Coprological survey of parasitic infections in pigs and cattle in slaughterhouse in Osaka, Japan.

A coprological survey was performed at a slaughterhouse in Osaka, Japan, from 2004 to 2007 on 129 pigs reared in 8 prefectures, and on 213 cattle reared in 21 prefectures. Eimeria spp., Trichuris suis, Ascaris suum and Metastrongylus spp. infections were found in 52 (40.3%), 32 (24.8%), 19 (14.7%) and 3 pigs (2.3%), respectively, while Eimeria spp., Capillaria bovis and Trichuris sp. infections were detected in 163 (76.5%), 15 (7.0%) and 8 cattle (3.8%), respectively. Our results suggest that environmentally resistant oocysts and eggs of parasites could be widespread at the farms examined.

Importance of subunit vaccine antigen of major Fli C antigenic site of Salmonella enteritidis II: a challenge trial.

Salmonella enterica subsp. enterica serovar Enteritidis (SE) infection in chickens shows a mild pathogenicity except for young ages, compared with other animals, and laying hens sometimes produce SE-contaminated eggs leading to public health concerns. To reduce the problem, SE bacterin in poultry farms has been applied. We previously demonstrated that a subunit antigen, g.m. part polypeptide in SE-Fli C (SEp 9), could be a candidate subunit antigen of SE vaccine which may show less side effects in chickens. In this study, we used SEp 9 along with an adjuvant to inoculate chickens, then the chickens were orally challenged with SE, and suppression of the SE count in the cecum was investigated. Chickens inoculated with a commercial SE vaccine were prepared as positive controls (vaccine group), and those with physiological saline (control group) for comparison of the bacterial count after challenge. Employing two types of antibody-detection ELISA coated with either de-flagellated SE or SEp 9, specific antibody levels in blood and the intestine were determined. The bacterial count was significantly lower 1 and 3 weeks after challenge in the SEp 9 than in the control group. Specific antibody only against SEp 9 in blood but not the intestine of these birds in the SEp 9 group was detected. This study confirmed that SEp 9 antigen is a major effective antigen in SE inactivated vaccine, and it is suggested that only the subunit vaccine antigen SEp 9 is needed to effectively suppress colonization in the chicken intestine, without the need for other SE component antigens.

Public health assessment of Salmonella enterica serovar enteritidis inactivated-vaccine treatment in layer flocks.

Although there have been several reports on the efficacy assessment of a Salmonella enterica serovar Enteritidis vaccine against intestinal and parenchymatous organ diseases of laying hens, no public health risk characterization of its long-term effect on eggs has been reported. In this study, we attempted to assess the public health effect of an inactivated S. enterica serovar Enteritidis vaccine against serovar Enteritidis contamination of chicken eggs. We analyzed serovar Enteritidis isolation test results from four windowless farms in which inactivated-vaccine administration was initiated based on the sanitary monitoring program of a farm. When flocks with and without S. enterica serovar Enteritidis vaccine treatments were mixed, the application of an inactivated serovar Enteritidis vaccine decreased the most probable number (MPN) of bacteria by at least 100-fold in broken (liquid) egg samples positive for serovar Enteritidis, although a statistical difference between those MPNs could not be obtained. The isolation frequency after the vaccine application was less than 1/10 (P < 0.01). No S. enterica serovar Enteritidis bacteria were isolated approximately 1 year after all of the chickens had received the inactivated serovar Enteritidis vaccine. It was suggested that an adequate administration of an inactivated serovar Enteritidis vaccine reduced the contamination risk of eggs (the number of isolated serovar Enteritidis cells and detection frequency) compared to the contamination risk of eggs laid by nonvaccinated hens.

Genetical survey of novel type of Cryptosporidium andersoni in cattle in Japan.

Previously, we reported that an isolate of novel type of Cryptosporidium andersoni detected in cattle in Japan contained Type A (identical to C. andersoni reported previously) and Type B (having a thymine nucleotide insertion unlike the Type A) genotypes in the 18S rRNA gene. Here, we conducted an extensive investigation of Cryptosporidium infections in adult cattle in Japan from 2004 to 2007. Consequently, Cryptosporidium sp. were detected in 12 of the 205 cattle examined (5.9%), and partial sequences of the Cryptosporidium oocyst wall protein (COWP) gene in all isolates were identical to those of the previously reported data for C. andersoni whereas two signals were observed in the sequence of the partial 18S rRNA gene in all the isolates. In transmission studies using five of the isolates, they all infected SCID mice. Modified multiplex PCR using DNA of a single oocyst isolated from the infected SCID mice revealed that the partial sequences in the 18S rRNA gene of 40-80% of 10 isolates were identical to the Type A genotype of C. andersoni and those of other samples were identical to the Type B genotype. These results suggested that the C. andersoni novel type is widespread in cattle throughout Japan, and have multiple copies (Types A and B) in the 18S rRNA gene.

Importance of the major Fli C antigenic site of Salmonella enteritidis as a subunit vaccine antigen.

Our previous study indicated that the antibody against the major antigenic site of SE Fli C (g.m. region) is characteristically produced after the application of SE bacterin, however, the antibody is not produced in chickens after SE infection. In the present study, we determined histologically if the major antigenic site could be a candidate antigen for SE subunit vaccine. When Layermune SE, a commercial SE bacterin, was injected subcutaneously into the shoulder region as a positive control, the following histological changes were observed: formation of epithelioid granuloma with epithelioid cells and multinuclear giant cells surrounding necrotic sites and oil cysts (Indicator 1); a perivascular accumulation of lymphocytes near the granulation tissue (Indicator 2); peripheral fibroplasia encapsulating the granulation tissue (Indicator 3). On the other hand, at the injection site from the incomplete Freund adjuvant as a negative control antigen, there was only hyperplasia of the connective tissues around oil cysts. By using these indicators, the histological changes induced by injection of major antigenic site (SEp9) of Fli C, Fli C, and SE somatic antigen were evaluated. Histological changes after the injection with SEp9 demonstrated Indicators 2 and 3. The injection with SE Fli C demonstrated all three indicators. Contrarily, de-flagellated SE antigen injection induced only Indicator 3. The present results suggest that the antigen g.m. site of SE Fli C (SEp9) may play an important role as a subunit vaccine not only for including continuous immunological reaction in SE infection in chickens but also for antigen presentation.

Improvement of DNA extraction method for dried blood spots and comparison of four PCR methods for detection of Babesia gibsoni (Asian genotype) infection in canine blood samples.

To eradicate canine babesiosis in epidemic areas, mass-screening of the infection situation of Babesia gibsoni including occult infection is necessary. The development of cost-effective method for storage and transport of blood samples is required. A highly efficient DNA extraction procedure from dried blood spots (DBS) onto Whatman 3MM filter paper was developed for the diagnosis of B. gibsoni infection in dog by PCR. In 3 extraction methods, Chelex-based method in combination with saponin washing and phenol-chloroform-isoamyl alcohol extraction (Saponin-PCI method) provided the best results. Sensitivity of the 4 previously described PCR methods for detection of B. gibsoni infection was also compared using serially diluted blood samples of B. gibsoni-infected dogs. The PCR method using Gib599F/Gib1270R primer pair provided the best performance. To evaluate the stability of DNA in DBS, DBS of B. gibsoni-infected dogs stored at room temperature for 2 months. The stability was superior to whole blood samples stored at -20 degrees C for 2 months. This highly efficient DNA extraction method on DBS using Whatman 3MM filter paper has potential to be cost-effective and high performance tool for storage, and molecular diagnosis of clinical blood sample from dog. This procedure in combination with the PCR method using Gib599F/Gib1270R primer pair may greatly assist in diagnosis of B. gibsoni infection in dog populations that are geographically distant.

Characterization of monoclonal antibodies that recognize the Eimeria tenella microneme protein MIC2.

The apicomplexan pathogens of Eimeria cause coccidiosis, an intestinal disease of chickens, which has a major economic impact on the poultry industry. Members of the Apicomplexa share an assortment of unique secretory organelles (rhoptries, micronemes and dense granules) that mediate invasion of host cells and formation and modification of the parasitophorous vacuole. Among these, microneme protein 2 from Eimeria tenella(EtMIC2) has a putative function in parasite adhesion to the host cell to initiate the invasion process. To investigate the role of EtMIC2 in host parasite interactions, the production and characterization of 12 monoclonal antibodies (mabs) produced against recombinant EtMIC2 proteins is described. All mabs reacted with molecules belonging to the apical complex of sporozoites and merozoites of E. tenella, E. acervulina and E. maxima in an immunofluorescence assay. By Western blot analysis, the mabs identified a developmentally regulated protein of 42 kDa corresponding to EtMIC 2 and cross-reacted with proteins in developmental stages of E. acervulina. Collectively, these mabs are useful tools for the detailed investigation of the characterization of EtMIC2 related proteins in Eimeria species.

Detection of a mixed infection of a novel Cryptosporidium andersoni and its subgenotype in Japanese cattle.

Cryptosporidium oocysts were detected in the feces of cattle in Saga, Japan. Isolates were morphologically large. We attempted to identify the species or genotypes of the isolates by analyzing the partial sequences of the 18S rRNA and Cryptosporidium oocyst wall protein (COWP) genes, and measuring the infectivity in mice. The isolates showed 100% homology with Cryptosporidium andersoni in the COWP gene sequence and it could be transmitted to mice, but in the 18S rRNA gene, there was an additional signal in the ABI sequence chromatogram. To examine the additional signal, we analyzed both the 18S rRNA and the COWP gene sequences of a single oocyst passaged from mice using a modified multiplex PCR that was able to amplify both genes. As a result, it was revealed that two distinct genotypes (Types A and B) of a novel C. andersoni type existed in the 18S rRNA gene, whereas the COWP gene sequences of both oocysts were identical to C. andersoni. Although the sequence of the 18S rRNA gene of Type A was identical to that of C. andersoni, that of Type B had a thymine insertion and was not identical to any sequence registered with GenBank. Here we report that this is a new type of C. andersoni.

An epidemiological analysis of Salmonella enteritidis contamination in a rat-infested chicken layer farm, an egg processing facility, and liquid egg samples by pulsed-field gel electrophoresis.

In order to determine the epidemiological link between the Salmonella Enteritidis contamination in a rat-infested chicken layer farm, an attached egg processing facility and liquid egg samples, several S. Enteritidis isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and bacteriophage typing. A total of 33 S. Enteritidis strains were isolated from a total of 4,081 samples. Similar pulsed-field patterns were generated by S. Enteritidis isolates from liquid eggs, rats and effluent water. Additionally, only two phage types were detected among the S. Enteritidis isolates, PT 1b and PT 6. These results suggest that S. Enteritidis isolates from rats, egg processing facility, and liquid eggs are genetically related. Furthermore, S. Enteritidis infection in rats in layer farms poses a serious public health concern and should be included in future epidemiological studies.

Effects of Salmonella enterica subsp. enterica serovar Enteritidis vaccination in layer hens subjected to S. Enteritidis challenge and various feed withdrawal regimens.

Levels of Salmonella enterica subsp. enterica serovar Enteritidis infection and serum S. Enteritidis antibodies after experimental S. Enteritidis challenge and feed withdrawal were investigated in S. Enteritidis-vaccinated and unvaccinated hens. The results were used to determine whether formalin-inactivated S. Enteritidis vaccination can protect layer hens from S. Enteritidis challenge during feed withdrawal periods. S. Enteritidis infection rates were evaluated from cloacal swabs, eggs and organs. Serum antibody titers to deflagellated S. Enteritidis whole cells (DEWC) and S. Enteritidis FliC-specific 9-kDa polypeptide (SEp 9) were examined by commercial ELISA kits. Cloacal S. Enteritidis recovery rates were lower in the vaccinated than unvaccinated group. Recovery rates of S. Enteritidis from samples increased after feed withdrawal and decreased after re-introduction of feed. S. Enteritidis counts in cloacal swabs were lower in the vaccinated than in the unvaccinated group (P<0.05). More S. Enteritidis-positive eggs were detected from the unvaccinated group. Before S. Enteritidis challenge, the DEWC ELISA titer of the vaccinated group was higher (P<0.05) than the unvaccinated group; subsequently, the S. Enteritidis DEWC ELISA titers of both groups increased gradually. In contrast, only the vaccinated group elicited high SEp-9 antibody titer during post-challenge and feed withdrawal. Additionally, vaccinated hens yielded negative S. Enteritidis isolation rates from egg contents. There is a correlation between negative S. Enteritidis isolation rates and high SEp 9 titers in vaccinated layer hens challenged with S. Enteritidis and subjected to feed withdrawal regimens. These findings suggest the S. Enteritidis vaccination of pullets may protect against S. Enteritidis infection during forced molting and that SEp 9 titer could be a potential indicator of antibody protection against S. Enteritidis infection. The potential of the SEp 9 peptide as an antigen for S. Enteritidis vaccination in the future is worth noting.

Monoclonal antibodies for the diagnosis of canine mastocytoma.

Mastocytomas are the most common malignant neoplasm in the dog; they are more aggressive than the mast cell tumors of other species. Therefore, it is imperative to develop a highly sensitive and specific immunoassay for clinical diagnosis of canine mastocytoma. The production and characterization of new mouse monoclonal antibodies (MAb 9-3 and MAb 80) directed against canine mastocytoma are reported here. By immunohistochemistry using fresh frozen tissue of tissue impression smears, we observed that the antigen recognized by MAb 9-3 is expressed exclusively on the surface and cytoplasmic granules of canine mastocytoma but not on the mast cells in normal canine skin. MAb 80 did not compete for binding to mast cells in normal canine skin. Western blot assays performed with canine mastocytoma indicated that MAb 9-3 recognized the 74 kDa band, and MAb 80 recognized the 167 and 248 kDa bands. We studied the immunostaining pattern of impression smears with MAb 9-3 from 36 benign and malignant canine masses, including eight samples of mastocytoma that were positive and other tumor samples that were negative by MAb 9-3. This report for the first time precisely characterizes a monoclonal antibody specific for canine mastocytoma, facilitating clinical and molecular investigation of canine mastocytoma.

Histidine decarboxylases and their role in accumulation of histamine in tuna and dried saury.

Histamine-producing bacteria (HPB) such as Photobacterium phosphoreum and Raoultella planticola possess histidine decarboxylase (HDC), which converts histidine into histamine. Histamine fish poisoning (HFP) is attributable to the ingestion of fish containing high levels of histamine produced by HPB. Because freezing greatly decreases the histamine-producing ability of HPB, especially of P. phosphoreum, it has been speculated that HFP is caused by HDC itself from HPB cells autolyzing during frozen storage, even when HPB survive frozen storage. Here we constructed recombinant HDCs of P. phosphoreum, Photobacterium damselae, R. planticola, and Morganella morganii and investigated the ability of HDCs to produce sufficient histamine to cause HFP. To elucidate the character of these HDCs, we examined the specific activity of each recombinant HDC at various temperatures, pH levels, and NaCl concentrations. Further, we also investigated the stability of each HDC under different conditions (in reaction buffer, tuna, and dried saury) at various temperatures. P. damselae HDC readily produced sufficient histamine to cause HFP in fish samples. We consider that if HDC is implicated as an independent cause of HFP in frozen-thawed fish, the most likely causative agent is HDC of P. damselae.

Therapeutic effects of beta-thujaplicin eardrops on canine Malassezia-related otitis externa.

An eardrop solution of beta-thujaplicin was examined for therapeutic effects on canine Malassezia-related otitis externa. Half to one ml of beta-thujaplicin solution of 100 microg/ml including DMSO 2% was injected everyday into both external ear canals of 31 cases for test-of-cure agreement. Fifteen score phases were established from the symptoms and cerumen smear biopsy findings, and score changes were recorded at least once a week. The means of the second and the third inspection day scores decreased significantly more than the previous value of each. In addition, the numbers of yeast-like organisms clearly decreased. These results suggest that beta-thujaplicin eardrops are effective for Malassezia-related otitis externa in dogs.

Specific adhesion and invasion of Salmonella Enteritidis in the vagina of laying hens.

Salmonella Enteritidis is the predominant serovar associated with egg-borne salmonellosis in humans. The colonization of S. Enteritidis in the vagina may play a role in the production of S. Enteritidis-contaminated eggs. In the first experiment, the in vitro adhesion of S. Enteritidis in vaginal and follicular explants was compared with that of S. Typhimurium by bacteriological isolation methods. The mean number of S. Enteritidis associated with vaginal explants was significantly (P < 0.05) higher than S. Typhimurium associated with vaginal explants and both serovars associated with follicular explants. In the second experiment, the in vitro adhesion and invasion of S. Enteritidis strains in the vaginal epithelium was compared with that of several strains of S. Agona, S. Infantis, S. Hadar, S. Heidelberg, S. Montevideo and S. Typhimurium, by immunohistochemical methods. The mean number of Salmonella in the vaginal epithelium depended on their lipopolysaccharide (LPS) type, with the rank order as follows: LPS type O9 (S. Enteritidis) > LPS type O4 (S. Agona, S. Typhimurium and S. Heidelberg) > LPS type O7 (S. Montevideo and S. Infantis) and LPS type O8 (S. Hadar). This rank order of Salmonella invasiveness is in accordance with the frequency of Salmonella outbreaks involving contaminated eggs. These findings suggest that S. Enteritidis has a higher ability to colonize the vaginal epithelium than other serovars, and the Salmonella LPS type may play an essential role in tropism of the reproductive tract.

Differential responses of macrophages to Salmonella enterica serovars Enteritidis and Typhimurium.

Macrophages are major effectors against Salmonella infection, and also transport bacteria between host tissues and provide a protected site for intracellular bacterial replication. We hypothesized that differences in chicken macrophage responses to Salmonella enterica serovar Enteritidis (SE) and serovar Typhimurium (ST) played a role in preferential infection of eggs by SE compared with ST. To test this hypothesis, we determined bacterial phagocytosis and intracellular viability and macrophage nitric oxide (NO) production following in vitro infection with SE or ST in the presence or absence of interferon-gamma (IFN-gamma). The effects of bacterial components, lipopolysaccharide (LPS), outer membrane proteins (OMP) and flagella, on NO production were also assessed. Our results showed: (1) in the presence or absence of IFN-gamma, the percentage macrophages phagocytizing SE and ST was similar; (2) the number of intracellular viable SE was significantly reduced compared with ST in the presence or absence of IFN-gamma; (3) increased macrophage necrosis was seen in the presence of IFN-gamma and ST; (4) Salmonella infection acted synergistically with IFN-gamma in induction of nitric oxide production; and (5) in the absence of IFN-gamma, macrophages produced significantly greater NO following treatment with SE outer membrane protein or flagella compared with ST OMP or flagella, while in the presence of IFN-gamma significantly less NO was produced following treatment with SE-LPS compared with ST-LPS. These results suggest that differential responses of chicken macrophages to SE versus ST may result in increased macrophage death with ST, which could result in an increased inflammatory response as compared to SE.

Molecular characterization of crane Coccidia, Eimeria gruis and E. reichenowi, found in feces of migratory cranes.

Eimeria gruis and E. reichenowi have lethal pathogenicity to a number of species of cranes. These parasites develop at multiple organs or tissues in infected cranes, thus lacking the specificity of infection sites shown by other Eimeria spp. in spite of morphologic similarity. To date, there have been many reports of crane Eimeria infections, however, genetic examinations of these parasites have never been published. In the present study, we isolated oocysts of E. gruis and E. reichenowi from crane feces at a wintering area in Japan. By phylogenic analysis, we first demonstrated that partial sequences of the isolates formed their own cluster, located separately from other Eimeria spp.

Proliferative responses to canine thyroglobulin of peripheral blood mononuclear cells from hypothyroid dogs.

The immune responses of hypothyroid dogs to canine thyroglobulin (cTg) were evaluated for the proliferative ability of peripheral blood mononuclear cells (PBMC). PBMC from three hypothyroid dogs with high titers of thyroglobulin autoantibody (TgAA) and 3 clinically normal dogs were cultured with 5, 10, or 20 microg/ml of cTg for 72 hr. The proliferative responses of the cells were determined by the level of incorporated BrdU. The numbers of cells expressing Thy-1, CD4, CD8 and IgG in the PBMC were counted by the immunofluorescence method. Proliferative responses to cTg were observed in the cells from hypothyroid dogs. The number of cells expressing IgG and CD8 in the hypothyroid dogs tended to be high compared with the clinically normal dogs. The CD4+ cells in cultures from hypothyroid dogs increased depending upon the amount of cTg. There was a significant (P<0.05) positive correlation between the number of CD4+ cells and the concentration of cTg in the cultures from hypothyroid dogs. These findings suggest a possible relationship between canine hypothyroidism and cellular immunity. Loss of self tolerance to thyroid antigens in CD4+ T cells may play an important role in the development of canine hypothyroidism.

Infectivity of a novel type of Cryptosporidium andersoni to laboratory mice.

Previously, we reported 'a novel type' of Cryptosporidium andersoni detected from cattle in Japan, and showed that the isolate was infective to mice. In the present study, we examined the patterns of oocyst shedding in both immunocompromised and immunocompetent mice, as well as pathological lesions in the infected mice. After oral inoculation with 1 x 10(6) oocysts, all five severe combined immunodeficiency (SCID) mice began to shed endogenously produced oocysts on day 6 post-inoculation (p.i.). The number of oocysts per day (OPD) reached 1 x 10(6) on day 17 p.i., and an OPD level of 1 x 10(6) to 10(7) was maintained until 91 days p.i. when the mice were sacrificed. In the five immunocompetent mice inoculated with 1 x 10(6) oocysts, the pre-patent and patent periods were 6 and 19 days, respectively, and the maximal OPD level was 1.5 x 10(5) on average. On histological examinations of infected SCID mice, a large number of parasites were present on the surface of the gastric glands of the stomach, but not in other organs examined. In conclusion, the novel type of C. andersoni, which genetically coincides with C. andersoni reported in other countries, is infective to mice, but susceptibility was lower than that of Cryptosporidium muris infecting rodents from the perspective of infectivity to immunocompetent mice.

Cross-reactivities with Cryptosporidium spp. by chicken monoclonal antibodies that recognize avian Eimeria spp.

In a previous study, we have developed several chicken monoclonal antibodies (mAbs) against Eimeria acervulina (EA) in order to identify potential ligand molecules of Eimeria. One of these mAbs, 6D-12-G10, was found to recognize a conoid antigen of EA sporozoites and significantly inhibited the sporozoite invasions of host T lymphocytes in vitro. Furthermore, some of these chicken mAbs showed cross-reactivities with several different avian Eimeria spp. and the mAb 6D-12-G10 also demonstrated cross-reactivities with the tachyzoites of Neospora caninum and Toxoplasma gondii. Cryptosporidium spp. are coccidian parasites closely related to Eimeria spp., and especially C. parvum is an important cause of diarrhea in human and mammals. In the present study, to assess that the epitopes recognized by these chicken mAbs could exist on Cryptosporidium parasites, we examined the cross-reactivity of these mAbs with Cryptosporidium spp. using an indirect immunofluorescent assay (IFA) and Western blotting analyses. In IFA by chicken mAbs, the mAb 6D-12-G10 only showed a immunofluorescence staining at the apical end of sporozoites of C. parvum and C. muris, and merozoites of C. parvum. Western blotting analyses revealed that the mAb 6D-12-G10 reacted with the 48-kDa molecular weight band of C. parvum and C. muris oocyst antigens, 5D-11 reacted the 155 kDa of C. muris. Furthermore, these epitopes appeared to be periodate insensitive. These results indicate that the target antigen recognized by these chicken mAbs might have a shared epitope, which is present on the apical complex of apicomplexan parasites.

Effects of beta-thujaplicin on anti-Malassezia pachydermatis remedy for canine otitis externa.

The antifungal activity of beta-thujaplicin was evaluated against 51 Malassezia pachydermatis strains isolated from canine ear canals with or without otitis externa. For comparison, sensitivity tests were performed on M. pachydermatis isolates for nystatin, ketoconazole, and terbinafine HCl, all clinically available antifungal agents. The minimal inhibition concentrations over 50% of the tested isolates (MIC50) were 3.13 microg/ml for beta-thujaplicin and nystatin, 0.016 microg/ml for ketoconazole, and 1.56 microg/ml for terbinafine HCl. The antifungal effect for M. pachydermatis of beta-thujaplicin compared favorably with commercial antifungal agents. None of the 51 M. pachydermatis isolates showed resistance against any of the tested antibiotics investigated in this study. Ten representative isolates of M. pachydermatis were subcultured for 30 generations at concentrations close to the MIC levels of beta-thujaplicin, nystatin, ketoconazole, and terbinafine HCl, and examined to determine whether they had acquired resistance to each drug. As a result, M. pachydermatis was found to achieve resistance more easily for ketoconazole and terbinafine HCl than for beta-thujaplicin or nystatin. The MIC50 of beta-thujaplicin did not change during the course of subculture, and it is thought that the potential development of a resistant strain is low, even with continuous infusion for otitis externa therapy. beta-Thujaplicin is an inexpensive and safe treatment with anti-inflammatory and deodorant effects that can be recommended as an effective remedy for canine otitis externa.