无菌或非抗生素反应性脓疱性皮炎及犬利什曼病-14例病例描述及242 ...

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Sterile or nonantibiotic-responsive pustular dermatitis and canine leishmaniosis: a 14 case series description and a statistical association study on 2420 cases

无菌或非抗生素反应性脓疱性皮炎及犬利什曼病-14例病例描述及2420例统计相关性研究

作者：Mar Bardag , Moira Monaco and Dolors Fondevila

翻译：李诗如

 

Background – No striking clinical and histopathological features of pustular dermatitis (PustD) in dogs suffering from canine leishmaniosis (CanL) have been identified; an association between CanL and PustD has not been demonstrated.

Objectives – To characterize a series of dogs affected by CanL and pruritic PustD, and to evaluate a possible association between the two conditions.

Animals – Fourteen dogs and a case log of 2,420 cases.

Methods and materials – A clinical and histopathological retrospective study was conducted on 14 dogs. Leishmania immunohistochemistry (IHC) and real-time PCR for L. infantum on skin biopsies were performed. An association of PustD with CanL was tested with Fisher’s exact test using medical records of 2,420 cases.

Results – Dogs were characterized: clinically by generalized pruritic sterile or antibiotic-unresponsive papulo-pustular dermatitis; and histopathologically by neutrophilic pustules and neutrophilic and mastocytic perivascular dermatitis with vasodilation and oedema. IHC showed Leishmania amastigotes in six cases and PCR was positive in three of eight IHC-negative cases. Anti-Leishmania and immunomodulatory treatments controlled clinical signs. The difference in the frequency of PustD in dogs with CanL and without CanL was statistically significant (P < 0.001).

Conclusions – An association exists between PustD and CanL. At least in Leishmania-endemic areas, CanL should be ruled out before attempting an immunosuppressive treatment in dogs with PustD with the aforementioned characteristics. Staging of CanL through diagnostic procedures besides IHC and PCR is recommended. Anti-Leishmania treatment and short-to-medium courses of low-dose anti-inflammatory or immunomodulatory drugs are effective in controlling the clinical signs of PustD.

 

背景 – 犬利什曼病(CanL)的脓疱性皮炎(PustD)的临床和组织病理学特征尚不明确;CanL和PustD之间的相关性仍未被证明。

目的–描述一系列犬利什曼病和瘙痒性PustD病例,并评估这两种疾病之间可能的相关性。

动物– 14只犬及2420份病历记录。

方法和材料–对14只犬进行临床和组织病理学回顾性研究。采用利什曼原虫免疫组化(IHC)和实时荧光定量 PCR技术,检测皮肤活组织中的婴儿利什曼原虫。用费希尔精确试验对2420份病历记录检测PustD与CanL的相关性。

结果–犬的特征:临床表现为全身瘙痒性无菌或抗生素无效的丘疹-脓疱性皮炎;组织学表现为中性粒细胞性脓疱,以及中性粒细胞和肥大细胞性血管周皮炎,伴血管扩张和水肿。6例IHC发现婴儿利什曼原虫, IHC 阴性的8个病例中,有3例PCR阳性。抗利什曼和免疫调节治疗控制临床症状。伴有或不伴有CanL的PustD发生率有显著性差异(P < 0.001)。

结论– PustD和CanL之间存在相关性。至少在利什曼病流行地区,在尝试对具有上述特征的犬进行免疫抑制治疗之前,应排除CanL。建议通过免疫组化和PCR诊断CanL。抗利什曼病治疗和中短期低剂量抗炎或免疫调节药物治疗,对控制PustD的临床症状有效。

 

Introduction

介绍

Cutaneous problems represent the most commonly reported clinical signs of canine leishmaniosis (CanL).These problems are diverse and include scaling, ulcerative, papular, nodular dermatitis, onychopathy, nasodigital hyperkeratosis, multifocal alopecia and pustular dermatitis (PustD). Scaling dermatitis and ulcerative lesions on bony prominences are considered frequent and suggestive signs, whereas the other signs are less frequent and/ or less typical. Pustular dermatitis has long been a controversial clinical presentation for CanL. In 1988, PustD was described in a dog with leishmaniosis; further cases of superficial neutrophilic sterile pustular dermatitis, including histopathological features, have since been reported.In 2015, PustD was described in a series of 22 dogs affected by CanL. That study included dogs with variably pruritic, multifocal to generalized PustD, characterized histologically by subcorneal neutrophilic pustules accompanied by variable degrees of acantholysis. Skin lesions were unresponsive to antibiotic therapy; bacteria were not detected on skin cytological evaluation in any dogs, bacterial cultures were not performed in all cases. Leishmania immunohistochemistry (IHC) was performed in 13 of 22 cases and was positive for only four. A favourable outcome was reported in 50% of the dogs treated with anti-Leishmanial therapy along with immunosuppressive therapies; a clear association between CanL and PustD could not be demonstrated.

犬利什曼病（CanL）最常见的临床症状是皮肤问题。这些问题是多种多样的，包括皮屑、溃疡、丘疹、结节性皮炎、甲病、鼻趾过度角化、多灶性脱毛和脓疱性皮炎（PustD）。骨性隆起处的皮屑性皮炎和溃疡性病变被认为是常见的提示性症状，而其他症状则不太常见和/或不太典型。脓疱性皮炎长期以来一直是CanL的一个有争议的临床表现。1988年，在一只利什曼病患犬身上发现了脓疱性皮炎，此后又报告了浅表性中性粒细胞性无菌性脓疱性皮炎的病例 ，包括组织病理学特征。2015年，在22只患CanL的犬身上发现了脓疱性皮炎。这项研究包括犬的不同程度的瘙痒，多灶性至全身性脓疱，其组织学特征是角质层下中性粒细胞脓疱，伴随不同程度的棘层松懈。皮肤病变对抗生素治疗无反应；在任何犬的皮肤细胞学检查中均未检测到细菌，所有病例均未进行细菌培养。22例中13例进行了利什曼原虫免疫组化（IHC）检测，仅4例阳性。50%的抗利什曼原虫治疗加免疫抑制治疗的犬有良好的结果；CanL和PustD之间没有明确的联系。

 

Based on the authors’ observation that in dogs with CanL, PustD can have a striking clinical and histopathological presentation, the primary objective of this retrospective study was to characterize clinically and histopathologically cutaneous problems in a series of 14 dogs affected by CanL accompanied by a sterile (negative bacterial culture) or antibiotic-unresponsive (positive bacterial culture with no response to antibiotics selected based on susceptibility test results) generalized pruritic PustD. A second objective was to evaluate a possible association between the two conditions. To test this association, a second study was performed using the medical records of 2,420 cases.

根据作者的观察，在犬CanL中，PustD可以有显著的临床和组织病理表现，本次回顾性研究的主要目的是描述一系列14只犬的临床和组织病理皮肤问题，这些犬患CanL，伴有无菌（阴性细菌培养）或抗生素无反应（阳性细菌培养，根据药敏试验结果选择抗生素无反应）全身性瘙痒性脓疱性皮炎。第二个目标是评估这两种疾病之间的可能联系。为了检验这种联系，我们使用2420例患犬的病历进行了第二项研究。

 

Methods and materials

方法和材料

Clinical and histopathological study

临床和组织病理学研究

Case selection

病例选择

The study population was dealt with by veterinarians in several referral or first opinion practices between 2006 and 2016. Criteria for enrolment were that dogs must present with generalized sterile or antibiotic-unresponsive PustD accompanied by pruritus and they also must have leishmaniosis diagnosed according to published criteria.The diagnosis of PustD had to be confirmed histopathologically: it was defined as sterile, in case of negative results of bacterial cultures (performed from intact pustules after pricking or from underneath the crusts) or antibiotic-unresponsive, if not responsive to antibiotic treatment selected according to susceptibility test results (Kirby Bauer test or minimum I nhibitory concentration determination performed in diagnostic laboratories). No personal identifiable data were used, and clinical pictures were obtained after verbal informed consent of the animal owners.

研究对象为2006年至2016年期间兽医进行了多次转诊或初诊病例。登记的标准是，犬必须出现全身无菌或抗生素无反应性脓疱性皮炎伴瘙痒，他们还必须根据公布的标准诊断利什曼病。脓疱性皮炎的诊断必须经组织病理学证实：定义为无菌，如果细菌培养结果为阴性（从完整的脓疱穿刺后或从结痂下进行）或抗生素无反应，如果对根据药敏试验结果选择的抗生素治疗无效（在诊断实验室进行的Kirby-Bauer试验或最低抑制浓度测定）。未使用个人身份资料，经动物主人口头知情同意后获得临床照片。

 

Clinical data collection

临床资料收集

Medical records were reviewed retrospectively. Data collected were signalment and type and distribution of skin lesions, and, whenever available, duration of skin disease, drugs administered before diagnosis, skin cytological and bacterial culture results, anti-Leishmania antibody levels and CanL stage at the time of diagnosis. Treatment and outcome also were recorded and long-term follow-up data were collected through review of medical records or telephone contact with referring veterinarians or dog owners.

回顾性病历分析。收集的数据包括皮肤病变特征、类型和分布，以及皮肤病的持续时间、诊断前用药、皮肤细胞学和细菌培养结果、诊断时抗利什曼原虫抗体水平和CanL分期。治疗和结果也被记录下来，并通过查阅医疗记录或与转诊的兽医或养犬人电话联系收集长期随访数据。

 

Histopathological study

组织病理学研究

Skin biopsies stained with haematoxylin and eosin were reviewed by two authors. The following histopathological changes were recorded：

i Epidermis: hyperplasia, hyperkeratosis, keratinocyteapoptosis, spongiosis, exocytosis, necrosis, crusts and pustules. Pustules were assessed taking into account their depth in the epidermis, size (involving one or more follicular units), cell content and presence of acantholytic cells.

ii Dermis: inflammatory cell distribution and types, oedema, vasodilation, red blood cell extravasation, presence of immature fibroblasts and adnexal lesions.

两位作者回顾了苏木精和伊红染色的皮肤活检。记录了以下组织病理学变化：

i表皮：增生、角化过度、角质形成细胞凋亡、海绵样水肿、细胞外排、坏死、结痂和脓疱。评估脓疱时考虑到它们在表皮的深度、大小（包括一个或多个毛囊单位）、细胞含量和棘层松懈细胞的存在。

ii真皮：炎性细胞分布和类型，水肿，血管扩张，红细胞外渗，存在未成熟成纤维细胞和附件损伤。

 

Immunohistochemistry and real-time PCR for Leishmania infantum

婴儿利什曼原虫的免疫组化和实时PCR检测

Leishmania IHC was performed and assessed in all skin biopsies of all included cases (n = 14). It was performed using a published avidin-biotin complex protocol with a polyclonal anti-Leishmania spp. antibody.

利什曼原虫IHC在所有病例的所有皮肤活检中进行并评估（n=14）。使用已发表的抗利什曼原虫多克隆抗体的抗生物素-生物素复合物方案。

 

A real-time quantitative (q)PCR TaqMan assay that targets the Leishmania kinetoplast minicircle DNA was performed in those samples that were IHC-negative (n = 8). DNA was extracted from the paraffin-embedded skin samples. Deparaffinization was performed as described previously; DNA extraction was carried out using the PureLink Genomic DNA Mini Kit (Invitrogen, Life Technologies; Carlsbad, CA, USA). The PCR assay has demonstrated a sensitivity of 0.001 parasites per PCR reaction, which corresponds to 0.2 parasites/mL. The eukaryotic 18S RNA Pre-Developed TaqMan Assay Reagents (Applied Biosystems; Foster City, CA, USA) was used as internal reference for canine genomic DNA amplification to ensure proper qPCR amplification of each sample and that negative results corresponded to true negative samples rather than to a problem with DNA loading, sample degradation or PCR inhibition. Relative quantification of Leishmania DNA was carried out based on the DCT results, using 18S RNA as an endogenous control. According to these DCT results, values were grouped as: low positive (>15); medium positive (5–15); high positive (<5); or very high positive (<0). If the Leishmania DNA was not determined, the result was considered negative.

在IHC阴性（n=8）的样品中，进行了以利什曼原虫运动细胞小圆DNA为靶点的实时定量（q）PCR-TaqMan分析。从石蜡包埋的皮肤样本中提取DNA。如前所述进行脱蜡；使用PureLink基因组DNA小型试剂盒（Invitrogen，Life Technologies；Carlsbad，CA，USA）进行DNA提取。PCR检测显示每个PCR反应的敏感性为0.001个寄生虫，相当于0.2个寄生虫/mL。真核18S RNA预先开发的TaqMan检测试剂（应用生物系统；加利福尼亚州福斯特市，（美国）被用作犬基因组DNA扩增的内部参考，以确保每个样本的qPCR扩增正确，并且阴性结果与真实阴性样本对应，而不是与DNA装载、样本降解或PCR抑制有关的问题。根据DCT结果，以18srna为内生对照，对利什曼原虫DNA进行了相对定量。根据这些DCT结果，值被分为：低阳性（>15）；中阳性（5-15）；高阳性（<5）；或非常高阳性（<0）。如果利什曼原虫的DNA没有确定，结果被认为是阴性的。

 

CanL and PustD statistical association study

CanL与PustD统计相关性研究

Case selection

病例选择

In order to assess if there was an association between pruritic, sterile or antibiotic-unresponsive PustD with an unidentified underlying cause and CanL, the small animal dermatology case log of one of the authors, comprising 2,420 cases seen during 11 years in a CanL-endemic area, was searched for dogs with these two entities. The association of PustD with CanL was tested using Fisher’s exact test. A P-value <0.05 was considered significant and the Odds Ratio (OR) with a confidence interval of 95% was calculated.

为了评估瘙痒性、无菌性或非抗生素反应性脓疱性皮炎是否与CanL有关联，作者之一的小动物皮肤科病例记录，包括在CanL流行区11年内发现的2420例病例，被搜索到有这两种实体的犬。用Fisher精确检验检验PustD与CanL的相关性。P值<0.05被认为是显著的，计算了置信区间为95%的优势比（OR）。

 

Results

结果

Clinical and histopathological study

临床和组织病理学研究

Signalment, history and clinical summary

特征、病史和临床总结

Fourteen cases were included; their signalment details (breed, age and sex), history and tests performed for the diagnosis of CanL and PustD are summarized in Table 1. The median age of the study cases when skin lesions appeared was 3.5 years (range 1–6 years). Eight dogs developed lesions in early adulthood (between 1 to 3 years of age) and six dogs in mid-adulthood (between 4 and 7 years of age), with only three dogs being 6-yearsold at the time of diagnosis.

包括14例，他们的特征细节（品种、年龄和性别）、病史和诊断CanL和PustD的测试总结在表1中。出现皮肤病变的研究病例的中位年龄为3.5岁（1-6岁）。8只犬在成年早期（1至3岁）出现病变，6只犬在成年中期（4至7岁）出现病变，只有3只犬在诊断时正好6岁。

 

Skin lesions affected haired (trunk, extremities and head) and sparsely haired areas such as abdomen and, typically, the concave aspect of the pinna, but they spared glabrous skin, including nasal planum, except in Case 4, in which footpad hyperkeratosis was observed. Lesions consisted of erythematous papules, pustules, markedly erythematous collarettes with occasional polycyclic or arciform configuration, and yellowish scales and crusts adherent to the skin surface (Figures 1 and 2). When affecting haired skin, these lesions were not necessarily associated with alopecia. In all dogs, lesions were accompanied by generalized intense pruritus, except in one case that presented with moderate pruritus. Total or partial alopecia due to severe pruritus also was observed (Figure 1). Two dogs presented with concurrent hyperthermia (40°C) (cases 12 and 14) and two other showed distal extremities oedema (cases 11 and 13).

皮肤病变影响毛发（躯干、四肢和头部）和毛发稀疏的区域，如腹部，通常是耳廓的凹部，但它们保留了无毛皮肤，包括鼻平面，除了病例4，在病例4中观察到足垫角化过度。病变包括发红丘疹、脓疱、明显的环形发红，偶尔有多环或弓形结构，皮肤表面粘附有淡黄色的皮屑和结痂（图1和图2）。当影响有毛皮肤时，这些损伤不一定与脱毛有关。除1例表现为中度瘙痒外，所有犬的皮肤病变均伴有全身性剧烈瘙痒。可见严重瘙痒引起的全部或部分脱毛（图1）。2只犬同时出现40℃高温（病例12和14），另外2只犬出现远端肢体水肿（病例11和13）。

 

Four dogs had been serologically tested for CanL before the onset of PustD: one was negative (three months before), one was low positive (one month before) and two were high positive (one four years before and the other one month before). Five dogs were known to have received no treatments before the onset of clinical signs. One dog (Case 2) with high anti-Leishmania antibody levels had been vaccinated with CaniLeish (Virbac Espana SA; Esplugues~ de Llobregat, Spain) three months before the onset of clinical signs, after testing negative with Speed–Leish K (Virbac Espana~ SA). One dog (Case 6) had received an annual booster (MLV-CPV vaccine) four days before developing PustD. One dog (Case 4) had been treated with allopurinol and doxycycline for the previous four weeks before the onset of PustD. Four dogs (cases 4, 8, 12 and 14) had been treated with antibiotics before the diagnosis of generalized PustD. The median duration of clinical signs at the point of diagnosis was one month, being one month or less in eight dogs and longer in five dogs (two years maximum).

4只犬在PustD发病前进行了CanL血清学检测：1只为阴性（3个月前），1只为弱阳性（1个月前），2只为强阳性（1年前和1个月前）。已知五只犬在出现临床症状之前没有接受任何治疗。一只抗利什曼原虫抗体水平高的犬（病例2）在用Speed-Leish K（Virbac Espana SA）检测阴性后，在出现临床症状前3个月接种了犬疫苗。其中一只犬（病例6）在患上PustD前4天接受了年度加强免疫（MLV-CPV疫苗）。1只犬（病例4）在PustD发病前4周用别嘌呤醇和多西环素治疗。4只犬（病例4、8、12、14）在确诊为全身性脓疱性皮炎前均用抗生素治疗。诊断时临床症状的中位持续时间为1个月，8只犬的中位持续时间为1个月或更短，5只犬的中位持续时间更长（最长两年）。

 

At the time of diagnosis of PustD, all dogs were staged II–III using the criteria of the LeishVet group.All dogs were serologically tested for anti-Leishmania antibody levels through enzyme-linked immunosorbent assay (ELISA) or immunofluorescence antibody test (IFAT). High antibody levels (defined as a 3–4-fold elevation above the cut-off level of the laboratory) were detected in 10 of 14 dogs. The presence of Leishmania in bone marrow or blood was demonstrated in six dogs; bone marrow PCR was positive in all tested cases, including those with low anti-Leishmania antibody levels (Table 1).

诊断为PustD时，所有犬均按LeishVet组标准分为II-III期，用酶联免疫吸附试验（ELISA）或免疫荧光抗体试验（IFAT）检测抗利什曼原虫抗体水平。14只犬中有10只检测到高抗体水平（定义为高于实验室截止水平3-4倍）。6只犬的骨髓或血液中发现利什曼原虫；所有受试病例的骨髓PCR均为阳性，包括抗利什曼原虫抗体水平较低的病例（表1）。

 

Cutaneous cytological investigation, performed from intact pustules after pricking or from underneath the crusts, results were available for eight dogs. Neutrophils were observed in all cases, eosinophils were present in four (cases 1, 2, 4 and 6), macrophages in three (cases 4, 12 and 14), and lymphocytes and plasma cells in cases 2 and 4 respectively. Bacteria (cocci) were observed in two dogs (cases 3 and 13) and free nucleated keratinocytes in one dogs (Case 6). Bacterial cultures were performed from skin lesions in 11 cases and in four yielded a negative result. In the remaining cases meticillin-sensitive (n = 5) or meticillin-resistant (n = 2) staphylococci were isolated. In one dog (Case 14) a beta-haemolytic Streptococcus also was isolated (Table 1).

从完整的脓疱或从结痂下刺入进行皮肤细胞学检查，得到了八只犬的结果。所有病例均可见中性粒细胞，其中嗜酸性粒细胞4例（1、2、4、6），巨噬细胞3例（4、12、14），淋巴细胞、浆细胞2例、4例。在2只犬（病例3和13）中观察到细菌（cocci），在1只犬（病例6）中观察到游离有核角质形成细胞。皮肤病变细菌培养11例，阴性4例。其余病例均分离到对甲氧西林敏感（n=5）或对甲氧西林耐药（n=2）的葡萄球菌。在一只犬（病例14）中，也分离出β溶血性链球菌（表1）。

 

Histopathological findings

组织病理学表现

All 14 cases presented with epidermal hyperplasia; in four it was accompanied by orthokeratotic or parakeratotic hyperkeratosis. Spongiosis was observed in 11 cases and neutrophilic exocytosis in 12 cases, together with eosinophils (two cases) and lymphocytes (one case). Intracorneal and/or subcorneal pustules involving more than one follicular ostium were present in all cases. Pustules contained degenerated neutrophils (n = 14), keratin (n = 12), proteinaceous material (n = 7), eosinophils (n = 3), cocci and hairs (two cases each). Moreover, sparse intrapustular acantholytic keratinocytes were observed in 11 of 14 cases. Crusts were present in seven of 14, five of which formed waves of alternating degenerated polymorphonuclear cells and keratin. In nine of 14 cases, necrosis of the epidermis underneath the pustules was observed (Figure 3).

14例均表现为表皮增生，4例伴有正角化性或角化不全性角化过度。观察11例海绵样水肿，12例中性粒细胞增多，2例嗜酸性粒细胞增多，1例淋巴细胞增多。所有病例均出现涉及一个以上毛囊单位的角质层内和/或角质层下脓疱。脓疱内有退行性中性粒细胞（n=14）、角蛋白（n=12）、蛋白样物质（n=7）、嗜酸性粒细胞（n=3）、球菌和毛发（各2例）。14例中11例可见稀疏的表皮内棘层松懈角质形成细胞。结痂在14例中有7例出现，其中5例形成了退行性多核细胞细胞和角蛋白交替表现。14例中有9例脓疱下表皮坏死（图3）。

 

Regarding dermal findings, all 14 cases showed a perivascular to interstitial mixed inflammatory infiltrate in the superficial and mid-dermis which was more marked underneath pustules. This infiltrate was composed of neutrophils (13 of 14), mast cells (13 of 14), plasma cells (seven of 14), lymphocytes (eight of 14), histiocytes (six of 14) and eosinophils (four of 14); mild peri-isthmal granulomas were observed only in one case. In the superficial dermis, vasodilation (13 of 14), oedema (12 of 14), erythrocyte extravasation (nine of 14) and immature fibroblasts (10 of 14) also were observed (Figure 3).

关于真皮的发现，所有14例病例均在真皮浅表和中层出现血管周围至间质的混合炎症浸润，在脓疱下方更为明显。浸润由中性粒细胞（13/14）、肥大细胞（13/14）、浆细胞（7/14）、淋巴细胞（8/14）、组织细胞（6/14）和嗜酸性粒细胞（4/14）组成；仅1例可见轻度毛囊周围肉芽肿。在浅层真皮中，还观察到血管扩张（14例中的13例）、水肿（14例中的12例）、红细胞外渗（14例中的9例）和未成熟成纤维细胞（14例中的10例）（图3）。

 

IHC and PCR results

IHC和PCR结果

The IHC and PCR results are summariszed in Table 1. IHC showed Leishmania amastigotes in six cases (Figure 4); in one case a single amastigote was visualized (Case 9). In the eight IHC-negative cases and the one with a single amastigote, qPCR was performed from the paraffinembedded skin and yielded the following results: four positive cases (three medium positive and one highly positive, the latter corresponding to Case 9, with one amastigote positive to the IHC); and five negative cases of which two were negative for the internal reference for canine genomic DNA (repeated twice).

IHC和PCR结果总结见表1。IHC显示6例利什曼原虫无鞭毛体（图4）；1例显示单个无鞭毛体（病例9）。8例IHC阴性和1例IHC阳性者，取石蜡包埋皮肤行qPCR，结果：4例阳性（3例中阳性，1例强阳性，后者对应9例，1例IHC阳性）；5例阴性，其中2例为犬基因组DNA内参照阴性（重复2次）。

 

Clinical management and follow-up

临床管理与随访

Data on PustD treatment and follow-up are shown in Table S1. Antibiotic treatment (including cefalexin, amoxicillin-clavulanate, enrofloxacin, doxycycline and rifampicin) based on susceptibility testing results did not lead to clinical improvement in any case (Table 1). Thirteen dogs received glucocorticoids to control pruritus and skin lesions. Prednisone at a dose of 0.2 to 2 mg/kg/day (mean 1 mg/kg/day) was used in ten cases and methylprednisolone at a dose of 0.3–1 mg/kg/day (mean 0.58 mg/kg/ day) in four cases (case 7 received both prednisone and methylprednisolone). The total duration of glucocorticoid treatment was known for 12 patients, it ranged between one and seven months (mean of 2 months). A minimum of one week of glucocorticoids was needed to induce remission of the lesions. Four dogs (cases 3, 5, 12 and 13) received hydrocortisone aceponate spray (Cortavance, Virbac Espana SA) once daily (cases 3 and 5) or every other~ day (cases 12 and 13) to lower the dose of systemic glucocorticoids at the beginning of the treatment (Case 5) or to treat residual pustular lesions during corticosteroid maintenance on alternating days (cases 3, 12 and 13). Treatment with hydrocortisone aceponate spray lasted one week in Case 3, three months in Case 5 and 15 days in Case 12; the duration was unknown in Case 13.

PustD治疗和随访数据见表S1。基于药敏试验结果的抗生素治疗（包括头孢氨苄、阿莫西林克拉维酸钾、恩诺沙星、多西环素和利福平）在所有病例中均未得到临床改善（表1）。13只犬接受糖皮质激素来控制瘙痒和皮肤病变。泼尼松0.2～2mg/kg/天（平均1mg/kg/天）10例，甲基泼尼松龙0.3～1mg/kg/天（平均0.58mg/kg/天）4例（7例同时使用泼尼松和甲基泼尼松龙）。据了解，糖皮质激素治疗的总持续时间为12例，介于1至7个月（平均2个月）之间。至少需要一周的糖皮质激素来诱导病变的缓解。四只犬（病例3、5、12和13）接受氢化可的松醋丙酯喷雾剂（皮乐美）每日一次（病例3和5）或每隔日一次（病例12和13），以在治疗开始时降低全身糖皮质激素的剂量（病例5）或在交替的皮质类固醇维持期间治疗残余脓疱性皮炎（病例3、12和13）。氢化可的松醋丙酯喷雾剂治疗3例1周，5例3个月，12例15天，13例持续时间未知。

 

Besides glucocorticoids, tetracycline and azathioprine were employed as immunomodulatory-immunosuppressive treatment. Five dogs (cases 2, 3, 4, 5 and 11) were treated with tetracyclines, with/without nicotinamide (Table S1). One dog (Case 3) had a flare-up of the PustD (on cytological evaluation, only neutrophils were detected) two months after the lesions cleared and was controlled with doxycycline (5 mg/kg twice daily) and nicotinamide (500 mg three times a day) for four months. Another dog (Case 5) was maintained with tetracycline and nicotinamide (500 mg each three times a day) for one year to keep PustD in remission without the need of glucocorticoids. One dog (Case 7) was treated with azathioprine (1 mg/kg once daily for two weeks then every 48 h) together with glucocorticoids. All cases needed anti-inflammatory-immunomodulatory treatment to control clinical signs except Case 1, which had less generalized clinical signs and less severe pruritus. This dog responded completely to allopurinol and meglumine antimoniate. Standard doses of allopurinol and meglumine antimoniate2 were administered in all dogs and the duration of the treatment was known for ten dogs. Allopurinol was given long term in all dogs but three (cases 4, 8 and 14), and meglumine antimoniate for one to two months in all dogs but three (cases 8, 9 and 11). In two dogs, meglumine antimoniate treatment was administered until Leishmania bone marrow qPCR was negative (seven and five weeks, respectively, in cases 2 and 13). In two dogs (cases 9 and 11) miltefosine was administered.

除糖皮质激素外，四环素和硫唑嘌呤也被用作免疫调节性免疫抑制治疗。5只犬（病例2、3、4、5和11）分别是用四环素类药物治疗，含/不含烟酰胺（表S1）。一只犬（病例3）在病变清除两个月后出现PustD（细胞学检查仅检测到中性粒细胞），并用多西环素（5 mg/kg，每天两次）和烟酰胺（500 mg，每天三次）控制4个月。另一只犬（病例5）用四环素和烟酰胺（每天三次，每次500毫克）维持一年，以保持脓疱性皮炎得以缓解，而不需要糖皮质激素。一只犬（病例7）用硫唑嘌呤（1 mg/kg，每日一次，持续两周，然后每48小时）和糖皮质激素治疗。除1例临床症状较轻，瘙痒较轻外，其余均需抗炎免疫调节治疗。这只犬对别嘌呤醇和锑葡胺完全有效。所有犬均服用标准剂量的别嘌呤醇和抗氨葡胺，已知10只犬的治疗持续时间。除3只（病例4、8、14）外，所有犬均长期服用别嘌呤醇；除3只（病例8、9、11）外，所有犬均长期服用抗氨葡胺1～2个月。在两只犬中，给予葡甲胺锑酸盐治疗，直到利什曼原虫骨髓qPCR阴性（在病例2和13中分别为7周和5周）。在两只犬（病例9和11）中，使用了米替福新。

 

One case was lost to follow-up and 11 of the remaining 13 cases responded well to treatment with resolution of PustD and pruritus. Two dogs responded partially to treatment: Case 14, euthanized at owner request due to sudden clinical worsening and Case 8 that died of renal failure 2.5 months after diagnosis. Case 7, a responder dog, also died of renal failure seven months after diagnosis. Nine dogs (69%) did not show relapses and were free of clinical signs at the end of the follow-up period, which was between eight months and four years. Of these nine cases, six were known to be receiving allopurinol at the time of follow-up contact.

1例失访，其余13例中11例治愈，脓疱、瘙痒消失。两只犬对治疗有部分反应：病例14，因临床突然恶化而应主人要求实施安乐死，病例8在诊断后2.5个月死于肾功能衰竭。病例7，一只有反应的犬，在确诊7个月后也死于肾衰竭。9只犬（69%）没有复发，在8个月到4年的随访期结束时没有临床症状。在这9例患者中，有6例在随访时已知正在接受别嘌呤醇治疗。

 

CanL and PustD statistical association study

CanL与PustD统计相关性研究

Of the 2,420 dogs of the small animal dermatology case log, 16 had a pruritic, nonantibiotic-responsive or sterile PustD with unidentified underlying cause [excluding pemphigus foliaceous (PF)], 12 were dogs with leishmaniosis and four were dogs without leishmaniosis (Table 2). The frequency of PustD in dogs with CanL was 11.65% (12 of 103) and frequency of PustD in dogs without CanL was 0.2% (four of 2,317) [OR 76.25 (24.12–241.02)]. This difference between the two groups was statistically significant (P < 0.001).

在小动物皮肤病学病例记录的2420只犬中，有16只患有瘙痒、非抗生素反应性或无菌性脓疱性皮炎，其根本原因不明[不包括落叶型天疱疮（PF）），12只犬患有利什曼病，4只犬没有利什曼病（表2）。有CanL的犬PustD发生率为11.65%（12/103），无CanL的犬PustD发生率为0.2%（4/2317）[或76.25（24.12-241.02）]。两组间差异有统计学意义（P<0.001）。

 

Discussion

讨论

Our primary objective was to clinically and histologically characterize dogs with pruritic PustD and CanL. Accordingly, all cases reported herein had papular, pustular and/ or crusting lesions together with pruritus. However, in agreement with previous studies, PustD in dogs with concomitant CanL also may present without pruritus. In fact, half of the dogs in a previous study had nonpruritic PustD,and the first reported cases of PustD in dogs suffering from leishmaniosis were not pruritic; clinical and histological overlap with PF was observed occasionally in the cases reported.

我们的主要目标是犬瘙痒脓疱和CanL的临床和组织学特征。所有报告的病例都有丘疹、脓疱和/或结痂性病变并伴有瘙痒。然而，与先前的研究一致，伴发CanL的犬的脓疱性皮炎也可能出现无瘙痒。事实上，在之前的一项研究中，一半的犬有非瘙痒性PustD，首次报告的利什曼病犬PustD病例不是瘙痒性的；在所报告的病例中，偶尔观察到临床和组织学上与PF重叠。

 

The distribution of lesions in the PustD described herein was more suggestive of immune-mediated superficial pustular disease, for example, subcorneal pustular dermatosis or superficial pustular drug reaction, rather than of papulo-pustular dermatitis due to follicular-oriented bacterial infections. Lesions in fact involved the face, both sides of the ear pinnae and the distal extremities, body regions not usually affected by canine pyoderma. In addition, pustular lesions were not always associated with alopecia. Unlike what is reported for PF, most dogs suffering from this PustD were intensely pruritic and glabrous skin was generally not affected, with the exception of footpad hyperkeratosis in Case 4. The lack of response to appropriate antibiotic treatment chosen according to susceptibility testing results and the good response to glucocorticoids despite bacteria isolation may be explained by the presence of a superinfection on an immune-mediated process or by isolation of resident staphylococci not directly implicated in the pathogenesis of lesions. Nevertheless, the isolation of bacteria prompted us to refer to these cases as “antibiotic-unresponsive” instead of “sterile” pustular dermatitis.

本文描述的脓疱性皮炎的分布更提示免疫介导的浅表性脓疱性皮炎，例如，角质层下脓疱性皮肤病或浅表性脓疱药物反应，而不是毛囊导向细菌感染引起的丘疹性脓疱性皮肤炎。病变事实上累及面部、耳廓两侧和四肢远端，体表区域通常不受犬脓皮病的影响。此外，脓疱性皮炎并不总是与脱毛有关。与PF不同的是，大多数患有这种脓疱性皮炎的犬都有严重的瘙痒，无毛的皮肤一般不会受到影响，除了病例4中的足垫角化过度。根据药敏试验结果对适当的抗生素治疗无效，尽管细菌被分离，但对糖皮质激素的良好反应可能是由于免疫介导过程中存在双重感染或分离了与病程没有直接关系的常驻葡萄球菌引起的损伤。然而，细菌的分离促使我们将这些病例称为“抗生素无反应”而不是“无菌”脓疱性皮炎。

 

Histologically, the majority of cases were characterized by broad intracorneal or subcorneal pustules and crusts, few acantholytic cells, epidermal hyperplasia and a perivascular to interstitial mixed inflammatory infiltrates with predominant neutrophils and mast cells and, less commonly, eosinophils; thus, findings were comparable to PustD as described previously. However, histopathological lesions such as apoptotic keratinocytes or high number of free-floating epidermal cells (acantholytic cells), which are characteristic of canine PF,12 were not observed. We believe that epidermal spongiosis, vasodilation, dermal oedema, erythrocyte extravasation and necrosis underneath the pustules represent additional histological findings useful to orientate the suspicion of the pathologist towards a PustD possibly associated with CanL.

在组织学上，大多数病例的特征是广泛的角质层内或角质层下脓疱和结痂，少量棘层松解细胞，表皮增生，血管周围至间质混合炎性浸润，主要是中性粒细胞和肥大细胞，不常见的是嗜酸性粒细胞；因此，结果与前面描述的PustD相似。然而，未观察到具有犬PF特征的组织病理学损伤，如角质形成细胞凋亡或大量游离表皮细胞（棘层松解细胞），12例。我们认为，脓疱下方的表皮海绵样水肿，血管扩张，皮肤水肿，红细胞外渗和坏死代表了有助于病理学家怀疑可能与CanL相关的PustD的其他组织学发现。

 

It is worth pointing out that eight cases had a negative IHC and five of these IHC-negative cases also were negative to qPCR performed on paraffin-embedded skin. Two cases were negative due to insufficient amount of DNA/ RNA in the sample and three due to lack of Leishmania DNA. Considering that qPCR is a technique with a sensitivity of 0.001 parasites per PCR reaction, the two latter cases could be negative because the Leishmania organisms were indeed absent or very low in number. Alternatively, it might be hypothesized that skin processing to obtain the paraffin blocks destroyed the Leishmania DNA.

值得指出的是，8例IHC阴性，其中5例对石蜡包埋皮肤qPCR阴性。2例因DNA/RNA含量不足而呈阴性，3例因利什曼原虫DNA缺乏而呈阴性。考虑到qPCR是一种每PCR反应敏感度为0.001个寄生虫的技术，后两个病例可能是阴性的，因为利什曼原虫确实不存在或数量非常少。另一种可能的假设是，获取石蜡切片的皮肤加工破坏了利什曼原虫的DNA。

 

Degradation of nucleic acids through processing also could explain the lack of amplification of canine 18S RNA in the other two samples. Our results suggest that either Leishmania IHC or, less commonly, qPCR can be negative in skin biopsies from dogs with PustD and CanL. As a consequence, other procedures commonly suggested to confirm or rule out the diagnosis of CanL and to stage the disease,such as serological investigation, are recommended.

加工过程中核酸的降解也可以解释另外两个样本中犬18srna缺乏扩增的原因。我们的结果表明，利什曼原虫IHC或不太常见的qPCR在PustD和CanL犬的皮肤活检中可能是阴性的。因此，建议采用血清学调查等其他方法来确定或排除CanL的诊断和分期。

 

Even though no histological signs suggestive of drug reaction, such as keratinocyte apoptosis, were observed in any of the cases, the possibility of an adverse drug reaction cannot be completely excluded at least in six cases, including the two vaccinated dogs (cases 2 and 6), the dog that had been receiving allopurinol and doxycycline for the previous four weeks (Case 4) and the three dogs that had received cefalexin, sulphametopirazine or enrofloxacin before PustD diagnosis (cases 8, 12 and 14, respectively).

尽管在任何病例中均未观察到任何提示药物反应的组织学症状，如角质形成细胞凋亡，但至少有6例（包括两条免疫犬（病例2和6））不能完全排除药物不良反应的可能性，前4周接受别嘌呤醇和多西环素治疗的犬（病例4）和诊断前接受头孢氨苄、磺胺嘧啶或恩诺沙星治疗的犬（病例8、12和14）。

 

Treatment of PustD in dogs with CanL can be challenging because anti-inflammatory or immunosuppressive treatment needs to be given to a dog with a systemic infectious disease. In a previous case series of 22 cases, treatment of PustD involved anti-Leishmania drugs plus long-term immunomodulatory treatment with corticosteroids, azathioprine or ciclosporin and a favourable response was observed in 50% of the dogs. In the present case series, treatment involved anti-Leishmania drugs as well, but shorter courses and lower doses of glucocorticoids were administered. In addition, tetracyclines with/without nicotinamide were used as immunomodulatory drugs and low-dose azathioprine was used only in Case 7. Good response to treatment was observed in 12 (85%) dogs, nine of which (69%) were alive and free of clinical signs for eight months to four years follow-up. Accordingly, treatment with low anti-inflammatory doses of glucocorticoids without addition of immunosuppressive drugs seemed to be associated with a more favourable prognosis. Nevertheless, the small number of cases included in the study does not allow a definitive conclusion to be drawn.

在患有CanL的犬身上治疗PustD可能是一个挑战，因为对于患有全身性传染病的犬，需要进行抗炎或免疫抑制治疗。在先前的22例病例中，PustD的治疗包括抗利什曼原虫药物和皮质类固醇、硫唑嘌呤或环孢素的长期免疫调节治疗，在50%的犬中观察到良好的反应。在本病例系列中，治疗也涉及抗利什曼原虫药物，但疗程较短，使用的糖皮质激素剂量较低。另外，四环素类药物加/不加烟酰胺作为免疫调节药物，小剂量硫唑嘌呤仅用于病例7。12只犬（85%）对治疗有良好反应，其中9只（69%）存活，8个月至4年随访无临床症状。因此，在不加免疫抑制药物的情况下，用低剂量的糖皮质激素抗炎治疗似乎与更有利的预后有关。尽管如此，这项研究中包含的少数病例并不能得出明确的结论。

 

In this study, we also attempted to find out, as a second objective, if there was any association between CanL and PustD characterized by the aforementioned features. The statistical analysis performed herein suggests that this association exists and, at least in Leishmania-endemic areas, points to a 76 times greater risk of finding both diseases concomitantly rather than PustD without CanL. There are no studies or hypotheses linking a pustular eruption to CanL. Two scenarios could be proposed: (i) an idiopathic or drug-related immune-mediated PustD develops in a Leishmania-infected dog, causes an immune dysregulation that favours the switching of Leishmania infection to a disease state (CanL); or (ii) an immune-mediated PustD develops, by means of different and still unknown mechanisms, secondary to immune dysregulation due to CanL. Also, human sterile neutrophilic dermatoses are associated with several underlying systemic diseases. Moreover, neutrophils, which are able to phagocytose Leishmania amastigotes, are considered to play a first and essential role in leishmaniosis transmitted by sand flies and Leishmania promastigotes can induce neutrophil migration by releasing the potent chemotactic factor LCF. Furthermore, activated Toll-like receptors, especially those on dermal dendritic cells, result in the production of interleukin (IL)8, which induces neutrophil recruitment at the site of Leishmania promastigote inoculation.Accordingly, a neutrophil dysregulation directly associated with the presence of Leishmania also could be proposed. However, because PustD is seen in a small numbers of dogs with leishmaniosis, other still unknown underlying factors are needed to explain the development of PustD in CanL.

在这项研究中，我们还试图找出，作为第二个目标，CanL和PustD的特点是否有任何关联，上述特点。本文所做的统计分析表明，这种关联存在，并且至少在利什曼原虫流行区，指出同时发现这两种疾病的风险比没有CanL的PustD高76倍。没有研究或假说将脓疱疹与CanL联系起来。可以提出两种情况：（i）在利什曼原虫感染的犬中发展出特发性或药物相关的免疫介导的PustD，引起有利于利什曼原虫感染转变为疾病状态（CanL）的免疫失调；或（ii）免疫介导的PustD通过不同且仍然未知的机制发展，继发于CanL引起的免疫失调。此外，人类无菌性中性粒细胞性皮肤病与一些潜在的系统性疾病有关。此外，中性粒细胞可以吞噬利什曼原虫无鞭毛体，被认为是沙蝇传播利什曼病的首要和必要因素，利什曼原虫可以通过释放有效的趋化因子LCF诱导中性粒细胞迁移。此外，激活的Toll样受体，特别是真皮树突状细胞上的Toll样受体，导致白细胞介素（IL）8的产生，从而诱导利什曼原虫前鞭毛体接种部位的中性粒细胞募集，因此，也可以认为与利什曼原虫存在直接相关的中性粒细胞失调。然而，由于PustD见于少数利什曼病犬，还需要其他未知的潜在因素来解释PustD在CanL的发展。

 

Inherent with performing a retrospective study, limitations encountered while analyzing the data included a lack of predetermined standardization in diagnostic investigations, treatment protocols, dog follow-up, medical documentation and criteria used to make therapeutic changes.

由于进行回顾性研究，在分析数据时遇到的局限性包括在诊断调查、治疗方案、犬的随访、医疗文件和用于进行治疗改变的标准方面缺乏预先确定的标准。

 

In conclusion, a statistical association of antibiotic-unresponsive or sterile PustD with the aforementioned characteristics exist. Accordingly, PustD should be included in the broad clinical spectrum of CanL. Therefore, at least in Leishmania-endemic areas, CanL should be ruled out before attempting an immunosuppressive treatment in dogs with this kind of PustD. When diagnosing CanL, it should be taken into account that even though Leishmania can be detected in skin sections through IHC or PCR, results of these techniques can be negative. As a consequence, diagnosis of CanL in dogs with PustD through other procedures (e.g. serological investigation) is recommended. Anti-Leishmania treatment, along with short-tomedium courses of low-dose anti-inflammatory or immunomodulating drugs, is effective in controlling the PustD clinical signs. In the majority of dogs of this case series the PustD did not recur.

总之，抗生素无反应或无菌性PustD与上述特征存在统计关联。从而，PustD应纳入广泛的CanL临床谱。因此，至少在利什曼原虫流行区，在尝试用免疫抑制剂治疗这种脓疱性皮炎的犬之前，应该排除CanL。在诊断CanL时，应考虑到即使皮肤切片可以通过IHC或PCR检测到利什曼原虫，但这些技术的结果可能是阴性的。因此，建议通过其他程序（如血清学调查）诊断患有PustD的犬的CanL。抗利什曼原虫治疗，加上短疗程的小剂量抗炎或免疫调节药物，对控制PustD的临床症状是有效的。在本病例组的大多数犬中，脓疱性皮炎没有复发。

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 1. Signalment, history and diagnostic tests for 14 dogs with canine leishmaniosis (CanL) and sterile or antibiotic-unresponsive pustular dermatitis (PustD) Case number and signalment History Previous CanL tests- Previously administered drugs/vaccines Duration of clinical signs of PustD Tests results of CanL and PustD   Leishmania tests Leishmania in skin biopsy (IHC, real-time PCR) Bacterial culture and response to antibiotics 1 Cross-breed, F, 2 yo None None 3 weeks ELISA high + IHC – PCR + Negative 2 Labrador retriever, SF, 4 yo Negative Speed– Leish K (3 months earlier) CaniLeish (3 months earlier) 1 month ELISA high + IHC + Negative 3 Great Dane, M, 6 yo Diagnosis of CanL (4 years earlier) No treatments during the last year 3 weeks ELISA high + IHC + MSSP; no response to cefalexin 4 Spanish water dog, F, 1 yo Diagnosis of CanL (1 month earlier). Allopurinol and doxycycline during 1 month 1 week ELISA high + IHC – PCR + Not performed 5 None None 2.5 months ELISA high + IHC – PCR – Negative Cross-breed, SF, Blood qPCR 4 yo high + 6 Labrador retriever, M, 1.5 yo None Annual booster vaccination (4 days earlier) 1 week ELISA high + IHC – PCR – (PCR genomic DNA -) Negative 7 Unknown Unknown 9 months IFAT + BM IHC – PCR – MSSP; partial Brittany, M, cytological (PCR response to 3.5 yo results + genomic cefalexin and DNA –) enrofloxacin 8 Labrador retriever, M, 3 yo None Cefalexin for pinnal erythema and oedema (1 month earlier) with no response. 1 month IFAT high + BM PCR + IHC – PCR + MSS; no response to amoxicillinclavulanate 9 None None 2 months ELISA + BM IHC + (1 Not performed, Dalmatian cross, PCR + amastigote) antibiotic NM, 3 yo PCR + (unknown); unresponsive 10 Westphalian Dachsbracke, F, 3 yo Unknown Unknown 2 years IFAT low + BM cytological results+ IHC – PCR – Not performed Antibiotic (unknown); unresponsive 11 Unknown Unknown 2 months IFAT high + IHC + MSSP; no response Cross-breed, M, to clindamycin 6 yo 12 Pointer, SF, 6 yo None Sulphametopyrazine for muzzle alopecia (1 month earlier) with no response 1 month IFAT high + IHC + MSSP; no response to cefalexin 13 Low positive ELISA None 2 months ELISA high + IHC - PCR - MDR-MRSP; no Rhodesian ridge- (1 month earlier) response to back, M, 3 yo doxycycline and rifampicin 14 Jack Russell terrier, F, 2 yo None Enrofloxacin for papular-PustD (3 months earlier) with no response 3 months ELISA low + BM PCR + IHC + MDR-MRSP and beta-haemolytic Streptococcus; no response to enrofloxacin BM, bone marrow; ELISA, enzyme-linked immunosorbent assay; F, female; IFAT, indirect fluorescent antibody test; IHC, immunohistochemistry; M, male; MSS, meticillin-susceptible Staphylococcus; MSSP, meticillin-susceptible Staphylococcus pseudintermedius; MRSP, meticillin-resistant Staphylococcus pseudintermedius; MDR, multidrug-resistant; NM, neutered male; qPCR, quantitative PCR; SF, spayed female; #, number; +, posi tive; -, negative; yo, year old.                             表1.14只犬利什曼病（CanL）和无菌或抗生素无反应性脓疱性皮炎（PustD）的特征、病史和诊断试验 病例号及特征 病史 以前的CanL测试- 以前服用的药物/疫苗 临床症状持续时间  CanL 和 PustD测试结果   利什曼原虫试验 皮肤活检利什曼原虫 （IHC，实时 聚合酶链反应）     细菌培养及耐药试验 1 杂交品种,  F, 2yo 无 无 3周 ELISA high + IHC – PCR + 阴性 2 拉布拉多犬，SF,4yo 阴性Speed– Leish K® (3个月之前) CaniLeish®  (3 个月前) 1 月 ELISA high + IHC + 阴性 3 大丹犬,M, 6 yo CanL的诊断 （4年前） 去年没有治疗 3 周 ELISA high + IHC + MSSP; 头孢氨苄无反应 4 西班牙水犬,  F, 1 yo CanL的诊断 (1个月前). 别嘌呤醇和多西环素1个月 1 周 ELISA high + IHC – PCR + 未执行 5 无 无           2.5 月        ELISA high +       IHC – PCR –         阴性 杂交品种, SF,4 yo                                                                  血液 qPCR high + 6 拉布拉多犬,  M, 1.5 yo 无 年度强化疫苗接种（提前4天） 1 周 ELISA high + IHC – PCR – (PCR 基因组DNA -) 阴性 7 未知 未知        9 月 IFAT + BM IHC – PCR –          MSSP; 对头孢氨苄 布列塔尼,                                                                                                        和恩诺沙星有反应 M,3.5 yo 细胞学结果+    (PCR基因组 DNA –) 8 拉布拉多犬, M, 3yo 无 头孢氨苄治疗耳廓红斑水肿（1个之前）无反应。 1月 IFAT high + BM PCR + IHC – PCR + MSS; 对阿莫西林无反应 9 无 无  2 月 ELISA + BM      IHC + (一个          未执行, 达尔马提亚杂交, PCR + 无鞭毛体)         抗生素(未知); NM, 3 yo  PCR +           无反应的 10 达切斯勃拉克犬, F,3 yo 未知 未知 2 years IFAT low + BM 细胞学结果+ IHC – PCR – 未执行,抗生素(未知);无反应的 11 未知 未知 2 months IFAT high + IHC +                   MSSP; 杂交品种, M,6 yo 对克林霉素无反应 12 指示犬, SF, 6 yo 无 磺胺吡嗪治疗口鼻部脱发（1个月前）无反应 1 month IFAT high + IHC + MSSP; 对头孢氨苄无反应 13 弱阳 ELISA 无 2 months ELISA high + IHC - PCR -      MDR-MRSP; 对多西环素 罗得西亚背脊犬， (1 个月前)     和利福平无反应  M, 3 yo r 14 杰克·拉塞尔梗,  F, 2yo 无 恩诺沙星治疗丘疹性脓疱性皮炎（3个月前）无反应 3 months ELISA low + BM PCR + IHC + MDR-MRSP 和β溶血性链球菌；对恩诺沙星无反应 BM, bone marrow; ELISA, enzyme-linked immunosorbent assay; F, female; IFAT, indirect fluorescent antibody test; IHC, immunohistochemistry; M, male; MSS, meticillin-susceptible Staphylococcus; MSSP, meticillin-susceptible Staphylococcus pseudintermedius; MRSP, meticillin-resistant Staphylococcus pseudintermedius; MDR, multidrug-resistant; NM, neutered male; qPCR, quantitative PCR; SF, spayed female; #, number; +, positive; -, negative; yo, year old. BM，骨髓；ELISA，酶联免疫吸附试验；F，雌性；IFAT，间接荧光抗体试验；IHC，免疫组化； M、 雄性；MSS，甲氧西林敏感葡萄球菌；MSSP，甲氧西林敏感假中间型葡萄球菌；MRSP，耐甲氧西林假中间型葡萄球菌；MDR，多重耐药；NM，去势雄性；qPCR，定量PCR；SF，绝育雌性；#，个数；+，阳性；-，阴性；yo，岁。         Table 2. Contingency table summarizing the results of the two variables studied: sterile or antibiotic-unresponsive pruritic pustular dermatitis (PustD) and canine leishmaniosis (CanL) in 2,420 dogs. 表2.列联表总结了两个研究变量的结果：2420只犬的无菌或抗生素无反应性瘙痒性脓疱性皮炎（PustD）和犬利什曼病（CanL）。   CanL No CanL Total PustD 12 4 16 No PustD 91 2,313 2,404 Total总计 103 2,317 2,420         PustD: sterile or antibiotic-unresponsive pruritic pustular dermatitis excluding cases of pemphigus foliaceous. 脓疱性皮炎：无菌或抗生素无效的瘙痒性脓疱性皮炎，不包括落叶型天疱疮。

 

 

Figure 1. Skin lesions of dogs with sterile or antibiotic-unresponsive pustular dermatitis and canine leishmaniosis.

(a,b,c,d) Case 3: alopecia and crusts on the abdomen and extremities (a), yellowish crusts overlying erythematous papules (b,c) and a broad pustule enclosing more than one follicular unit (d). (e,f,g,h) Case 4: self-induced alopecia due to severe pruritus affecting the trunk, abdomen and extremities (e); erythematous papules and yellowish crusts can be observed in the alopecic areas (f,g) and in the inner part of the right pinna (h).

图1.无菌或抗生素无反应性脓疱性皮炎和犬利什曼病的皮肤病变。

（a，b，c，d）病例3：脱毛和腹部及四肢结痂（a），覆盖在红色丘疹（b，c）上的淡黄色结痂和包围一个以上毛囊单位（d）的广泛脓疱。（e，f，g，h）病例4：因严重瘙痒影响躯干、腹部和四肢引起的自损性脱毛（e）；脱毛区（f，g）和右耳廓内侧（h）可见发红丘疹和黄色结痂。

 

 

Figure 2. Skin lesions of dogs with sterile or antibiotic-unresponsive pustular dermatitis and canine leishmaniosis.

(a) Case 5: alopecia and crusting affecting the face. (b) Case 13: yellowish crusts on the outer side of the right pinna. (c) Case 6: pustules surrounded by an erythematous halo on the abdomen. (d) Case 8: arciform-polycyclic configuration of skin lesions.

图2.无菌或抗生素无反应性脓疱性皮炎和犬利什曼病的皮肤病变。

（a） 病例5：影响面部的脱毛和结痂。（b） 病例13：右侧耳廓外侧的黄色结痂。（c） 病例6：腹部脓疱周围皮肤发红。（d） 病例8：皮肤病变呈弓形多环结构。

 

 

Figure 3. Photomicrographs of skin sections of dogs affected by sterile or antibiotic-unresponsive pustular dermatitis and canine leishmaniosis. (a–f) Case 4: (a) broad intracorneal and subcorneal pustule with epidermal hyperplasia; (b) detail of (a), perivascular to interstitial inflammatory infiltrate in the superficial dermis underneath the pustule; (c) detail of (b), closer view of the pustule with polymorphonuclear epidermal exocytosis; (d) detail of (c), necrosis of the epidermis underneath the pustule; (e) large pustule involving a minimum of five follicular units; (f) detail of (e), crust with degenerated polymorphonuclear cells and acantholytic keratinocytes; in the superficial dermis there is marked erythrocyte extravasation. (g, h) Case 5: (g) intra- and subcorneal pustule with marked spongiosis and exocytosis; in the superficial dermis there are vasodilation, intense oedema and interstitial inflammatory infiltrate; (h) detail of (g), eosinophil exocytosis and superficial epidermal necrosis. Haematoxylin and eosin.

图3.无菌或抗生素无反应性脓疱性皮炎和犬利什曼病犬皮肤切片的显微照片。（a–f）病例4:（a）广泛的伴有表皮增生的角质层内和角质层下脓疱；（b）脓疱下浅真皮血管周围至间质的炎性浸润；（c）细节（b），表皮分叶中性粒细胞外渗的近距离视图；（d）细节（c），脓疱下面的表皮坏死；（e）大脓疱，至少包含五个毛囊单位；（f）细节（e），表皮有退行性分叶中性粒细胞和棘层松解细胞；在浅层真皮有明显的红细胞外渗。（g，h）病例5:（g）有明显海绵样水肿和胞外分泌的角质层内和角质层下脓疱；真皮浅层有血管扩张、严重水肿和间质炎性浸润；（h）详细（g）、嗜酸性粒细胞外排和表皮浅层坏死。苏木精和曙红。

Figure 4. Photomicrograph of the immunohistochemical findings in a dog with canine leishmaniosis and sterile or antibiotic-unresponsive pustular dermatitis (Case 11).

(a) Immunohistochemistry for Leishmania spp. with a macrophagic infiltrate in the superficial dermis and presence of few Leishmania amastigotes in their cytoplasm (arrowheads) (asterisk marks the epidermis); (b) detail of (a), Leishmania amastigotes (arrows) (bar, 50 lm). Avidin–biotin–peroxidase complex method, Mayer’s haematoxylin counterstain.

图4。犬利什曼病和无菌或抗生素无反应性脓疱性皮炎的免疫组化结果的显微照片（病例11）。

（a） 利什曼原虫的免疫组织化学研究。在浅层真皮中有巨噬细胞浸润，细胞质中有少量利什曼原虫无鞭毛体（箭头）（星号标记表皮）；（b）（a），利什曼原虫无鞭毛体（箭头）（bar，50 lm）。抗生物素-生物素-过氧化物酶复合物法，梅耶苏木精复染。

 

 

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