Calcium bicarbonate as an antimicrobial, antiviral, and prion-inhibiting agent (Review).

Calcium bicarbonate does not act as a disinfectant at neutral pH; however, it exerts strong antimicrobial activity after it is placed in a high-voltage electric field, whereby it assumes an alkaline pH (12.4). Moreover, the microbicidal activity of the resulting solution (named CAC-717) is not influenced by the presence of organic material or resistance of the agent to inactivation. When sprayed on the skin surface, the pH of CAC-717 decreases rapidly to 8.84. CAC-717 comprises fine particles of 50-500 nm. When these mesoscopic crystals are dissolved in water, they destroy the genomes of bacteria or viruses and neutralize the infectious properties of abnormal prion proteins produced in ScN2a cells. The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic has resulted in unprecedented international demand for disinfectants. A small titer of SARS-CoV-2 remains infectious even after 30 sec in growth medium at pH 12.4. CAC-717 has exhibited a strong virucidal effect (3.6 to 4.4 log10 decrease) against all examined SARS-CoV-2 isolates, including mutant forms. Similarly, human noroviruses also remain intact at pH 12.4; however, CAC-717 has been shown to cause a 3.25 log10 reduction in norovirus genomic RNA compared to untreated samples. Existing evidence suggests that an unidentified mechanism controls the virucidal activity of CAC-717.

Heterogeneous IgE reactivities to Staphylococcus pseudintermedius strains in dogs with atopic dermatitis, and the identification of DM13-domain-containing protein as a bacterial IgE-reactive molecule.

Staphylococcus pseudintermedius is one of the major pathogens causing canine skin infection. In canine atopic dermatitis (AD), heterogeneous strains of S. pseudintermedius reside on the affected skin site. Because an increase in specific IgE to this bacterium has been reported, S. pseudintermedius is likely to exacerbate the severity of canine AD. In this study, the IgE reactivities to various S. pseudintermedius strains and the IgE-reactive molecules of S. pseudintermedius were investigated. First, examining the IgE reactivities to eight strains of S. pseudintermedius using 141 sera of AD dogs, strain variation of S. pseudintermedius showed 10-63% of the IgE reactivities. This is different from the expected result based on the concept of Staphylococcus aureus clonality in AD patients. Moreover, according to the western blot analysis, there were more than four proteins reactive to IgE. Subsequently, the analysis of the common IgE-reactive protein at ∼15 kDa confirmed that the DM13-domain-containing protein was reactive in AD dogs, which is not coincident with any S. aureus IgE-reactive molecules. Considering these, S. pseudintermedius is likely to exacerbate AD severity in dogs, slightly different from the case of S. aureus in human AD.

IgE reactivity to milk components in dogs with cutaneous adverse food reactions.

We investigated the IgE reactivity to crude and purified milk antigens in the sera of 112 dogs with cutaneous adverse food reactions (CAFRs). Of the 112 dogs, 33 (29%) had specific IgE for crude milk antigens. In the dogs with milk-specific IgE, IgE reactivity to casein, bovine serum albumin (BSA), α-lactalbumin, ß-lactoglobulin, and bovine IgG were 81%, 85%, 39%, 27%, and 35%, respectively. Casein and BSA may be important allergens in dogs with CAFRs. Some canine vaccines contain casein hydrolysate as a stabilizer and the pooled serum with anti-casein IgE showed IgE reactivity to the vaccines containing it. Information about IgE reactivity to casein in dogs with CAFRs could be useful for predicting adverse reactions to the vaccines including casein hydrolysate.

Results of food challenge in dogs with cutaneous adverse food reactions.

BACKGROUND: Elimination diet trials and food challenge tests remain the gold standard for diagnosing cutaneous adverse food reaction (CAFR) in dogs. However, food challenge tests are difficult to perform owing to a lack of definite information on expected clinical signs and the time frame within which they develop after challenge. OBJECTIVES: To report the results of the food challenge test carried out in client-owned dogs under observation. METHODS AND MATERIALS: A retrospective evaluation of the medical records of dogs presenting with pruritus was performed. Skin infections and/or parasites were treated before inclusion in the study. Dogs that underwent an elimination diet with clinical improvement and relapse with food challenge tests were included in this study. A pruritus Visual Analog scale score was recorded as an index of pruritus before and at the end of the elimination diet trial before challenge. The onset of time to relapse of clinical signs and the body sites affected after food challenge testing were recorded by the owners. RESULTS: We examined the medical records of 310 dogs with pruritus. Of these, 46 dogs with a diagnosis of CAFR were enrolled in this study. Significant clinical improvement was observed after the elimination diet trial (P < 0.0001). All dogs exhibited pruritus, and none exhibited anaphylaxis after food challenge testing. Among these dogs, 60.9% developed pruritus within 12 h of food challenge. Notably, 23.9% of the dogs developed pruritus within 3-6 h. The limbs exhibited the highest frequency of pruritus (56.5%), followed by the face (26.1%). CONCLUSIONS AND CLINICAL RELEVANCE: This study is the first to report details of the time to onset of clinical signs and body site affected after food challenge testing in dogs. These results will assist veterinarians and dog owners in the decision-making process regarding food challenge testing.

Association analysis of non-synonymous polymorphisms of interleukin-4 receptor-α and interleukin-13 genes in canine atopic dermatitis.

Interleukin-4 (IL4) and interleukin-13 (IL13) are involved in the initial response of T helper 2 lymphocytes through the activation of the IL4 receptor alpha (IL4RA), which is a common receptor chain for these cytokines. In humans, several single-nucleotide polymorphisms (SNPs) identified in the IL4R and in interleukin coding genes were associated with atopic disorders. However, the association between canine IL4R polymorphisms and atopic disorders has not been investigated yet. This study aimed to determine the associations between four non-synonymous SNPs and canine atopic dermatitis (CAD) in shiba inu and miniature dachshund populations. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis were used to genotype four polymorphisms of canine IL4R and IL13 in 34 shiba inu and 19 miniature dachshund patients with CAD, as well as 29 shiba inu and 39 miniature dachshund patients without the condition. Results from miniature dachshunds revealed a potential association between the presence of minor A allele rs24378020 and CAD (odds ratio, 0.10; 95% confidence interval, 0.01-0.85; Poriginal=0.0062). This CAD resistance allele led to an amino acid substitution (Arg688Cys) that could impair IL4 and IL13 signaling. In shiba inu patients, rs24378020 was fixed by homozygosity of the major G allele. No association was found between the remaining three evaluated SNPs and CAD. Nevertheless, the study suggests that the IL4R Cys688 variant reduces the risk of CAD in miniature dachshunds.

IgE sensitivity to Malassezia pachydermatis and mite allergens in dogs with atopic dermatitis.

In this study, dogs with atopic dermatitis were separated into non-food-induced atopic dermatitis (NFIAD) group (n = 15) and food-induced atopic dermatitis (FIAD) group (n = 37) based on an elimination diet test. IgE reactivity for crude Malassezia pachydermatis (M. pachydermatis) and house dust mites (HDM) allergen extracts was investigated in the two groups using fluorometric enzyme-linked immunosorbent assay (ELISA) and intradermal skin test (IDST). Nine (60%) of the 15 dogs in NFIAD group and 6 (16%) of the 37 dogs in FIAD group showed specific IgE for M. pachydermatis (Mann-Whitney U-test, P < 0.01). By immunoblotting analysis, the pooled serum samples from dogs with IgE for M. pachydermatis showed IgE reactivity for 50 kDa protein of M. pachydermatis. Twelve (80%) of the 15 dogs in NFIAD group and 8 (22%) of the 37 dogs in FIAD group showed specific IgE for HDM (Mann-Whitney U-test, P < 0.01). In addition, the dogs in NFIAD group significantly show a positive IDST to M. pachydermatis and HDM extracts compared with the dogs in FIAD group. The results suggest that dogs with NFIAD are at increased risk of becoming sensitized to the normal commensal organism M. pachydermatis compared with dogs with FIAD, perhaps co-sensitization occurred due to an HDM protease antigen's, Der f 1 and/or Der p 1, proteolytic activity related epidermal skin barrier defects. Treatment to limit skin colonization may thus be especially important in NFIAD.

Inactivation of human norovirus and its surrogate by the disinfectant consisting of calcium hydrogen carbonate mesoscopic crystals.

Human norovirus is one of the major causes of foodborne gastroenteritis, and it can be easily transmitted from infected person, virus-contaminated foods and environmental surfaces. Effective disinfection method is needed to stop the transmission of human norovirus. CAC-717 is a new disinfectant consisting of calcium hydrogen carbonate mesoscopic crystals. We aimed to evaluate the efficacy of CAC-717 against human norovirus. This study used human norovirus derived from fecal specimens and cultured murine norovirus, which is one of the surrogate viruses for human norovirus. The disinfection effect against murine norovirus was estimated by infectivity assay and transmission electron microscopy. The inactivation effect against human norovirus was assessed by reverse transcription polymerase chain reaction. Disinfection effect of CAC-717 against the infectivity of murine norovirus was shown within 100 s after the CAC-717 treatment, presenting the destruction of viral capsids. The treatment of CAC-717 significantly reduced human norovirus genomic RNA (3.25-log reduction) by the presence of the mesoscopic structure of calcium hydrogen carbonate. CAC-717 stably inactivated human norovirus in stool suspensions. The inactivation effect of CAC-717 against human norovirus was less susceptible to organic substances than sodium hypochlorite. CAC-717 would be a useful alternative for disinfecting human norovirus in contaminated environmental surfaces.

Identification of bovine papillomavirus type 1 and 2 from bovine anogenital fibropapillomas.

Papillomavirus (PV) is a well-known pathogen associated with epithelial and mucosal neoplastic diseases. In contrast to human PVs, characterization of animal PVs from the aspect of anogenital neoplasm is still on a learning curve. In the present study, two vulval and one anal warts, histologically diagnosed as fibropapillomas, excised from dairy cattle were analyzed. PCR and sequencing revealed that bovine papillomavirus type 1 (BPV-1) and BPV-2 were detected from anal and vulval fibropapillomas, respectively. Immunohistochemistry detected PV antigen in a few differentiated keratinocytes of one vulval case. Reverse-transcriptase PCR detected the early region, but not the late region of BPV mRNA in all three cases. The present study will provide new insight into the relationship between BPV and anogenital papilloma in cattle.

Spontaneous regression of canine papillomavirus type 2-related papillomatosis on footpads in a dog.

Persistent papillomatosis on footpads related to canine papillomavirus type 2 (CPV-2) infection has been described in dogs with immunocompromised condition. A 9-year-old, male French bulldog was presented with cauliflower-like nodules on the footpads of his left front leg. Histopathological examination revealed multiple finger-like projections of squamous epithelium with intranuclear inclusion bodies. Immunohistochemistry using an anti-bovine papillomavirus antibody demonstrated immunostaining in the keratinocytes. Partial genome DNA of CPV-2 was amplified from the lesion. Full genome sequence of CPV-2 in the subject showed 99.95% nucleotide identity with that of CPV-2 from the reference data. Two weeks after a biopsy, the skin lesion spontaneously regressed without any specific treatment. In non-immunocompromised dogs, CPV-2-related footpad papillomatosis could spontaneously resolve after a biopsy.

Full genome analysis of bovine papillomavirus type 1 derived from a calf with severe cutaneous multiple papillomatosis.

Severe papillomatosis occasionally causes astasia leading to euthanizing cattle. There are currently a limited number of reports on virologic approach in severe bovine papillomatosis. Here we report a full genome characterization of bovine papillomavirus type 1 (BPV-1) from the case of severe papillomatosis. A calf developed numerous papillomas on the skin and some nodules in the upper gastrointestinal tract at seven months old. The skin lesion was diagnosed as the epithelial papilloma with BPV antigen expression, while the gastrointestinal lesions were diagnosed as the fibropapilloma without BPV antigen. Full genome analysis revealed that BPV-1s detected in all the lesions were exactly the same. Compared with the reference BPV-1 sequence, there was a single nucleotide insertion in the upstream regulatory region.

Detection of felis catus papillomavirus type 3 and 4 DNA from squamous cell carcinoma cases of cats in Japan.

Association of felis catus papillomaviruses (FcaPVs) with feline squamous cell carcinoma (SCC) has been reported worldwide, while there is limited information about FcaPVs in Asia. In this study, 21 feline SCC biopsy samples from cats in Japan were analyzed by PCR with PV consensus primers and type-specific primers for FcaPV type 2 (FcaPV-2), FcaPV-3 and FcaPV-4 designed in this study. Sequence analysis revealed that one sample was FcaPV-3, and two were FcaPV-4. In both FcaPV-4 positive samples, 334th tryptophan in L1 ORF was deleted compared with the reference sequence. Moreover, immunohistochemistry showed that p16 protein was positive in both FcaPV-4 detected samples. This study would contribute to the molecular epidemiological and pathological understanding of FcaPV in Japan.

Adsorption of Staphylococcus viruses S13' and S24-1 on Staphylococcus aureus strains with different glycosidic linkage patterns of wall teichoic acids.

The group of phages belonging to the family Podoviridae, genus P68virus, including Staphylococcus viruses S13' and S24-1, are important because of their benefits in phage therapy against Staphylococcus aureus infections. The O-glycosidic linkage patterns of wall teichoic acids (WTAs) in S. aureus cell walls seem to be important for adsorption of this phage group. In this study, the adsorption of Staphylococcus viruses S13' and S24-1 to S. aureus was examined using strains with modified WTA glycosidic linkage patterns. We found that the ß-O-N-acetylglucosamine of WTAs was essential for S13' adsorption, while N-acetylglucosamine, regardless of the α- and ß-O-glycosidic linkages of the WTAs, was essential for S24-1 adsorption. Next, examining the binding activities of their receptor-binding proteins (RBPs) to cell walls with different WTA glycosidic patterns, the ß-O-N-acetylglucosamine of the WTAs was essential for S13' RBP binding, while N-acetylglucosamine, regardless of the α- and ß-O-glycosidic linkages of the WTAs, was essential for S24-1 RBP binding. Therefore, the results of the RBP binding assays were consistent with those of the phage adsorption assays. Bioinformatic analysis suggested that the RBPs of Staphylococcus viruses S13' and S24-1 were structurally similar to the RBPs of phage phi11 of thefamily Siphoviridae. Phylogenetic analysis of the RBPs indicated that two phylogenetic subclusters in the family Podoviridae were related to the glycosidic linkage patterns required for phage adsorption, possibly mediated by RBPs. We hope that this study will encourage the future development of therapeutic phages.

Screening of KHP30-like prophages among Japanese Helicobacter pylori strains, and genetic analysis of a defective KHP30-like prophage sequence integrated in the genome of the H. pylori strain NY40.

We have recently reported the active Helicobacter pylori bacteriophages (phages), KHP30 and KHP40, the genomic DNAs of which exist as episomes in host bacterial strains isolated in Japan (i.e. pseudolysogeny). In this study, we examined the possibility of the lysogeny of active KHP30-like phages in Japanese H. pylori strains, because their genomes contain a putative integrase gene. Only the NY40 strain yielded partial detection of a KHP30-like prophage sequence in PCR among 174 Japanese H. pylori isolates, except for strains producing the above active phages. Next, according to the genomic analysis of the NY40 strain, the KHP30-like prophage sequence was found to be located from ca. 524 to 549 kb in the host chromosome. The attachment sites, attL and attR, in the NY40 genome showed almost the same genomic location and sequence as those detected in a French isolate B38, suggesting that an active parental KHP30-like phage had integrated into the ancestral NY40 genome in a site-specific manner. The prophage found in the NY40 genome was assumed to have been genetically modified, after site-specific integration. These, together with the data in the KHP30-like prophages of other H. pylori genomes, suggest that the lysogenic state of the KHP30-like phages is generally unstable.

IgE reactivity to hen egg white allergens in dogs with cutaneous adverse food reactions.

Dogs with cutaneous adverse food reactions (CAFR) often have specific IgE to food allergens. Egg white, which is majorly composed of ovomucoid, ovalbumin, ovotransferrin, and lysozyme, is a food allergen in dogs. Information of the IgE reactivity to purified egg white allergens supports accurate diagnosis and efficiency treatment in humans. However, to the best of our knowledge, there have been no studies on the IgE reactivity to purified egg white allergens in dogs. Here, we investigated the IgE reactivity to crude and purified allergens of hen egg white in dogs with CAFR. First, when we examined serum samples from 82 dogs with CAFR for specific IgE to crude egg white by ELISA, 9.8% (8/82) of the dogs with CAFR showed the IgE reactivity to crude egg white. We then used sera from the eight dogs with positive IgE reactivity to crude egg white to examine the IgE reactivity to four purified allergens, ovomucoid, ovalbumin, ovotransferrin, and lysozyme, by ELISA. We found that 75% (6/8) of the dogs showed IgE reactivity to both ovomucoid and ovalbumin, and that 37.5% (3/8) of the dogs showed IgE reactivity to ovotransferrin. None (0/8) showed IgE reactivity to lysozyme. Moreover, validating these results, the immunoblot analyses were performed using the sera of the three dogs showing the highest IgE reactivity to crude egg white. Both anti-ovomucoid and anti-ovalbumin IgE were detected in the sera of these dogs, while anti-ovotransferrin IgE was not detected. Considering these, ovomucoid and ovalbumin appears to be the major egg white allergens in dogs with CAFR.

Analyses of Short-Term Antagonistic Evolution of Pseudomonas aeruginosa Strain PAO1 and Phage KPP22 (Myoviridae Family, PB1-Like Virus Genus).

UNLABELLED: Pseudomonas aeruginosa causes serious intractable infections in humans and animals. Bacteriophage (phage) therapy has been applied to treat P. aeruginosa infections, and phages belonging to the PB1-like virus genus in the Myoviridae family have been used as therapeutic phages. To achieve safer and more effective phage therapy, the use of preadapted phages is proposed. To understand in detail such phage preadaptation, the short-term antagonistic evolution of bacteria and phages should be studied. In this study, the short-term antagonistic evolution of bacteria and PB1-like phage was examined by studying phage-resistant clones of P. aeruginosa strain PAO1 and mutant PB1-like phages that had recovered their infectivity. First, phage KPP22 was isolated and characterized; it was classified as belonging to the PB1-like virus genus in the Myoviridae family. Subsequently, three KPP22-resistant PAO1 clones and three KPP22 mutant phages capable of infecting these clones were isolated in three sets of in vitro experiments. It was shown that the bacterial resistance to phage KPP22 was caused by significant decreases in phage adsorption and that the improved infectivity of KPP22 mutant phages was caused by significant increases in phage adsorption. The KPP22-resistant PAO1 clones and the KPP22 mutant phages were then analyzed genetically. All three KPP22-resistant PAO1 clones, which were deficient for the O5 antigen, had a common nonsense mutation in the wzy gene. All the KPP22 mutant phage genomes showed the same four missense mutations in the open reading frames orf060, orf065, and orf086 The information obtained in this study should be useful for further development of safe and efficient phage therapy. IMPORTANCE: Pseudomonas aeruginosa causes serious intractable infections in humans and animals; bacteriophage (phage) therapy has been utilized to treat P. aeruginosa infections, and phages that belong to the PB1-like virus genus in the family Myoviridae have been used as therapeutic phages. The preadapted phage is trained in advance through the antagonistic evolution of bacteria and phage and is proposed to be used to achieve safer and more effective phage therapy. In this study, to understand the phage preadaptation, the in vitro short-term antagonistic evolution was studied using P. aeruginosa strain PAO1 and the newly isolated PB1-like phage KPP22. Phage KPP22 was characterized, and the molecular framework regarding the phage preadaptation of KPP22 was elucidated. The importance of study of antagonistic evolution of bacteria and phage in phage therapy is discussed.

Prevalence of food-responsive enteropathy among dogs with chronic enteropathy in Japan.

There have been limited reports on the prevalence of adverse food reactions among dogs suffering from chronic enteropathy (CE) in Japan. We examined the prevalence and histological features of food-responsive enteropathy (FRE) in a total of 32 dogs with history of CE. Fourteen of 18 cases (56.2%) diagnosed as FRE had lymphocytic-plasmacytic enteritis or eosinophilic enteritis by histopathological examination. Characteristic histopathological changes indicating FRE were not identified in 18 cases, though 4 cases did not show any abnormalities. Results collected from this study provided important information that can help to change the way dogs with CE are treated in the future.

Cross-reactivity between major IgE core epitopes on Cry j 2 allergen of Japanese cedar pollen and relevant sequences on Cha o 2 allergen of Japanese cypress pollen.

BACKGROUND: Cry j 2 and Cha o 2 are major allergens in Japanese cedar (Cryptomeria japonica; CJ) and Japanese cypress (Chamaecyparis obtusa; CO) pollen, respectively. Here, we assessed the epitopes related to the cross-reactivity between Cry j 2 and Cha o 2 using in vitro analyses. METHODS: Peptides were synthesized based on Cry j 2 sequential epitopes and relevant Cha o 2 amino acid sequences. Four representative monoclonal antibodies (mAbs) against Cry j 2 were used according to their epitope recognitions. Serum samples were collected from 31 patients with CJ pollinosis. To investigate cross-reactivity between Cry j 2 and Cha o 2, ELISA and inhibition ELISA were performed with mAbs and sera from patients with CJ pollinosis. RESULTS: Two of four mAbs had reactivity to both Cry j 2 and Cha o 2. Of these two mAbs, one mAb (T27) recognized the amino acid sequence (169)KVVNGRTV(176) on Cha o 2. This is related to the core epitope (169)KWVNGREI(176) on Cry j 2, which is an important IgE epitope. In addition, we found that these correlative sequences and purified allergens showed cross-reactivity between Cry j 2 and Cha o 2 in IgE of CJ patients. CONCLUSIONS: We demonstrated the importance of (169)KVVNGRTV(176) in Cha o 2 for cross-reactivity with the Cry j 2 epitope (169)KWVNGREI(176), which plays an important role in allergenicity in CJ pollinosis. Our results are useful for the development of safer and more efficient therapeutic strategies for the treatment of CJ and CO pollen allergies.

Characterization of Pseudomonas aeruginosa phage KPP21 belonging to family Podoviridae genus N4-like viruses isolated in Japan.

Bacteriophages (phages) belonging to the family Podoviridae genus N4-like viruses have been used as therapeutic agent in phage therapy against Pseudomonas aeruginosa infections. P. aeruginosa phage KPP21 was isolated in Japan, and phylogenetically investigated the phages belonging to this viral genus. Morphological and genetic analyses confirmed that phage KPP21 belongs to the family Podoviridae genus N4-like viruses. Moreover, phylogenetic analyses based on putative DNA polymerase and major virion protein showed that P. aeruginosa phages belonging to the genus N4-like viruses are separated into two lineages and that phage KPP21 is in the same clade as phage LUZ7.

Analysis of conformational and sequential IgE epitopes on the major allergen Cry j 2 of Japanese cedar (Cryptomeria japonica) pollen in humans by using monoclonal antibodies for Cry j 2.

PURPOSE: Japanese cedar (Cryptomeria japonica; CJ) pollinosis is a type I allergy induced by CJ pollen, and Cry j 2 is one of the major allergens in this pollen. In a previous study, we analyzed IgE epitopes on Cry j 2 in humans by using synthetic peptides. The main purpose of this study was to identify B-cell epitopes on Cry j 2 in patients with CJ pollinosis by using monoclonal antibodies (mAbs) for Cry j 2. METHODS: We used ELISA with mAbs for the epitope analysis. Sera samples were collected from 80 patients with CJ pollinosis, and allergenic epitopes for mAbs and human IgE were identified using ELISA with synthetic peptides. The importance of the epitopes for human IgE was analyzed using an inhibition ELISA. RESULTS: Four independent epitopes (epitope #1, #2, #3, and #4) were identified on Cry j 2 with the use of mAbs. Epitope #3 and #4, corresponding to peptides No. 25 and No. 33, respectively, were newly determined as epitopes for mAbs and human IgE. Inhibition ELISA showed that not only epitope #2 (sequential) but epitope #1 (conformational) may play an important role in the CJ pollinosis. CONCLUSIONS: Our results revealed 4 epitopes, including two new ones, on Cry j 2. We also found that inhibition ELISA with appropriate mAbs could be a viable method of evaluating the importance of the conformational and sequential epitopes for human IgE. These results are beneficial for the development of safer and more efficient therapeutic strategies for treating CJ pollinosis.

IgE reactivity to a Cry j 3, an allergen of Japanese cedar (Cryptomeria japonica) pollen in dogs with canine atopic dermatitis.

The present study investigated IgE reactivity to a new Cryptomeria japonica pollen allergen (Cry j 3) in dogs with atopic dermatitis by using a fluorometric ELISA. Serum samples from 15 dogs that showed IgE sensitivity to crude C. japonica pollen allergen by ELISA were tested for specific IgE to each allergen, individually. All 15 dogs had anti-Cry j 1 IgE, 6 (40%) had anti-Cry j 2 IgE, and 11 (73%) had anti-Cry j 3 IgE. Further, we found that these anti-Cry j 3 IgE reacted to Cry j 3 with immunoblotting analysis. These findings indicate that Cry j 3 may be a major allergen in dogs.