Clinical efficacy of recombinant canine interferon-gamma therapy in dogs with cutaneous epitheliotropic T-cell lymphoma.

BACKGROUND: The antitumour effects of interferon-gamma (IFN-γ) in humans with cutaneous epitheliotropic T-cell lymphoma (CETCL) have been described; however, the efficacy of IFN-γ in dogs has not been investigated. HYPOTHESIS/OBJECTIVES: The aim of this study was to evaluate the efficacy of recombinant canine IFN-γ (rCaIFN-γ) therapy in dogs with CETCL. ANIMALS: Twenty dogs with CETCL recruited from seven veterinary clinics were enrolled in the study. MATERIALS AND METHODS: Fifteen dogs were treated with rCaIFN-γ, and five control dogs were treated with prednisolone. We evaluated survival time, skin lesions (erythema, nodules, ulcers and bleeding), pruritus and general condition (sleep, appetite and body weight). In the rCaIFN-γ group, a questionnaire regarding the therapy was administered to owners after the dogs died. RESULTS: No significant differences existed in the median survival time between the rCaIFN-γ and control groups (log-rank test: p = 0.2761, Wilcoxon's rank sum test: p = 0.4444). However, there were significant differences in ulcer, bleeding, pruritus, sleep, appetite and body weight between the groups (Wilcoxon-Mann-Whitney U-test: p = 0.0023, p = 0.0058, p = 0.0005, p = 0.0191, p = 0.0306 and p = 0.0306, respectively). Two (40%) of five dogs were euthanised in the control group, compared with none in the rCaIFN-γ group. Fourteen questionnaires were collected, and owners reported that they were satisfied with the rCaIFN-γ treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Although the median survival time was not prolonged, rCaIFN-γ could be helpful in maintaining good quality of life for dogs with CETCL.

Azole and terbinafine susceptibility testing of Malassezia pachydermatis in Japan.

Canine Malassezia dermatitis and otitis externa are generally treated by antifungal drugs. However, multi-drug-resistant strains of Malassezia pachydermatis have been reported worldwide. Given the presence of these multi-drug-resistant strains, it is unclear which antifungal agent is the most effective for canine Malassezia dermatitis and canine otitis. In this study, we attempted to determine the most effective drug against azole-resistant M. pachydermatis. Susceptibility to azoles and terbinafine (TBF) was assessed using a modified broth microdilution method. In all tested isolates, the minimum inhibitory concentration at 90% of organisms (MIC90) were 16 to >32 µg/mL for the azoles, and 2 µg/mL for TBF. All of the strains that showed low susceptibility to both itraconazole and miconazole were also relatively susceptible to TBF.

Rapid Molecular Detection of Antifungal-Resistant Strains of Malassezia pachydermatis.

Azole resistance in Malassezia pachydermatis has been reported in isolates from canine skin worldwide. Decreased susceptibility of M. pachydermatis to azoles has been hypothesized to potentially result from mutations in the ERG11 gene, which encodes lanosterol 14α-demethylase. To sequence the mutation hotspots of ERG11 in the isolates, we prepared primers (MPERG11hot2S and MPERG11hot2R) based on the conserved sequences of M. pachydermatis ERG11. DNA samples from azole-resistant and -susceptible strains were amplified by PCR using the primer pair. PCR amplicons were sequenced and analyzed for single-nucleotide polymorphisms (SNPs) in the target gene. Seven of the tested azole-resistant strains (16 strains) harbored ERG11 SNPs at nucleotide 904 (G→A) or 905 (C→T), resulting in the replacement of Ala 302 with Thr or Val (Ala302Thr or Val). None of the tested azole-susceptible strains had a mutation at either of those residues. Our PCR method detected SNPs at the nucleotide-905 (C→T) hotspot mutation site in M. pachydermatis ERG11. Moreover, we discovered an additional hot spot site at nucleotide 904 (G→A).

Molecular typing and antifungal drug susceptivity profile of Rhodotorula mucilaginosa from canine skin and ear canal.

Rhodotorula mucilaginosa are saprophytic yeast, and opportunistic infections known as human rhodotorulosis are increasing in immunocompromised patients. In this study, we isolated R. mucilaginosa from pet dogs in Japan and determined the minimum inhibitory concentrations (MICs) of antifungal drugs on these isolates to investigate the drug susceptibility pattern. All 10 isolates according to the broth microdilution (BM) assay of the Clinical and Laboratory Standards Institute (CLSI) M27-A2 were resistance to azoles and genetically close to fluconazole (FLZ)-resistant human isolates of R. mucilaginosa. Due to resistance, it is expected that treatment will be difficult if they infect humans.

Prevalence of antimicrobial-resistant staphylococci in nares and affected sites of pet dogs with superficial pyoderma.

Currently, antimicrobial-resistant staphylococci, particularly methicillin-resistant Staphylococcus pseudintermedius (MRSP), are frequently isolated from canine superficial pyoderma in Japan. However, little is known regarding the nasal prevalence of MRSP in pet dogs. Here, we determined the prevalence of antimicrobial-resistant staphylococci in nares and affected sites of pet dogs with superficial pyoderma. Of the 125 nares and 108 affected sites of pet dogs with superficial pyoderma, 107 (13 species) and 110 (eight species) staphylococci strains, respectively, were isolated. The isolation rate of S. pseudintermedius from pyoderma sites (82/110 strains, 74.5%) was significantly higher than that from nares (57/107 strains, 53.3%) (P<0.01). Notably, the prevalence of MRSP (18/57 strains, 31.6%) in nares was equivalent to that in pyoderma sites (28/82 strains, 34.1%). Furthermore, the phenotypes and genotypes of antimicrobial resistance in MRSP strains from nares were similar to those from pyoderma sites. Our findings revealed that the prevalence of antimicrobial-resistant staphylococci in the nares of pet dogs with superficial pyoderma is the same level as that in affected sites. Therefore, considerable attention should be paid to the antimicrobial resistance of commensal staphylococci in companion animals.

High multi-azole-resistant Malassezia pachydermatis clinical isolates from canine Malassezia dermatitis.

Malassezia pachydermatis, a lipophilic and aerobic yeast, is a causative agent of Malassezia dermatitis, a common skin mycosis in dogs and cats. This fungus is also responsible for zoonotic fungal infections in human neonates. Ravuconazole (RVZ) is an antifungal azole compound and the active metabolite of fosravuconazole, which was approved for use in humans in Japan in 2018. In the present study, in vitro RVZ susceptibility and multi-azole resistance of 13 clinical M. pachydermatis strains was investigated using the modified Clinical and Laboratory Standards Institute M27-A3 test. The minimum inhibitory concentrations (MICs) for the 13 isolates ranged from 0.094 to >32 mg/L for itraconazole (ITZ) and from 0.5 to >32 mg/l for RVZ. Similarly, MICs for ITZ- or RVZ-resistant strains (MICs >32 mg/l) were also >32 mg/l for clotrimazole (CTZ), >32 mg/l for miconazole (MCZ), and 0.25 to >32 mg/L for voriconazole (VRZ). BLAST analysis using the NCBI database showed that ERG11 cDNA of the RVZ-resistant strain encoded Gly at codon 461 and Asp in cytochrome p450 encoded by M. pachydermatis ERG11 mRNA. This work is the first report to describe that an RVZ-resistant M. pachydermatis strain contains ERG11 mutations. The affinity of the protein encoded by ERG11 for RVZ may differ from that of ITZ. Therefore, RVZ has considerable therapeutic potential for treating ITZ-resistant canine Malassezia dermatitis. However, RVZ-resistant strains already exist in canine Malassezia dermatitis in Japan.

Transcription profile of chemokine receptors, cytokines and cytotoxic markers in peripheral blood of dogs with epitheliotropic cutaneous lymphoma.

BACKGROUND: Mycosis fungoides (MF) is the most common form of canine epitheliotropic cutaneous lymphoma, which is characterized by the accumulation of neoplastic CD8(+) T cells. Given that multifocal skin lesions are commonly seen in MF, neoplastic lymphocytes may actively migrate into the blood circulation. HYPOTHESIS/OBJECTIVES: Cytotoxic T cells with a skin-homing phenotype could be increased in the blood circulation of dogs with MF. ANIMALS: Ten dogs with MF and 10 age-matched healthy dogs were included. METHODS: The transcription levels of chemokine receptors, cytokines and cytotoxic markers in peripheral blood of dogs with MF were quantified by real-time RT-PCR. RESULTS: The dogs with MF had lower transcription levels of chemokine receptors associated with skin homing (CCR4), epitheliotropism (CXCR3), lymph node homing (CCR7), a type-1 cytokine (LT-α) and cytotoxic markers (perforin and granzyme B) in the circulation than healthy control dogs (P < 0.05). CONCLUSIONS AND CLINICAL IMPORTANCE: The present results suggest that the number of peripheral cytotoxic T cells with a skin-homing phenotype could be decreased in the peripheral blood of dogs with MF, which might be due to the sequestration of cytotoxic T cells in the lesional skin.

In vitro antiseptic susceptibilities for Staphylococcus pseudintermedius isolated from canine superficial pyoderma in Japan.

BACKGROUND: Topical therapy, particularly with chlorhexidine, is becoming increasingly common as a treatment option for canine pyoderma; however, there are limited studies on the susceptibility of Staphylococcus pseudintermedius to chlorhexidine compounds. OBJECTIVES: To determine the in vitro susceptibility of both meticillin-resistant and meticillin-susceptible S. pseudintermedius isolates to chlorhexidine and other antiseptic agents and the presence of multidrug efflux pump genes. SAMPLES: One hundred S. pseudintermedius isolates from 23 initial and 77 recurrent cases of canine pyoderma. METHODS: After bacterial identification and mecA testing, minimal inhibitory concentrations (MICs) of antiseptic agents were determined. Multidrug efflux pump genes, including qacA, qacB and smr, were identified. RESULTS: Of the 100 isolates, 57 were identified as meticillin-resistant S. pseudintermedius. The MIC(90) of chlorhexidine acetate, chlorhexidine gluconate, acriflavine, ethidium bromide and benzalkonium chloride were 1, 1, 2, 0.5 and 2 µg/mL, respectively. Multidrug efflux pump genes qacA, qacB and smr were not detected in any of the isolates. CONCLUSIONS AND CLINICAL IMPORTANCE: The MICs for chlorhexidine and other antiseptics remain low, and multidrug efflux pump genes were not found in the tested isolates.

Gene transcription analysis in lesional skin of canine epitheliotropic cutaneous lymphoma using quantitative real-time RT-PCR.

Canine epitheliotropic cutaneous lymphoma (cECL) is characterized by infiltration of neoplastic lymphocytes in the skin with a specific tropism for the epidermis. Migration of lymphocytes is strictly controlled by interactions between chemokines and chemokine receptors, which may be involved in the pathogenesis of cECL. In this study, we investigated mRNA transcription levels of several chemokines (CCL17, CCL19, CCL21, CCL22, CCL27, CCL28 and CXCL10) and chemokine receptors (CCR4, CCR7, CCR10 and CXCR3) in lesional skin of cECL by quantitative real-time RT-PCR. To examine the subsets of accumulating neoplastic lymphocytes, we also investigated transcription levels of type-1 (IFN-γ, IL-12p35, IL-12p40 and LT-α) and type-2 (IL-4 and IL-13) cytokines and cytotoxic markers (perforin and granzyme B). We found that the lesional skin had higher mRNA transcription of CCL19, CXCL10, CCR4, CCR7, CCR10 and CXCR3 and lower transcription of CCL27 than healthy dog skin (p<0.05). In addition, transcription levels of type-1 cytokine and cytotoxic markers in lesional skin were significantly higher than those in healthy dog skin. These results indicate that the transcription of some chemokines and chemokine receptors, which are necessary for skin-homing, epitheliotropism and peripheral segregation of T-cells, is upregulated in the lesional skin of cECL. In addition, our results also indicate that the subset of neoplastic lymphocytes in cECL is most likely type-1 cytotoxic T-cells.

Dose assessment of 2% chlorhexidine acetate for canine superficial pyoderma.

The dose of 2% chlorhexidine acetate (2CA; Nolvasan(®) Surgical Scrub; Fort Dodge Animal Health, Fort Dodge, IA, USA) for canine superficial pyoderma was evaluated. The first trial compared three doses (group 1, 57 mL/m(2) body surface area; group 2, 29 mL/m(2) body surface area; and group 3, 19 mL/m(2) body surface area) in a randomized, double-blind, controlled fashion. Twenty-seven dogs with superficial pyoderma were treated with 2CA at the allocated doses every 2 days for 1 week. The owners and investigators subjectively evaluated the dogs, and investigators scored skin lesions, including erythema, papules/pustules, alopecia and scales, on a 0-4 scale. There were no significant differences in response between the treatment groups. The second trial established a practical dose-measuring method for 2CA. Sixty-eight owners were asked to apply 2CA on their palm in an amount corresponding to a Japanese ¥500 coin, 26.5 mm in diameter. This yielded an average dose of 0.90±0.40 mL. Mathematically, the doses used in groups 1, 2 and 3 can be represented as one coin per approximately one-, two- and three-hand-sized lesions, respectively. The results therefore suggest that owners instructed to apply one coin of the product per two-hand-sized areas of superficial pyoderma would use the range of doses evaluated in this trial.

Clinical comparison of human and canine atopic dermatitis using human diagnostic criteria (Japanese Dermatological Association, 2009): proposal of provisional diagnostic criteria for canine atopic dermatitis.

Atopic dermatitis (AD) is a common skin disease encountered in both humans and dogs. Canine AD can be used in the analysis of naturally occurring AD; however, details of clinical comparison have been lacking. The purpose of this study is to compare those clinical features using the human diagnostic criteria (Japanese Dermatological Association, 2009). Fifty-one dogs with canine AD were evaluated by the human criteria. Prior to this study, canine AD was basically diagnosed by the fulfillment of two authentic canine AD criteria and a positive reaction against Dermatophagoides farinae in serum immunoglobulin E levels and/or in intradermal tests. Among the human AD criteria items, behavior corresponding to pruritus was observed in all 51 dogs. Skin lesions corresponding to eczematous dermatitis were seen in 50 dogs, and symmetrical distribution of skin lesions was noted in all 51 dogs. A chronic or chronically relapsing course was observed in 50 dogs. Based on these results, the concordance rate for the criteria was 96% (49/51). Differential diagnoses of AD were also investigated in the same manner. The concordance rate for the criteria was 0% (0/69) in scabies, 2% (1/50) in pyoderma, 0% (0/50) in demodicosis, 0% (0/9) in cutaneous lymphoma, 0% (0/2) in ichthyosis, 25% (2/7) in flea allergy, 48% (24/50) in seborrheic dermatitis and 75% (3/4) in food allergy. Canine AD is thus indicated as a valuable counterpart to human AD in clinical aspects. In addition, the human AD criteria could be applicable, with some modification, as provisional diagnostic criteria for canine AD.

Sarcoptes scabiei var. canis refractory to ivermectin treatment in two dogs.

A 10-year-old castrated male Shih Tzu presented with severe generalized pruritus. Skin scrapings revealed the presence of Sarcoptes scabiei var. canis. A Yorkshire terrier in the same household simultaneously developed pruritus due to scabies. Both dogs were treated with 300 µg/kg ivermectin, at first orally and then subcutaneously at 14 day intervals. However, live mites were still found on day 35, and the skin condition deteriorated in both dogs. These findings suggested that the S. scabiei in these dogs was clinically refractory to ivermectin. The pruritus in both dogs rapidly and completely disappeared following topical fipronil administration. This appears to be the first report of canine scabies refractory to ivermectin treatment.

Identification of a novel Staphylococcus pseudintermedius exfoliative toxin gene and its prevalence in isolates from canines with pyoderma and healthy dogs.

Staphylococcal exfoliative toxins are involved in some cutaneous infections in mammals by targeting desmoglein 1 (Dsg1), a desmosomal cell-cell adhesion molecule. Recently, an exfoliative toxin gene (exi) was identified in Staphylococcus pseudintermedius isolated from canine pyoderma. The aim of this study was to identify novel exfoliative toxin genes in S. pseudintermedius. Here, we describe a novel orf in the genome of S. pseudintermedius isolated from canine impetigo, whose deduced amino acid sequence was homologous to that of the SHETB exfoliative toxin from Staphylococcus hyicus (70.4%). The ORF recombinant protein caused skin exfoliation and abolished cell surface staining of Dsg1 in canine skin. Moreover, the ORF protein degraded the recombinant extracellular domains of canine Dsg1, but not Dsg3, in vitro. PCR analysis revealed that the orf was present in 23.2% (23/99) of S. pseudintermedius isolates from dogs with superficial pyoderma exhibiting various clinical phenotypes, while the occurrence in S. pseudintermedius isolates from healthy dogs was 6.1% (3/49). In summary, this newly found orf in S. pseudintermedius encodes a novel exfoliative toxin, which targets a cell-cell adhesion molecule in canine epidermis and might be involved in a broad spectrum of canine pyoderma.

Antimicrobial susceptibility and methicillin resistance in Staphylococcus pseudintermedius and Staphylococcus schleiferi subsp. coagulans isolated from dogs with pyoderma in Japan.

To understand species distribution, trends of antimicrobial susceptibility and prevalence of methicillin resistance in canine staphylococci in Japan, 190 coagulase-positive staphylococci (CoPS) were isolated from dogs with pyoderma in 2 Japanese veterinary referral hospitals. Using a multiplex polymerase chain reaction (M-PCR) method, two CoPS species were identified: 170 Staphylococcus pseudintermedius (89.5%) and 20 S. schleiferi subsp. coagulans isolates (10.5%). In these isolates, susceptibility to 7 antimicrobial agents was determined. Overall, the levels of susceptibility to cefalexin (CEX), amoxicillin/clavulanic acid (CVA/AMPC), minocycline (MINO), ofloxacin (OFLX), norfloxacin (NFLX), lincomycin (LCM) and clindamycin (CLDM) in S. pseudintermedius isolates were 38.2, 52.4, 34.7, 31.2, 34.1, 1.2 and 11.2%, respectively. In S. schleiferi subsp. coagulans isolates, 55% demonstrated susceptibility to CEX, 80% to CVA/AMPC, 70% to MINO, 45% to OFLX or NFLX and 30% to CLDM. None of S. schleiferi subsp. coagulans isolates was susceptible to LCM. To determine the prevalence of methicillin-resistant strains, we used a PCR method, which enabled detection of the fragment of mecA gene in 66.5% (113 of 170) in S. pseudintermedius and 30.0% (6 of 20) in S. schleiferi subsp. coagulans isolates. The frequencies of susceptibility to CEX, CVA/AMPC, OFLX, NFLX and CLDM were significantly lower in methicillin-resistant CoPS than in methicillin-susceptible CoPS isolates. These data suggest a high level of methicillin resistance in staphylococci isolated from dogs with pyoderma in Japan.

Efficacy of a surgical scrub including 2% chlorhexidine acetate for canine superficial pyoderma.

The clinical efficacy of a surgical scrub containing 2% chlorhexidine acetate (2CA; Nolvasan® Surgical Scrub; Fort Dodge Animal Health, USA) was evaluated for the topical management of canine superficial pyoderma. The first study was a randomized, double-blind, controlled trial. The control was a shampoo containing 4% chlorhexidine gluconate (4CG; Skin Clinic Shampoo; CHD MEDICS, Goyang, Korea). Ten dogs with symmetrical lesions of canine superficial pyoderma were allocated to receive either 2CA or the control shampoo applied to either side of the body twice weekly for 1 week. Both the owners and the investigators subjectively scored skin lesions including pruritus, erythema, crusted papules and scales on a scale of 0-3. The 2CA and 4CG resulted in almost the same degree of improvement of skin lesions, and there were no significant differences between the two groups. The second study was an open trial of 2CA monotherapy in eight dogs with cefalexin-resistant Staphylococcus intermedius group-associated superficial pyoderma. The 2CA monotherapy was applied every 2 days for 2 weeks. Five dogs improved with 2CA monotherapy, one partially improved and two did not. No adverse reactions were seen in either trial. This suggests that a 2CA surgical scrub could be a useful and safe topical adjunct therapy for dogs with superficial pyoderma involving cefalexin-resistant Staphylococcus intermedius group.

A case of superficial suppurative necrolytic dermatitis of miniature schnauzers with identification of a causative agent using patch testing.

A 9-year-old, castrated male, miniature schnauzer presented with malaise, anorexia, fever and severe inflammatory skin lesions on the dorsum, thighs and pinnae. The lesions developed 2 days after bathing with a commercial shampoo. Histopathological examination of skin samples revealed neutrophilic exocytosis, parakeratosis, epidermal hyperplasia and neutrophilic infiltration in the superficial dermis. Skin lesions resolved completely after 14 days of treatment with prednisolone and ofloxacin. Patch testing performed on the patient and a clinically healthy dog showed erythema at the site exposed to the culprit shampoo 48 h later only on the patient. Histopathological findings of the erythematous reaction were similar to those of the spontaneous skin lesions. Based on these findings, the dog was diagnosed with superficial suppurative necrolytic dermatitis of miniature schnauzers. The patch test results suggested that contact dermatitis to a commercial shampoo played a role in the pathogenesis of this disease.