for identifying the aetiology of some diseases and for finding new therapeutic strategies. In this field, the zebrafish provides a convenient and complete biological system that could help to deepen in the knowledge of TRAIM-mediated mechanisms in pathogen-host interactions.

Functional characterization of α-Gal producing lactic acid bacteria with potential probiotic properties.

The possibility of exploiting the human immune response to glycan α-Gal for the control of multiple infectious diseases has been the objective of recent investigations. In this field of research, the strain of Escherichia coli O86:B7 has been at the forefront, but this Gram-negative microorganism presents a safety concern and therefore cannot be considered as a probiotic. To address this challenge, this study explored the identification of novel lactic acid bacteria with a safe history of use, producing α-Gal and having probiotic potential. The lactic acid bacteria were isolated from different traditionally fermented foods (kununn-zaki, kindirmo, and pulque) and were screened for the production of α-Gal and some specific probiotic potential indicators. The results showed that Ten (10) out of forty (40) [25%] of the tested lactic acid bacteria (LAB) produced α-Gal and were identified as Limosilactobacillus fermentum, Levilactobacillus brevis, Agrilactobacillus composti, Lacticaseibacillus paracasei, Leuconostoc mesenteroides and Weissella confusa. Four (4) LAB strains with highest levels of α-Gal were further selected for in vivo study using a mouse model (α1,3GT KO mice) to elucidate the immunological response to α-Gal. The level of anti-α-Gal IgG observed were not significant while the level of anti-α-Gal IgM was lower in comparison to the level elicited by E. coli O86:B7. We concluded that the lactic acid bacteria in this study producing α-Gal have potential probiotic capacity and can be further explored in α-Gal-focused research for both the prevention and treatment of various infectious diseases and probiotic development.

Current and Future Strategies for the Diagnosis and Treatment of the Alpha-Gal Syndrome (AGS).

The α-Gal syndrome (AGS) is a pathognomonic immunoglobulin E (IgE)-mediated delayed anaphylaxis in foods containing the oligosaccharide galactose-α-1,3-galactose (α-Gal) such as mammalian meat or dairy products. Clinical presentation of AGS can also comprise immediate hypersensitivity due to anticancer therapy, gelatin-containing vaccines or mammalian serum-based antivenom. The IgE initial sensitization is caused by hard-bodied tick bites and symptomatic individuals typically develop delayed pruritus, urticaria, angioedema, anaphylaxis, malaise or gut-related symptoms. Due to inapparent presentation, delayed reactions and a wide variety of patients´ clinical history, the AGS diagnosis and treatment remain challenging. This review covers not only current diagnostic methods used for AGS such as the skin prick test (SPT), the oral food challenge (OFC), anti-α-Gal IgE levels measurement and the basophil activation test (BAT), but also potentially relevant next-generation diagnostic tools like the mast cell activation test (MAT), the histamine-release (HR) assay, omics technologies and model-based reasoning (MBR). Moreover, it focuses on the therapeutical medical and non-medical methods available and current research methods that are being applied in order to elucidate the molecular, physiological and immune mechanisms underlying this allergic disorder. Lastly, future treatment and preventive tools are also discussed, being of utmost importance for the identification of tick salivary molecules, with or without α-Gal modifications, that trigger IgE sensitivity as they could be the key for further vaccine development. Bearing in mind climate change, the tick-host paradigm will shift towards an increasing number of AGS cases in new regions worldwide, which will pose new challenges for clinicians in the future.

Assessment of the Safety and Efficacy of an Oral Probiotic-Based Vaccine Against Aspergillus Infection in Captive-Bred Humboldt Penguins ().

Aspergillosis is a fungal infection caused mainly by that often results in respiratory disease in birds. Aspergillosis is a major cause of morbidity and mortality in captive-bred penguin species. Currently, there is no registered vaccine to prevent aspergillosis. Recent research demonstrated that oral administration of gram-negative bacteria expressing high levels of galactose-α-1,3-galactose (α-Gal) modulates anti-α-Gal immunity and protects turkeys from clinical aspergillosis caused by experimental infection. The role of anti-α-Gal immunity in penguins has not been studied. Here, we tested the distribution of α-1,3-galactosyltransferase (α1,3GT) genes in the fecal microbiome of Humboldt penguins (). The occurrence of natural anti-α-Gal antibodies (Abs) in sera and eggs of healthy Humboldt penguins was also assessed. A trial was then conducted to test whether oral administration of Nissle, expressing high α-Gal levels, modulates anti-α-Gal immunity in a colony of Humboldt penguins. Animals in the vaccination and placebo groups were evaluated before the trial and followed for one year for aspergillosis detection using a diagnostic panel including computed tomography scans, capillary zone electrophoresis, 3-hydroxybutyrate levels, and anti- Abs. Anti-α-Gal Abs were detected in sera (IgM and IgY) and eggs (IgY) of healthy penguins. Microbiota analysis and functional predictions revealed the presence of α1,3GT genes in the microbiota of Humboldt penguins and other penguin species. A strong decrease in anti-α-Gal IgM levels was observed in all animals in the placebo group three months after vaccination protocol. This decrease was not observed in Nissle-treated penguins. After the vaccination protocol, we found a positive correlation between anti- IgY and anti-α-Gal IgY in the Nissle group, suggesting a correlation between the presence of the bacteria and these Abs. During the study period, three penguins exhibited respiratory signs consistent with aspergillosis. Two were from the placebo group whose symptoms resolved with specific treatments, while a single vaccinated individual developed fatal respiratory aspergillosis eight months after the trial. We conclude that Nissle represents a safe potential probiotic with a protective effect against aspergillosis in Humboldt penguins that deserves to be further explored for therapeutic uses in these animals.

Exploring the Ecological Implications of Microbiota Diversity in Birds: Natural Barriers Against Avian Malaria.

Natural antibodies (Abs), produced in response to bacterial gut microbiota, drive resistance to infection in vertebrates. In natural systems, gut microbiota diversity is expected to shape the spectrum of natural Abs and resistance to parasites. This hypothesis has not been empirically tested. In this 'Hypothesis and Theory' paper, we propose that enteric microbiota diversity shapes the immune response to the carbohydrate α-Gal and resistance to avian malaria. We further propose that anti-α-Gal Abs are transmitted from mother to eggs for early malaria protection in chicks. Microbiota modulation by anti-α-Gal Abs is also proposed as a mechanism favoring the early colonization of bacterial taxa with α1,3-galactosyltransferase (α1,3GT) activity in the bird gut. Our preliminary data shows that bacterial α1,3GT genes are widely distributed in the gut microbiome of wild and domestic birds. We also showed that experimental infection with the avian malaria parasite induces anti-α-Gal Abs in bird sera. The bird-malaria-microbiota system allows combining field studies with infection and transmission experiments in laboratory animals to test the association between microbiota composition, anti-α-Gal Abs, and malaria infection in natural populations of wild birds. Understanding how the gut microbiome influences resistance to malaria can bring insights on how these mechanisms influence the prevalence of malaria parasites in juvenile birds and shape the host population dynamics.

Functional Food for the Stimulation of the Immune System Against Malaria.

Drug resistance has become a threat to global health, and new interventions are needed to control major infectious diseases. The composition of gut microbiota has been linked to human health and has been associated with severity of malaria. Fermented foods contribute to the community of healthy gut bacteria. Despite the studies connecting gut microbiota to the prevention of malaria transmission and severity, research on developing functional foods for the purpose of manipulating the gut microbiota for malaria control is limited. This review summarizes recent knowledge on the role of the gut microbiota in malaria prevention and treatment. This information should encourage the search for lactic acid bacteria expressing α-Gal and those that exhibit the desired immune stimulating properties for the development of functional food and probiotics for malaria control.

Tick-human interactions: from allergic klendusity to the α-Gal syndrome.

Ticks and the pathogens they transmit, including bacteria, viruses, protozoa, and helminths, constitute a growing burden for human and animal health worldwide. The ability of some animal species to acquire resistance to blood-feeding by ticks after a single or repeated infestation is known as acquired tick resistance (ATR). This resistance has been associated to tick-specific IgE response, the generation of skin-resident memory CD4+ T cells, basophil recruitment, histamine release, and epidermal hyperplasia. ATR has also been associated with protection to tick-borne tularemia through allergic klendusity, a disease-escaping ability produced by the development of hypersensitivity to an allergen. In addition to pathogen transmission, tick infestation in humans is associated with the α-Gal syndrome (AGS), a type of allergy characterized by an IgE response against the carbohydrate Galα1-3Gal (α-Gal). This glycan is present in tick salivary proteins and on the surface of tick-borne pathogens such as Borrelia burgdorferi and Anaplasma phagocytophilum, the causative agents of Lyme disease and granulocytic anaplasmosis. Most α-Gal-sensitized individuals develop IgE specific against this glycan, but only a small fraction develop the AGS. This review summarizes our current understanding of ATR and its impact on the continuum α-Gal sensitization, allergy, and the AGS. We propose that the α-Gal-specific IgE response in humans is an evolutionary adaptation associated with ATR and allergic klendusity with the trade-off of developing AGS.

Characterization of the anti-α-Gal antibody profile in association with Guillain-Barré syndrome, implications for tick-related allergic reactions.

Humans evolved by losing the capacity to synthesize the glycan Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal), which resulted in the capacity to develop a protective response mediated by anti-α-Gal IgM/IgG antibodies against pathogens containing this modification on membrane proteins. As an evolutionary trade-off, humans can develop the alpha-Gal syndrome (AGS), a recently diagnosed disease mainly associated with allergic reactions to mammalian meat consumption. The etiology of the AGS is the exposure to tick bites and the IgE antibody response against α-Gal-containing glycoproteins and glycolipids. The objective of this study was to characterize the anti-α-Gal antibody response in association with the immune-mediated peripheral neuropathy, Guillain-Barré syndrome (GBS), and compare it with different factors known to modulate the antibody response to α-Gal such as exposure to tick bites and development of allergic reactions in response to tick bites. The results showed a significant decrease in the IgM/IgG response to α-Gal in GBS patients when compared to healthy individuals. In contrast, the IgM/IgG levels to α-Gal did not change in patients with allergic reactions to tick bites. The IgE response was not affected in GBS patients, but as expected, the IgE levels significantly increased in individuals exposed to tick bites and patients with tick-associated allergies. These results suggest that the immune pathways of anti-α-Gal IgM/IgG and IgE production are independent. Further studies should consider the susceptibility to allergic reactions to tick bites in GBS patients.

Probiotic Bacteria with High Alpha-Gal Content Protect Zebrafish against Mycobacteriosis.

Mycobacteriosis affects wild fish and aquaculture worldwide, and alternatives to antibiotics are needed for an effective and environmentally sound control of infectious diseases. Probiotics have shown beneficial effects on fish growth, nutrient metabolism, immune responses, disease prevention and control, and gut microbiota with higher water quality. However, the identification and characterization of the molecules and mechanisms associated with probiotics is a challenge that requires investigation. To address this challenge, herein we used the zebrafish model for the study of the efficacy and mechanisms of probiotic interventions against tuberculosis. First, bacteria from fish gut microbiota were identified with high content of the surface glycotope Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) that has been shown to induce protective immune responses. The results showed that probiotics of selected bacteria with high α-Gal content, namely Aeromonas veronii and , were biosafe and effective for the control of Mycobacterium marinum. Protective mechanisms regulating immunity and metabolism activated in response to α-Gal and probiotics with high α-Gal content included modification of gut microbiota composition, B-cell maturation, anti-α-Gal antibodies-mediated control of mycobacteria, induced innate immune responses, beneficial effects on nutrient metabolism and reduced oxidative stress. These results support the potential of probiotics with high -Gal content for the control of fish mycobacteriosis and suggested the possibility of exploring the development of combined probiotic treatments alone and in combination with -Gal for the control of infectious diseases.

Immunity to glycan α-Gal and possibilities for the control of COVID-19.

Characterization of tick salivary gland and saliva alphagalactome reveals candidate alpha-gal syndrome disease biomarkers.

Ticks are obligate hematophagous arthropods that synthesize the glycan Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) associated with the alpha-gal syndrome (AGS) or allergy to mammalian meat consumption. In this study, we used a proteomics approach to characterize tick proteins in salivary glands (sialome SG), secreted saliva (sialome SA) and with α-Gal modification (alphagalactome SG and SA) in model tick species associated with the AGS in the United States () and Australia (). Selected proteins reactive to sera (IgE) from patients with AGS were identified to advance in the identification of possible proteins associated with the AGS. For comparative analysis, the α-Gal content was measured in various tick species. The results confirmed that ticks produce proteins with α-Gal modifications and secreted into saliva during feeding. Proteins identified in tick alphagalactome SA by sera from patients with severe AGS symptomatology may constitute candidate disease biomarkers. The results support the presence of tick-derived proteins with α-Gal modifications in the saliva with potential implications in AGS and other disorders and protective capacity against tick infestations and pathogen infection. Future research should focus on the characterization of the function of tick glycoproteins with α-Gal in tick biology and AGS.

The antibody response to the glycan α-Gal correlates with COVID-19 disease symptoms.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people worldwide. Characterization of the immunological mechanisms involved in disease symptomatology and protective response is important to progress in disease control and prevention. Humans evolved by losing the capacity to synthesize the glycan Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal), which resulted in the development of a protective response against pathogenic viruses and other microorganisms containing this modification on membrane proteins mediated by anti-α-Gal immunoglobulin M (IgM)/IgG antibodies produced in response to bacterial microbiota. In addition to anti-α-Gal antibody-mediated pathogen opsonization, this glycan induces various immune mechanisms that have shown protection in animal models against infectious diseases without inflammatory responses. In this study, we hypothesized that the immune response to α-Gal may contribute to the control of COVID-19. To address this hypothesis, we characterized the antibody response to α-Gal in patients at different stages of COVID-19 and in comparison with healthy control individuals. The results showed that while the inflammatory response and the anti-SARS-CoV-2 (Spike) IgG antibody titers increased, reduction in anti-α-Gal IgE, IgM, and IgG antibody titers and alteration of anti-α-Gal antibody isotype composition correlated with COVID-19 severity. The results suggested that the inhibition of the α-Gal-induced immune response may translate into more aggressive viremia and severe disease inflammatory symptoms. These results support the proposal of developing interventions such as probiotics based on commensal bacteria with α-Gal epitopes to modify the microbiota and increase α-Gal-induced protective immune response and reduce severity of COVID-19.

The α-Gal Syndrome and Potential Mechanisms.

The α-Gal syndrome is a complex allergic disease characterized by the development of specific IgE antibodies against the carbohydrate galactose-α-1,3-galactose (α-Gal), an oligosaccharide present in cells and tissues of non-primate mammals. Individuals with IgE antibodies to α-Gal suffer from a delayed form of anaphylaxis following red meat consumption. There are several features that make the α-Gal syndrome such a unique allergic disease and distinguish it from other food allergies: (1) symptoms causing IgE antibodies are directed against a carbohydrate moiety, (2) the unusual delay between the consumption of the food and the onset of the symptoms, and (3) the fact that primary sensitization to α-Gal occurs tick bites. This review takes a closer look at the immune response against α-Gal, in healthy and in α-Gal allergic individuals. Furthermore, the similarities and differences between immune response against α-Gal and against the other important glycan moieties associated with allergies, namely cross-reactive carbohydrate determinants (CCDs), are discussed. Then different mechanisms are discussed that could contribute to the delayed onset of symptoms after consumption of mammalian meat. Moreover, our current knowledge on the role of tick bites in the sensitization process is summarized. The tick saliva has been shown to contain proteins carrying α-Gal, but also bioactive molecules, such as prostaglandin E2, which is capable of stimulating an increased expression of anti-inflammatory cytokines while promoting a decrease in the production of proinflammatory mediators. Together these components might promote Th2-related immunity and trigger a class switch to IgE antibodies directed against the oligosaccharide α-Gal. The review also points to open research questions that remain to be answered and proposes future research directions, which will help to get a better understanding and lead to a better management of the disease.

Alpha-gal syndrome: challenges to understanding sensitization and clinical reactions to alpha-gal.

The α-Gal syndrome (AGS) is a type of allergy characterized by an IgE antibody response against the carbohydrate Galα1-3Galβ1-4GlcNAc-R (α-Gal). Tick bites are recognized as the most important cause of anti-α-Gal IgE antibody increase in humans. Several risk factors have been associated with the development of AGS, but their integration into a standardized disease diagnosis has proven challenging. Herein we discuss the current AGS diagnosis based on anti-α-Gal IgE titers and propose an algorithm that considers all co-factors in the clinical history of α-Gal-sensitized patients to be incorporated into the AGS diagnosis. The need for identification of host-derived gene markers and tick-derived proteins for the diagnosis of the AGS is also discussed. The current AGS diagnosis based on anti-α-Gal IgE titers has limitations because not all patients sensitized to α-Gal and with anti-α-Gal IgE antibodies higher than the cutoff (0.35 IU/ml) develop anaphylaxis to mammalian meat and AGS. The basophil activation test proposed to differentiate between patients with AGS and asymptomatic α-Gal sensitization cannot be easily implemented as a generalized clinical test. In coming years, the algorithm proposed here could be used in a mobile application for easier AGS diagnosis in the clinical practice.

Anti-Tick Microbiota Vaccine Impacts Performance during Feeding.

The tick microbiota is a highly complex ensemble of interacting microorganisms. Keystone taxa, with a central role in the microbial networks, support the stability and fitness of the microbial communities. The keystoneness of taxa in the tick microbiota can be inferred from microbial co-occurrence networks. Microbes with high centrality indexes are highly connected with other taxa of the microbiota and are expected to provide important resources to the microbial community and/or the tick. We reasoned that disturbance of vector microbiota by removal of ubiquitous and abundant keystone bacteria may disrupt the tick-microbiota homeostasis causing harm to the tick host. These observations and reasoning prompted us to test the hypothesis that antibodies targeting keystone bacteria may harm the ticks during feeding on immunized hosts. To this aim, in silico analyses were conducted to identify keystone bacteria in the microbiota of nymphs. The family Enterobacteriaceae was among the top keystone taxa identified in microbiota. Immunization of α-1,3-galactosyltransferase-deficient-C57BL/6 (α1,3GT KO) mice with a live vaccine containing the Enterobacteriaceae bacterium strain BL21 revealed that the production of anti- and anti-α-Gal IgM and IgG was associated with high mortality of nymphs during feeding. However, this effect was absent in two different strains of wild type mice, BALB/c and C57BL/6. This result concurred with a wide distribution of α-1,3-galactosyltransferase genes, and possibly α-Gal, in Enterobacteriaceae and other bacteria of tick microbiota. Interestingly, the weight of nymphs that fed on -immunized C57BL/6 was significantly higher than the weight of ticks that fed on C57BL/6 immunized with a mock vaccine. Our results suggest that anti-tick microbiota vaccines are a promising tool for the experimental manipulation of vector microbiota, and potentially the control of ticks and tick-borne pathogens.

Gut Microbiota Abrogates Anti-α-Gal IgA Response in Lungs and Protects against Experimental Infection in Poultry.

Naturally occurring human antibodies (Abs) of the isotypes IgM and IgG and reactive to the galactose-α-1,3-galactose (α-Gal) epitope are associated with protection against infectious diseases, caused by pathogens expressing the glycan. Gut microbiota bacteria expressing α-Gal regulate the immune response to this glycan in animals lacking endogenous α-Gal. Here, we asked whether the production of anti-α-Gal Abs in response to microbiota stimulation in birds, confers protection against infection by , a major fungal pathogen that expresses α-Gal in its surface. We demonstrated that the oral administration of O86:B7 strain, a bacterium with high α-Gal content, reduces the occurrence of granulomas in lungs and protects turkeys from developing acute aspergillosis. Surprisingly, the protective effect of O86:B7 was not associated with an increase in circulating anti-α-Gal IgY levels, but with a striking reduction of anti-α-Gal IgA in the lungs of infected turkeys. Subcutaneous immunization against α-Gal did not induce a significant reduction of lung anti-α-Gal IgA and failed to protect against an infectious challenge with . Oral administration of O86:B7 was not associated with the upregulation of lung cytokines upon infection. We concluded that the oral administration of bacteria expressing high levels of α-Gal decreases the levels of lung anti-α-Gal IgA, which are mediators of inflammation and lung damage during acute aspergillosis.

COVID-19 in the Developing World: Is the Immune Response to α-Gal an Overlooked Factor Mitigating the Severity of Infection?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), which has affected millions of people worldwide. Considerably lower prevalence and fatality rates resulting from COVID-19 are reported in Africa and Asia than in the industrialized world. In this Viewpoint, we discuss the possibility that this intriguing phenomenon could be, among other factors, due to protective immunity of the oligosaccharide galactose-α-1,3-galactose (α-Gal). The α-Gal immunity induced by gut microbiota that express the same glycan modification may prevent COVID-19 through the activation of different mechanisms involved in SARS-CoV-2 neutralization and the downregulation of the inflammatory response in the lungs of infected patients.

Infection with Inhibits the Production of IgE Antibodies to α-Gal in Humans: Towards a Conceptual Framework of the Hygiene Hypothesis?

α-Gal syndrome (AGS) is a type of anaphylactic reaction to mammalian meat characterized by an immunoglobulin (Ig)E immune response to the oligosaccharide α-Gal (Galα1-3Galβ1-4GlcNAc-R). Tick bites seems to be a prerequisite for the onset of the allergic disease in humans, but the implication of non-tick parasites in α-Gal sensitization has also been deliberated. In the present study, we therefore evaluated the capacity of helminths (, , ), protozoa (), and parasitic fungi () to induce an immune response to α-Gal. For this, different developmental stages of the infectious agents were tested for the presence of α-Gal. Next, the potential correlation between immune responses to α-Gal and the parasite infections was investigated by testing sera collected from patients with AGS and those infected with the parasites. Our results showed that and produce the terminal α-Gal moieties, but they were not able to induce the production of specific antibodies. By contrast, , and lack the α-Gal epitope. Furthermore, the patients with infection had significantly decreased anti-α-Gal IgE levels when compared to the healthy controls, suggesting the potential role of this nematode parasite in suppressing the allergic response to the glycan molecule. This rather intriguing observation is discussed in the context of the 'hygiene hypothesis'. Taken together, our study provides new insights into the relationships between immune responses to α-Gal and parasitic infections. However, further investigations should be undertaken to identify components with potent immunomodulatory properties and to assess their potential to be used in immunotherapy and control of AGS.

Tick and Host Derived Compounds Detected in the Cement Complex Substance.

Ticks are obligate hematophagous arthropods and vectors of pathogens affecting human and animal health worldwide. Cement is a complex protein polymerization substance secreted by ticks with antimicrobial properties and a possible role in host attachment, sealing the feeding lesion, facilitating feeding and pathogen transmission, and protection from host immune and inflammatory responses. The biochemical properties of tick cement during feeding have not been fully characterized. In this study, we characterized the proteome of salivary glands (sialome) and cement (cementome) together with their physicochemical properties at different adult female parasitic stages. The results showed the combination of tick and host derived proteins and other biomolecules such as α-Gal in cement composition, which varied during the feeding process. We propose that these compounds may synergize in cement formation, solidification and maintenance to facilitate attachment, feeding, interference with host immune response and detachment. These results advanced our knowledge of the complex tick cement composition and suggested that tick and host derived compounds modulate cement properties throughout tick feeding.

A dataset for the analysis of antibody response to glycan alpha-Gal in individuals with immune-mediated disorders.

Humans evolved by losing the capacity to synthesize the glycan Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal), which resulted in the development of a protective response mediated by anti-α-Gal IgM/IgG/IgA antibodies against pathogens containing this modification on membrane proteins. As an evolutionary trade-off, humans can develop the alpha-Gal syndrome (AGS), a recently diagnosed disease mediated by anti-α-Gal IgE antibodies and associated with allergic reactions to mammalian meat consumption and tick bites. However, the anti-α-Gal antibody response may be associated with other immune-mediated disorders such as those occurring in patients with COVID-19 and Guillain-Barré syndrome (GBS). Here, we provide a dataset (209 entries) on the IgE/IgM/IgG/IgA anti-α-Gal antibody response in healthy individuals and patients diagnosed with AGS, tick-borne allergies, GBS and COVID-19. The data allows correlative analyses of the anti-α-Gal antibody response with factors such as patient and clinical characteristics, record of tick bites, blood group, age and sex. These analyses could provide insights into the role of anti-α-Gal antibody response in disease symptomatology and possible protective mechanisms.

Allergic Reactions and Immunity in Response to Tick Salivary Biogenic Substances and Red Meat Consumption in the Zebrafish Model.

Ticks are arthropod ectoparasite vectors of pathogens and the cause of allergic reactions affecting human health worldwide. In humans, tick bites can induce high levels of immunoglobulin E antibodies against the carbohydrate Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) present in glycoproteins and glycolipids from tick saliva that mediate anaphylactic reactions known as the alpha-Gal syndrome (AGS) or red meat allergy. In this study, a new animal model was developed using zebrafish for the study of allergic reactions and the immune mechanisms in response to tick salivary biogenic substances and red meat consumption. The results showed allergic hemorrhagic anaphylactic-type reactions and abnormal behavior patterns likely in response to tick salivary toxic and anticoagulant biogenic compounds different from α-Gal. However, the results showed that only zebrafish previously exposed to tick saliva developed allergic reactions to red meat consumption with rapid desensitization and tolerance. These allergic reactions were associated with tissue-specific Toll-like receptor-mediated responses in types 1 and 2 T helper cells (T1 and T2) with a possible role for basophils in response to tick saliva. These results support previously proposed immune mechanisms triggering the AGS and provided evidence for new mechanisms also potentially involved in the AGS. These results support the use of the zebrafish animal model for the study of the AGS and other tick-borne allergies.

Tick Bites Induce Anti-α-Gal Antibodies in Dogs.

Due to the functional inactivation of the gene encoding for the enzyme that is involved in the oligosaccharide galactose-α-1,3-galactose (α-Gal) synthesis, humans and Old-World primates are able to produce a large amount of antibodies against the glycan epitope. Apart from being involved in the hyperacute organ rejection in humans, anti-α-Gal antibodies have shown a protective effect against some pathogenic agents and an implication in the recently recognized tick-induced mammalian meat allergy. Conversely, non-primate mammals, including dogs, have the ability to synthetize α-Gal and, thus, their immune system is not expected to naturally generate the antibodies toward this self-antigen molecule. However, in the current study, we detected specific IgG, IgM, and IgE antibodies to α-Gal in sera of clinically healthy dogs by an indirect enzyme-linked immunosorbent assay (ELISA) for the first time. Furthermore, in a tick infestation experiment, we showed that bites of induce the immune response to α-Gal in dogs and that the resulting antibodies (IgM) might be protective against . These findings may help lead to a better understanding of the underlying mechanisms involved in mammalian meat allergy and tick-host-pathogen interactions, but they also open up the question about the possibility that dogs could develop an allergy to mammalian meat after tick bites, similar to that in humans.

Clinical gamasoidosis and antibody response in two patients infested with Ornithonyssus bursa (Acari: Gamasida: Macronyssidae).

Blood-feeding ectoparasites constitute a growing burden for human and animal health, and animal production worldwide. In particular, mites (Acari: Gamasida) of the genera Dermanyssus (Dermanyssidae) and Ornithonyssus (Macronyssidae) infest birds and cause gamasoidosis in humans. The tropical fowl mite, Ornithonyssus bursa, is commonly found in tropical and subtropical countries but rarely reported in Europe. In this research we characterized the first two cases in Spain of clinical gamasoidosis diagnosed in patients infested with O. bursa, and investigated the IgE, IgM and IgG antibody response to mite proteins and the carbohydrate Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) involved in the tick-bite associated alpha-Gal syndrome (AGS). The results suggested that O. bursa is establishing across Mediterranean countries, and may increase the risk for gamasoidosis. The immune antibody response to mite proteins was higher for IgM and similar for IgE and IgG antibodies between patients and non-allergic control individuals exposed to mite or tick bites. The anti-α-Gal antibody levels were similar between patients and controls, a result supported by the absence of this carbohydrate in mites. These results suggested that mite bites do not correlate with antibody response to acarine proteins or α-Gal, and are not associated with the AGS.

Delayed hypersensitivity reaction to mammalian galactose-α-1,3-galactose (α-Gal) after repeated tick bites in a patient from France.

The α-Gal syndrome is a tick-associated and emerging IgE-mediated hypersensitivity reaction directed against the carbohydrate Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) epitope after red meat intake. Herein, we describe a clinical case of a 44-year-old French patient who suffered from recurrent anaphylactic reactions after mammalian meat consumption for five years before the final diagnosis of the α-Gal syndrome was established in 2018. The patient also reported multiple tick bites prior to symptom onset. This unique type of allergy has increasingly been reported across the world, but it is still unknown in many European countries. Therefore, the present clinical case should increase awareness among primary care practitioners and further improve the early diagnosis of the α-Gal syndrome in affected individuals.

The alpha-Gal syndrome: new insights into the tick-host conflict and cooperation.

This primer focuses on a recently diagnosed tick-borne allergic disease known as the alpha-Gal syndrome (AGS). Tick bites induce in humans high levels of IgE antibodies against the carbohydrate Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) present on tick salivary glycoproteins and tissues of non-catarrhine mammals, leading to the AGS in some individuals. This immune response evolved as a conflict and cooperation between ticks and human hosts including their gut microbiota. The conflict is characterized by the AGS that mediate delayed anaphylaxis to red meat consumption and certain drugs such as cetuximab, and immediate anaphylaxis to tick bites. The cooperation is supported by the capacity of anti-α-Gal IgM and IgG antibody response to protect against pathogens with α-Gal on their surface. Despite the growing diagnosis of AGS in all world continents, many questions remain to be elucidated on the tick proteins and immune mechanisms triggering this syndrome, and the protective response against pathogen infection elicited by anti-α-Gal antibodies. The answer to these questions will provide information for the evaluation of risks, diagnosis and prevention of the AGS, and the possibility of using the carbohydrate α-Gal to develop vaccines for the control of major infectious diseases.

Environmental and Molecular Drivers of the α-Gal Syndrome.

The α-Gal syndrome (AGS) is a type of allergy characterized by an IgE antibody (Ab) response against the carbohydrate Galα1-3Galβ1-4GlcNAc-R (α-Gal), which is present in glycoproteins from tick saliva and tissues of non-catarrhine mammals. Recurrent tick bites induce high levels of anti-α-Gal IgE Abs that mediate delayed hypersensitivity to consumed red meat products in humans. This was the first evidence that tick glycoproteins play a major role in allergy development with the potential to cause fatal delayed anaphylaxis to α-Gal-containing foods and drugs and immediate anaphylaxis to tick bites. Initially, it was thought that the origin of tick-derived α-Gal was either residual blood meal mammalian glycoproteins containing α-Gal or tick gut bacteria producing this glycan. However, recently tick galactosyltransferases were shown to be involved in α-Gal synthesis with a role in tick and tick-borne pathogen life cycles. The tick-borne pathogen increases the level of tick α-Gal, which potentially increases the risk of developing AGS after a bite by a pathogen-infected tick. Two mechanisms might explain the production of anti-α-Gal IgE Abs after tick bites. The first mechanism proposes that the α-Gal antigen on tick salivary proteins is presented to antigen-presenting cells and B-lymphocytes in the context of Th cell-mediated immunity induced by tick saliva. The second mechanism is based on the possibility that tick salivary prostaglandin E2 triggers Immunoglobulin class switching to anti-α-Gal IgE-producing B cells from preexisting mature B cells clones producing anti-α-Gal IgM and/or IgG. Importantly, blood group antigens influence the capacity of the immune system to produce anti-α-Gal Abs which in turn impacts individual susceptibility to AGS. The presence of blood type B reduces the capacity of the immune system to produce anti-α-Gal Abs, presumably due to tolerance to α-Gal, which is very similar in structure to blood group B antigen. Therefore, individuals with blood group B and reduced levels of anti-α-Gal Abs have lower risk to develop AGS. Specific immunity to tick α-Gal is linked to host immunity to tick bites. Basophil activation and release of histamine have been implicated in IgE-mediated acquired protective immunity to tick infestations and chronic itch. Basophil reactivity was also found to be higher in patients with AGS when compared to asymptomatic α-Gal sensitized individuals. In addition, host resistance to tick infestation is associated with resistance to tick-borne pathogen infection. Anti-α-Gal IgM and IgG Abs protect humans against vector-borne pathogens and blood group B individuals seem to be more susceptible to vector-borne diseases. The link between blood groups and anti-α-Gal immunity which in turn affects resistance to vector-borne pathogens and susceptibility to AGS, suggests a trade-off between susceptibility to AGS and protection to some infectious diseases. The understanding of the environmental and molecular drivers of the immune mechanisms involved in AGS is essential to developing tools for the diagnosis, control, and prevention of this growing health problem.

Tick galactosyltransferases are involved in α-Gal synthesis and play a role during Anaplasma phagocytophilum infection and Ixodes scapularis tick vector development.

The carbohydrate Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) is produced in all mammals except for humans, apes and old world monkeys that lost the ability to synthetize this carbohydrate. Therefore, humans can produce high antibody titers against α-Gal. Anti-α-Gal IgE antibodies have been associated with tick-induced allergy (i.e. α-Gal syndrome) and anti-α-Gal IgG/IgM antibodies may be involved in protection against malaria, leishmaniasis and Chagas disease. The α-Gal on tick salivary proteins plays an important role in the etiology of the α-Gal syndrome. However, whether ticks are able to produce endogenous α-Gal remains currently unknown. In this study, the Ixodes scapularis genome was searched for galactosyltransferases and three genes were identified as potentially involved in the synthesis of α-Gal. Heterologous gene expression in α-Gal-negative cells and gene knockdown in ticks confirmed that these genes were involved in α-Gal synthesis and are essential for tick feeding. Furthermore, these genes were shown to play an important role in tick-pathogen interactions. Results suggested that tick cells increased α-Gal levels in response to Anaplasma phagocytophilum infection to control bacterial infection. These results provided the molecular basis of endogenous α-Gal production in ticks and suggested that tick galactosyltransferases are involved in vector development, tick-pathogen interactions and possibly the etiology of α-Gal syndrome in humans.

Tick-host conflict: immunoglobulin E antibodies to tick proteins in patients with anaphylaxis to tick bite.

Tick-borne infectious diseases and allergies are a growing problem worldwide. Tick bite allergy has been associated with the direct effect of immunoglobulin E (IgE) response to tick salivary antigens, or secondary to the induction of allergy to red meat consumption through IgE antibodies against the carbohydrate α-Gal (Gal α 1-3Gal β 1-(3)4GlcNAc-R). However, despite the growing burden of this pathology, the proteins associated with anaphylaxis to tick bite have not been characterized. To address this question, a comparative proteomics approach was used to characterize tick proteins producing an IgE antibody response in a healthy individual with record of tick bites, which had not resulted in any allergic reactions, and two patients with anaphylactic reactions to Rhipicephalus bursa or Hyalomma marginatum tick bites. Both patients and the healthy individual were red meat tolerant. The results supported a patient-specific IgE antibody response to tick species responsible for the anaphylaxis to tick bite. Both patients and the healthy individual serologically recognized tick proteins with and without α-Gal modifications, with proteins differentially recognized by patients but not control sera. These proteins could be used as potential antigens for diagnostics, treatment and prevention of tick bite-induced allergies.

Effect of blood type on anti-α-Gal immunity and the incidence of infectious diseases.

The identification of factors affecting the susceptibility to infectious diseases is essential toward reducing their burden on the human population. The ABO blood type correlates with susceptibility to malaria and other infectious diseases. Due to the structural similarity between blood antigen B and Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal), we hypothesized that self-tolerance to antigen B affects the immune response to α-Gal, which in turn affects the susceptibility to infectious diseases caused by pathogens carrying α-Gal on their surface. Here we found that the incidence of malaria and tuberculosis, caused by pathogens with α-Gal on their surface, positively correlates with the frequency of blood type B in endemic regions. However, the incidence of dengue fever, caused by a pathogen without α-Gal, was not related to the frequency of blood type B in these populations. Furthermore, the incidence of malaria and tuberculosis was negatively correlated with the anti-α-Gal antibody protective response. These results have implications for disease control and prevention.

Salivary Prostaglandin E2: Role in Tick-Induced Allergy to Red Meat.

Tick-induced allergy to red meat is associated with anti-α-Gal IgE antibody levels. We propose that tick salivary prostaglandin E2 triggers antibody class switching in mature B cells, increasing the levels of anti-α-Gal IgE antibodies. Immune tolerance to α-Gal in blood type B individuals might reduce the risk to this allergy.

Prevalence of type I sensitization to alpha-gal in forest service employees and hunters: Is the blood type an overlooked risk factor in epidemiological studies of the α-Gal syndrome?

Immunity to α-Gal: The Opportunity for Malaria and Tuberculosis Control.

Control of vector-borne infectious diseases by human immunity against α-Gal.

Regulation of the Immune Response to α-Gal and Vector-borne Diseases.

Vector-borne diseases (VBD) challenge our understanding of emerging diseases. Recently, arthropod vectors have been involved in emerging anaphylactic diseases. In particular, the immunoglobulin E (IgE) antibody response to the carbohydrate Galα1-3Galβ1-(3)4GlcNAc-R (α-gal) following a tick bite was associated with allergies to red meat, cetuximab, and gelatin. By contrast, an anti-α-gal IgM antibody response was shown to protect against mosquito-borne malaria. Herein, we highlight the interplay between the gut microbiota, vectors, transmitted pathogens, and the regulation of the immune response as a model to understand the protective or allergic effect of α-gal. Establishing the source of α-gal in arthropod vectors and the immune response to vector bites and transmitted pathogens will be essential for diagnosing, treating, and ultimately preventing these emerging anaphylactic and other vector-borne diseases.