A tissue checkpoint regulates type 2 immunity.

Group 2 innate lymphoid cells (ILC2s) and CD4+ type 2 helper T cells (TH2 cells) are defined by their similar effector cytokines, which together mediate the features of allergic immunity. We found that tissue ILC2s and TH2 cells differentiated independently but shared overlapping effector function programs that were mediated by exposure to the tissue-derived cytokines interleukin 25 (IL-25), IL-33 and thymic stromal lymphopoietin (TSLP). Loss of these three tissue signals did not affect lymph node priming, but abrogated the terminal differentiation of effector TH2 cells and adaptive lung inflammation in a T cell-intrinsic manner. Our findings suggest a mechanism by which diverse perturbations can activate type 2 immunity and reveal a shared local-tissue-elicited checkpoint that can be exploited to control both innate and adaptive allergic inflammation.

Mast cell-derived prostaglandin D2 limits the subcutaneous absorption of honey bee venom in mice.

Mast cells play pivotal roles in innate host defenses against venom. Activated mast cells release large amounts of prostaglandin D2 (PGD2). However, the role of PGD2 in such host defense remains unclear. We found that c-kit-dependent and c-kit-independent mast cell-specific hematopoietic prostaglandin D synthase (H-pgds) deficiency significantly exacerbated honey bee venom (BV)-induced hypothermia and increased mortality rates in mice. BV absorption via postcapillary venules in the skin was accelerated upon endothelial barrier disruption resulting in increased plasma venom concentrations. These results suggest that mast cell-derived PGD2 may enhance host defense against BV and save lives by inhibiting BV absorption into circulation.

Basophil activation in insect venom allergy: comparison of an established test using liquid reagents with a test using 5-color tubes with dried antibody reagents.

BACKGROUND: Flow cytometry-based basophil activation tests (BAT) have been performed with various modifications, differing in the use of distinct identification and activation markers. Established tests use liquid reagents while a new development involves the use of tubes with dried antibody reagents. The aim of this pilot study was to compare these two techniques in patients with insect venom allergy. METHODS: Seventeen patients with an insect venom allergy were included in the study. The established "BAT 1" utilizes conventional antibody solutions of anti-CCR3 for basophil identification and anti-CD63 to assess basophil activation, whereas "BAT 2" uses dried anti-CD45, anti-CD3, anti-CRTH2, anti-203c and anti-CD63 for identification and activation measurement of basophils. Negative and positive controls as well as incubations with honey bee venom and yellow jacket venom at three concentrations were performed. RESULTS: Seven patients had to be excluded due to low basophil counts, high values in negative controls or negative positive controls. For the remaining 10 patients the overall mean (± SD) difference in activated basophils between the two tests was 0.2 (± 12.2) %P. In a Bland-Altman plot, the limit of agreement (LoA) ranged from 24.0 to -23.7. In the qualitative evaluation (value below/above cut-off) Cohen's kappa was 0.77 indicating substantial agreement. BAT 2 took longer to perform than BAT 1 and was more expensive. CONCLUSION: The BAT 2 technique represents an interesting innovation, however, it was found to be less suitable compared to an established BAT for the routine diagnosis of insect venom allergies.

Melittin-Phospholipase A2 Synergism Is Mediated by Liquid-Liquid Miscibility Phase Transition in Giant Unilamellar Vesicles.

The primary constituents of honeybee venom, melittin and phospholipase A2 (PLA2), display toxin synergism in which the PLA2 activity is significantly enhanced by the presence of melittin. It has been shown previously that this is accomplished by the disruption in lipid packing, which allows PLA2 to become processive on the membrane surface. In this work, we show that melittin is capable of driving miscibility phase transition in giant unilamellar vesicles (GUVs) and that it raises the miscibility transition temperature (Tmisc) in a concentration-dependent manner. The induced phase separation enhances the processivity of PLA2, particularly at its boundaries, where a substantial difference in domain thickness creates a membrane discontinuity. The catalytic action of PLA2, in response, induces changes in the membrane, rendering it more conducive to melittin binding. This, in turn, facilitates further lipid phase separation and eventual vesicle lysis. Overall, our results show that melittin has powerful membrane-altering capabilities that activate PLA2 in various membrane contexts. More broadly, they exemplify how this biochemical system actively modulates and capitalizes on the spatial distribution of membrane lipids to efficiently achieve its objectives.

Standardized guidelines for Africanized honeybee venom production needed for development of new apilic antivenom.

Africanized bees have spread across the Americas since 1956 and consequently resulted in human and animal deaths attributed to massive attacks related to exposure from Argentina to the USA. In Brazil, more than 100,000 accidents were registered in the last 5 years with a total of 303 deaths. To treat such massive attacks, Brazilian researchers developed the first specific antivenom against Africanized honey bee sting exposure. This unique product, the first of its kind in the world, has been safely tested in 20 patients during a Phase 2 clinical trial. To develop the antivenom, a standardized process was undertaken to extract primary venom antigens from the Africanized bees for immunization of serum-producing horses. This process involved extracting, purifying, fractionating, characterizing, and identifying the venom (apitoxin) employing mass spectrometry to generate standardized antigen for hyperimmunization of horses using the major toxins (melittin and its isoforms and phospholipase A2). The current guide describes standardization of the entire production chain of venom antigens in compliance with good manufacturing practices (GMP) required by regulatory agencies. Emphasis is placed upon the welfare of bees and horses during this process, as well as the development of a new biopharmaceutical to ultimately save lives.

1 H-NMR revealed pyruvate as a differentially abundant metabolite in the venom glands of Apis cerana and Apis mellifera.

As a common defense mechanism in Hymenoptera, bee venom has complex components. Systematic and comprehensive analysis of bee venom components can aid in early evaluation, accurate diagnosis, and protection of organ function in humans in cases of bee stings. To determine the differences in bee venom composition and metabolic pathways between Apis cerana and Apis mellifera, proton nuclear magnetic resonance (1 H-NMR) technology was used to detect the metabolites in venom samples. A total of 74 metabolites were identified and structurally analyzed in the venom of A. cerana and A. mellifera. Differences in the composition and abundance of major components of bee venom from A. cerana and A. mellifera were mapped to four main metabolic pathways: valine, leucine and isoleucine biosynthesis; glycine, serine and threonine metabolism; alanine, aspartate and glutamate metabolism; and the tricarboxylic acid cycle. These findings indicated that the synthesis and metabolic activities of proteins or polypeptides in bee venom glands were different between A. cerana and A. mellifera. Pyruvate was highly activated in 3 selected metabolic pathways in A. mellifera, being much more dominant in A. mellifera venom than in A. cerana venom. These findings indicated that pyruvate in bee venom glands is involved in various life activities, such as biosynthesis and energy metabolism, by acting as a precursor substance or intermediate product.

Exploring the antibiofilm effects on Escherichia coli biofilm associated with colon cancer and anticancer activities on HCT116 cell line of bee products.

The association between dysbiotic microbiota biofilm and colon cancer has recently begun to attract attention. In the study, the apitherapeutic effects of bee products (honey, bee venom, royal jelly, pollen, perga and propolis) obtained from the endemic Yigilca ecotype of Apis mellifera anatoliaca were investigated. Antibiofilm activity were performed by microplate assay using crystal violet staining to measure adherent biofilm biomass of Escherichia coli capable of forming biofilms. Bee venom showed the highest inhibition effect (73.98%) at 50% concentration. Honey, perga and royal jelly reduced biofilm formation by >50% at all concentrations. The antiproliferation effect on the HCT116 colon cancer cell line was investigated with the water­soluble tetrazolium salt­1 assay. After 48 h of honey application at 50% concentration, cell proliferation decreased by 86.51%. The high cytotoxic effects of royal jelly and bee venom are also remarkable. Additionally, apoptotic pathway analysis was performed by ELISA using caspase 3, 8 and 9 enzyme-linked immunosorbent assay kits. All bee products induced a higher expression of caspase 9 compared with caspase 8. Natural products that upregulate caspase proteins are promising therapeutic targets for proliferative diseases.

Hymenoptera venom immunotherapy: Safety and efficacy of an accelerated induction regimen with depot aluminum adsorbed extracts.

Introduction: Hymenoptera venom immunotherapy (VIT) is the only therapy that protects patients with Hymenoptera venom allergy by preventing systemic reactions after a new sting. Various extracts for VIT are available and used. VIT administration consists of an induction phase and a maintenance phase. Depot preparations of Hymenoptera VIT extracts are typically used for cluster and conventional protocols, and the maintenance phase. Many patients with Hymenoptera allergy need to achieve tolerance quickly because of the high risk of re-sting and possible anaphylaxis. Objective: Our study aimed to show the safety and efficacy of an accelerated regimen with depot preparations on aluminum hydroxide by using relatively high starting doses in a heterogeneous group of patients. Methods: The research focused on a group of patients with a history of severe systemic reactions to Hymenoptera stings, with the necessity of swift immunization due to high occupational risks. Aluminum hydroxide depot extracts either of Vepula species or Apis mellifera extracts were used. Results: The induction protocol was started with the highest concentration of depot venom extract of 100,000 standard quality unit and was well tolerated by 19 of 20 patients. Onne patient presented with a mild systemic reaction during the accelerated induction schedule, which was promptly treated with intravenous steroids and intramuscular H1 antihistamine; when switched to a conventional induction protocol, he had a similar reaction but finally reached maintenance with an H1-antagonist premedication. Conclusion: If validated, the accelerated induction protocol by using depot aluminum adsorbed extracts with the highest concentration of venom from the beginning could offer a streamlined and accessible treatment modality for patients diagnosed with anaphylaxis from bee and wasp venoms in need of rapid desensitization.

Prevalent bee venom genes evolved before the aculeate stinger and eusociality.

BACKGROUND: Venoms, which have evolved numerous times in animals, are ideal models of convergent trait evolution. However, detailed genomic studies of toxin-encoding genes exist for only a few animal groups. The hyper-diverse hymenopteran insects are the most speciose venomous clade, but investigation of the origin of their venom genes has been largely neglected. RESULTS: Utilizing a combination of genomic and proteo-transcriptomic data, we investigated the origin of 11 toxin genes in 29 published and 3 new hymenopteran genomes and compiled an up-to-date list of prevalent bee venom proteins. Observed patterns indicate that bee venom genes predominantly originate through single gene co-option with gene duplication contributing to subsequent diversification. CONCLUSIONS: Most Hymenoptera venom genes are shared by all members of the clade and only melittin and the new venom protein family anthophilin1 appear unique to the bee lineage. Most venom proteins thus predate the mega-radiation of hymenopterans and the evolution of the aculeate stinger.

Immunomodulatory activities and biomedical applications of melittin and its recent advances.

Melittin (MLT), a peptide containing 26 amino acids, is a key constituent of bee venom. It comprises ∼40%-60% of the venom's dry weight and is the main pricing index for bee venom, being the causative factor of pain. The unique properties of MLT extracted from bee venom have made it a very valuable active ingredient in the pharmaceutical industry as this cationic and amphipathic peptide has propitious effects on human health in diverse biological processes. It has the ability to strongly impact the membranes of cells and display hemolytic activity with anticancer characteristics. However, the clinical application of MLT has been limited by its severe hemolytic activity, which poses a challenge for therapeutic use. By employing more efficient mechanisms, such as modifying the MLT sequence, genetic engineering, and nano-delivery systems, it is anticipated that the limitations posed by MLT can be overcome, thereby enabling its wider application in therapeutic contexts. This review has outlined recent advancements in MLT's nano-delivery systems and genetically engineered cells expressing MLT and provided an overview of where the MLTMLT's platforms are and where they will go in the future with the challenges ahead. The focus is on exploring how these approaches can overcome the limitations associated with MLT's hemolytic activity and improve its selectivity and efficacy in targeting cancer cells. These advancements hold promise for the creation of innovative and enhanced therapeutic approaches based on MLT for the treatment of cancer.

Impact of bee venom supplement on productive performance, health status and economics of weaned male rabbits: Considering breed and dosage factors.

The objectives of the present study were to investigate the potential effects of purified bee venom (BV) on various aspects of growth, carcass, antioxidant, intestinal bacterial count and economic considerations in rabbits. A total of 240 male rabbits, comprising two distinct breeds (V-Line and New Zealand White [NZW]), 5 weeks old, with an average live body weight (BW) of 680 ± 20 g, were randomly divided into six groups, each containing 30 rabbits. Each group had five replicates, with six rabbits in each replicate. The allocation of animals to the groups followed a fully factorial design, incorporating two factors: breed (V-Line and NZW) and four levels of purified BV derived from Apis Mellifera. The control group (G1) received a basal diet without additives. The other three groups (G2, G3 and G4) received the basal diet with BV supplementation in their drinking water at 0.5, 1 and 2 mg/L respectively. The study results indicated that NZW rabbits significantly enhanced feed conversion ratio while maintaining consistent carcass attributes compared to the V-Line breed. Despite variations in growth parameters being less pronounced, the supplementation of BV at levels of 1-2 mg/L demonstrated significant improvements in various other parameters. Notably, the interaction between the BV supplement and the breed factor (p < 0.001) yielded notable distinctions in most production metrics, encompassing BW, weight gain, feed conversion, carcass attributes and blood parameters. Increasing levels of BV supplementation, particularly at 1 mg/L, led to substantial improvements in serum and tissue metabolites. Moreover, the levels of total bacterial count and Escherichia coli in the jejunum and colon were significantly diminished, while the population of Lactobacilli in the colon was augmented (p < 0.001) in rabbits from both breeds receiving BV supplementation (1-2 mg/L) compared to the control group. The results underscore the potential of the BV supplement to enhance final weights, bolster antioxidant status and mitigate the presence of pathogenic bacteria, thereby contributing to enhanced economic efficiency in rabbits. Further inquiries are warranted to comprehensively investigate BV supplementation's potential advantages and limitations across different breeds and dosage levels.

Allergy to stings and bites from rare or locally important arthropods: Worldwide distribution, available diagnostics and treatment.

Insect venom allergy is the most frequent cause of anaphylaxis in Europe and possibly worldwide. The majority of systemic allergic reactions after insect stings are caused by Hymenoptera, and among these, vespid genera induce most of the systemic sting reactions (SSR). Honey bees are the second leading cause of SSR. Depending on the global region, other Hymenoptera such as different ant genera are responsible for SSR. Widely distributed hornets and bumblebees or local vespid or bee genera rarely induce SSR. Hematophagous insects such as mosquitoes and horse flies usually cause (large) local reactions while SSR occasionally occur. This position paper aimed to identify either rare or locally important insects causing SSR as well as rarely occurring SSR after stings or bites of widely distributed insects. We summarized relevant venom or saliva allergens and intended to identify possible cross-reactivities between the insect allergens. Moreover, we aimed to locate diagnostic tests for research and routine diagnosis, which are sometimes only regionally available. Finally, we gathered information on available immunotherapies. Major allergens of most insects were identified, and cross-reactivity between insects was frequently observed. While some diagnostics and immunotherapies are locally available, standardized skin tests and immunotherapies are generally lacking in rare insect allergy.

Skin Tests versus Serology: Specific IgE May Suffice for Diagnosis of Vespid Venom Allergy and Follow-Up of Allergen Immunotherapy.

INTRODUCTION: In adults, allergic reactions to insect stings are among the most frequent causes of anaphylaxis, a potentially life-threatening condition. Recurrent anaphylaxis following vespid stings may be prevented by allergen immunotherapy (AIT). The aim of this study was to evaluate the benefit of measuring venom-induced wheal area in intracutaneous skin tests (ICT), in comparison to various serological and clinical parameters, for the diagnosis of severe vespid venom allergy and during follow-up of AIT. METHODS: We conducted a monocentric, retrospective evaluation of 170 patients undergoing AIT against vespid venoms. We scanned ICT wheals at baseline and at three time points after AIT initiation and measured wheal area using objective data analysis software. RESULTS: We found that ICT histamine-induced and venom-induced wheal areas did not correlate. In addition, the venom-induced wheal area was independent from the minimal venom concentration required to elicit a wheal in an ICT and all other parameters. No correlation was found between wheal area and the severity of anaphylaxis. Wheal area standardized to the application of 0.1 µg/mL venom inversely correlated with anaphylaxis severity and positively correlated with venom-specific IgE levels. During AIT, mean areas of venom-induced wheals did not change. In contrast, venom-specific IgG and IgG4 levels, and the minimal venom concentration required to induce a positive ICT result increased, while the venom wheal area standardized to 0.1 µg/mL venom application and specific IgE levels decreased over time. CONCLUSION: Wheal area evaluation did not provide additional information over specific IgE analysis. We therefore recommend that ICTs are used only as a secondary measure for confirming serological test results.

Wings and stings: Hymenoptera on vacation.

Traveling to different regions, one might encounter a species to which they have a known allergy, or other related and unrelated species. A first-time systemic reaction can occur while on vacation, even in those with previous asymptomatic stings. Three main groups of Hymenoptera are responsible for most sting reactions. Honey bee species are virtually identical around the world. Among social wasps (family Vespidae), the yellowjacket (genus Vespula and Dolichovespula) and hornet (genus Vespa) venoms have almost complete cross-reactivity, whereas paper wasp (genus Polistes) venoms show only partial cross-reactivity with other vespid venoms. Venom immunotherapy (VIT) confers 80% to 95% protection against related insects, though isolated species of paper wasps and yellowjackets exist in every country that may be distinct from the ones at home. Those allergic to imported fire ants (genus Solenopsis) in the United States should not react to other ant species around the world. Stinging ants belong to several unrelated subfamilies in different geographic regions, which do not have cross-reactive venom. The chances of encountering specific species of Hymenoptera at a traveler's destination vary by location, planned activities, and season. In this article, we discuss special considerations for traveling, including distribution of stinging insects around the world, risk factors for more severe reactions, ways to prepare for a trip, and when allergist examination or treatment may be helpful before travel.

Hymenoptera venom skin testing: Adopting an accelerated test protocol.

BACKGROUND: The standard method of Hymenoptera venom intradermal skin test is performed at a starting concentration of 0.001 to 0.01 µg/mL and increased by 10-fold concentrations until positive or a maximum concentration of 1 µg/mL. Accelerated methods that start at higher concentrations have been reported as safe; however, many institutions have not adopted this approach. OBJECTIVE: To compare the outcome and safety of standard and accelerated venom skin test protocols. METHODS: This was a retrospective chart review of patients with suspected venom allergy who underwent skin testing at 4 allergy clinics within a single health care system from 2012 to 2022. Demographic data, test protocol (standard vs accelerated), test results, and adverse reactions were reviewed. RESULTS: Of 134 patients who underwent standard venom skin test, 2 (1.5%) experienced an adverse reaction, whereas none of the 77 patients who underwent accelerated venom skin test experienced an adverse reaction. One patient, with a history of chronic urticaria, experienced urticaria. The other experienced anaphylaxis requiring an epinephrine although had tested negative to all venom concentrations. Within the standard testing protocol, more than 75% of the positive results occurred at concentrations of 0.1 or 1 µg/mL. Within the accelerated testing protocol, more than 60% of the positive results occurred at 1 µg/mL. CONCLUSION: The study underscores the overall safety of venom intradermal skin test. Most of the positive results occurred at 0.1 or 1 µg/mL. Adopting an accelerated approach would reduce time and cost associated with testing.

Investigation the effects of bee venom and H-dental-derived mesenchymal stem cells on non-small cell lung cancer cells (A549).

BACKGROUND: Lung cancer, one of the most common oncological diseases worldwide, continues to be the leading cause of cancer-related deaths. The development of new approaches for lung cancer, which still has a low survival rate despite medical advances, is of great importance. METHODS AND RESULTS: In this study, bee venom (BV), conditioned medium of MSCs isolated from dental follicles (MSC-CM) and cisplatin were applied at different doses and their effects on A549 cell line were evaluated. Dental follicles were used as a source of MSCs source and differentiation kits, and characterization studies (flow cytometry) were performed. Cell viability was measured by the MTT method and apoptosis was measured by an Annexin V-FITC/PI kit on flow cytometer. IC50 dose values were determined according to the 24th hour and were determined as 15.8 µg/mL for BV, 10.78% for MSC-CM and 5.77 µg/mL for cisplatin. IC50 values found for BV and MSC-CM were also given in combination and the effects were observed. It was found that the applied substances caused BV to decrease in cell viability and induced apoptosis in cells. In addition to the induction of apoptosis in BV, MSC-CM, and combined use, all three applications led to an increase in Bax protein expression and a decrease in Bcl-2 protein expression. The molecular mechanism of anticancer activity through inhibition of Bax and Bcl-2 proteins and the NF-κB signaling pathway may be suggested. CONCLUSION: Isolated MSCs in our study showed anticancer activity and BV and MSC-CM showed synergistic antiproliferative and apoptotic effects.

Determination of the Effects of Bee Venom on Triple Negative Breast Cancer Cells in Vitro.

Honeybees provide multiple products such as bee venom (BV) which are used for various nutritional and medicinal purposes. BV has received great attention due to its wide range of bioactive components with potential anti-cancer effects on different cancers. Triple negative breast cancer (TNBC) is defined as an aggressive type of breast cancer and new therapeutic targets are required for its treatment. In the current literature information is varied about the composition and quantity of BV bioactive compounds as well as the origin of BV and its significance. In this context, the cytotoxic and apoptotic effects of BV with a higher rate of mellitin from Apis mellifera anatoliaca (Mugla ecotype) on MDA-MB-231 cells was evaluated, in vitro. The cytotoxic, apoptotic and morphological effects of BV were determined by WST-1, Annexin V, cell cycle analysis and Acridine Orange staining. The results showed that BV caused apoptotic cell death in TNBC cells at a lower dose (0.47 µg/mL, p<0.01). This study suggests that BV could be developed as a potential therapeutic agent for cancer treatment. However, the mechanism of BV-induced apoptosis death should be clarified at the molecular level.

Bee Venom Induces the Interaction between Phosphorylated Histone Variant, H2AX, and the Intracellular Site of beta-Actin in Liver and Breast Cancer Cells.

Bee venom is a natural mixture and candidate anti-cancer agent with selective cytotoxic effect on some cancer cells. However, the cellular mechanisms of how bee venom selectively targets cancer cells remain elusive. The aim of this study was to reveal the genotoxic effect of bee venom in concordance with the location of ß-actin protein throughout the nucleus or/and cytoplasm. For this aim, the level of H2AX phosphorylation (γH2AX) and intracellular location of ß-actin were assessed by immunofluorescence in liver (HEPG2) and metastatic breast (MDA-MB-231) cancer cell lines compared to normal fibroblasts (NIH3T3) after bee venom treatment. Colocalisation profiles of γH2AX and ß-actin in each cell line were also analysed. The results showed that the levels of γH2AX staining decreased in normal cells but increased in cancer cells. The majority of ß-actin was localised within the cytoplasm of normal cells after bee venom treatment, but it was mostly accumulated within the nucleus in cancer cells. Colocalisation of ß-actin and γH2AX both in nucleus and cytoplasm was induced in each cancer cell by different patterns. The results showed that normal and cancerous cells had different responses against bee venom, and suggested that bee venom induced a cellular response by the interaction between γH2AX and ß-actin.

Extracorporeal membrane oxygenation support in refractory anaphylactic shock after bee stings: A case report.

An allergy to insect stings is one of the most frequent causes of anaphylactic reactions. Such reactions can be fatal, even on the first reaction, although it very rarely happens. The use of veno-arterial extracorporeal membrane oxygenation (VA ECMO) in refractory anaphylactic shock was previously described. We report a case of a 31-year-old female who presented with refractory anaphylactic shock after bee stings without the presence of cutaneous manifestations other than the rashes in her neck. The toxic component of bee venom and systemic allergic response plays a vital role in pathophysiology. She did not respond to conventional advanced life support, but following urgent VA ECMO, she survived neurologically intact. Despite an uncommon indication for anaphylaxis, ECMO support may be possible and effective in patients with refractory shock.

Modified rush venom immunotherapy in dogs with Hymenoptera hypersensitivity.

BACKGROUND: Hymenoptera envenomation occurs frequently in people and dogs and can trigger anaphylaxis. Venom immunotherapy (VIT) is the only preventive treatment for Hymenoptera hypersensitivity and is indicated for people with severe adverse reactions to insect stings. Rush VIT is an accelerated VIT protocol in people. This has not been reported in dogs. OBJECTIVES: The objective of the study was to evaluate the safety of modified rush VIT. ANIMALS: Twenty client-owned dogs with Hymenoptera hypersensitivity based on a history of adverse reactions to Hymenoptera envenomation and a positive intradermal test to honey bee and/or paper wasp venom. MATERIALS AND METHODS: Dogs received incremental doses of venom via subcutaneous injection one day per week for three consecutive weeks until the maintenance dose was achieved. Vital signs were recorded every 30 min prior to venom administration. Adverse reactions were categorised as localised or grade I-IV systemic reactions. RESULTS: Nineteen of 20 dogs (95%) completed rush VIT. One dog experienced a grade III systemic adverse reaction and was withdrawn from the study. No adverse reactions occurred in 10 of 20 dogs (50%). Localised and grade I-II systemic reactions occurred in nine of 20 dogs (45%), including nausea (n = 5), injection site pruritus (n = 3) and diarrhoea and lethargy (n = 1). CONCLUSIONS AND CLINICAL RELEVANCE: Modified rush VIT in dogs was well-tolerated and should be considered for dogs with Hymenoptera hypersensitivity. Larger studies are needed to evaluate the efficacy of VIT in dogs for preventing hypersensitivity reactions to insect stings.