一只感染汉氏巴尔通体患犬的耳缘血管炎

王帆

Bartonella henselae in a dog with ear tip vasculitis

一只感染汉氏巴尔通体患犬的耳缘血管炎

作者：Brittany L. Southern*, Pradeep Neupane, Marna E. Ericson， Jamie C. Dencklau, Keith E.Linder, Julie M. Bradley, Gabriel P. McKeon*, Charles T. Long and Edward B. Breitschwerdt

翻译：唐翔

Background – Bartonella henselae, a Gram-negative, zoonotic, alpha-proteobacteria has been previously implicated in association with cutaneous vasoproliferative lesions (bacillary angiomatosis), nodular panniculitis and multifocal erythema (erythema multiforme) in dogs.

Objective – Describe clinical, microbiological and histological lesions in a dog with ear margin vasculitis and B. henselae infection.

Animals – A 12-month-old, specific pathogen-free intact female beagle dog maintained in a vector-free laboratory animal resource facility.

Methods and materials – Bartonella and Rickettsia serological evaluation, Bartonella and Rickettsia PCR, Bartonella alpha-proteobacteria growth medium (BAPGM) enrichment blood culture/PCR,histopathological investigation and confocal immunohistochemical evaluation.

Results – Serological investigation (seroreversion) and PCR testing of aural tissue biopsies failed to support Rickettsia rickettsii as a cause of the aural vasculitis; however, B. henselae, genotype San Antonio 2 DNA was amplified and sequenced from both ear tip margins and from normal-appearing abdominal skin. Seroconversion to B. henselae was documented retrospectively by IFA testing. Bartonella henselae organisms were visualized by confocal immunostaining within all three biopsies. Histopathology revealed small vessel necrotizing vasculitis and dermal necrosis. Bartonella henselae seroreversion and complete resolution of skin lesions occurred in conjunction with administration of oral doxycycline and enrofloxacin for six weeks.

Conclusions and Clinical Importance – Bartonella henselae is an emerging zoonotic pathogen that has been associated with leucocytoclastic vasculitis in humans and may have had a contributing or causative role in the development of the cutaneous aural margin vasculitis in this beagle.

背景-汉氏巴尔通体，是一种革兰氏阴性、人畜共患的α-变形菌，曾被认为其与犬皮肤血管增生性病变（杆菌性血管瘤病）、结节性脂膜炎和多灶性红斑（多形红斑）有关。

目的-描述耳缘血管炎和感染汉氏巴通体患犬的临床表现、微生物学和组织学病变。

动物-一只12月龄、无特定病原体感染的未绝育雌性比格犬,饲养在无病媒实验室中。

方法和材料-进行巴尔通体和立克次体血清学评估,巴尔通体和立克次体PCR检测、巴尔通体α-变形菌生长培养基 (BAPGM) 富血培养/ PCR、组织病理学调查和共聚焦免疫组化评估。

结果-耳部组织活检的血清学检测(血清学转归)和PCR检测结果,证明立式立克次体不是导致耳部血管炎的病因，然而,从耳缘和正常腹部皮肤分离出的巴尔通体,圣安东尼奥2号DNA基因型被成功扩增并测序。经IFA测试回顾性研究证实巴尔通体血清转归。在三个活检样本的共聚焦免疫染色中,观察到汉氏巴尔通体病原体。组织病理学显示小血管坏死性血管炎和真皮坏死。口服多西环素和恩诺沙星六周后,汉氏巴尔通体血清学转归和皮肤病变的完全消退。

结论和临床价值-汉氏巴尔通体是一种新出现的人畜共患病原体,可引起人的白细胞分裂性血管炎,并且可能可能是这只比格犬耳缘皮肤血管炎的诱因或直接病因。

Introduction

介绍

Bartonella spp. cause chronic intra-erythrocytic and endotheliotropic infections in mammals, potentially spanning weeks, months or years in duration.Similar to other highly adaptive intracellular vector- transmitted pathogens, factors that result in disease manifestations are most likely multifactorial and variable among individual patients.

巴尔通体属在哺乳动物中造成慢性的红细胞内和嗜内皮性感染，可能会持续数周、数月或数年。与其他高度适应性细胞内载体传播病原体相似，导致疾病表现的因素很可能是多因素的，并且在个体患者中各不相同。

After the “rediscovery” of the genus Bartonella in association with the AIDS epidemic, research focused on the pathogenesis of vascular endothelial proliferative lesions such as bacillary angiomatosis, peliosis hepatis or peliosis splenis.Researchers have emphasized a dermal niche, as well as the previously described vascular niche, as an important component of the pathophysiology of Bartonella infections. We report a novel clinical presentation in a beagle infected with B. henselae genotype San Antonio 2 (BhSA2), while being maintained in a vector-free environment.

与艾滋病流行相关的巴尔通体属的“重新发现”后，研究重点在血管内皮增生性病变的发病机制，如杆菌性血管瘤病、肝紫癜病或脾紫癜病。研究人员强调了皮肤，以及之前描述的血管，作为巴尔通体感染病理生理学的重要组成部分。我们报道了饲养在无病媒环境中的比格犬感染汉氏巴尔通体基因型圣安东尼奥2型 (BhSA2) 的一种新的临床表现。

Case report

病例报告

A 12-month-old, intact female beagle dog was obtained from Covance Laboratories, a commercial vendor in Cumberland, VA, USA. Subsequently, the dog was maintained in our AAALAC-accredited facility in accordance with the Animal Welfare Act and Guide for the Care and Use of Laboratory Animals. All procedures for animal use were approved by the North Carolina State University (NCSU) Institutional Animal Care and Use committee. The dog was housed in an indoor, limited access facility, fed a commercial diet twice daily (Laboratory Canine Diet 5006, LabDiet; St Louis, MO, USA), and received daily environmental and social enrichment activities.

来自美国弗吉尼亚州坎伯兰市的商业供应商Covance实验室的一只12月龄健康未绝育雌性比格犬。随后，根据动物福利法和实验动物护理和使用指南，将犬饲养在我们的AAALAC认证机构。所有动物使用程序均获得了北卡罗来纳州立大学 (NCSU)机构动物护理和使用委员会的批准。将犬圈养在有限设施的室内生活，每日两次喂食商品化日粮，并每日进行环境和社会化丰容活动。

After an acclimation period, six dogs were infected intradermally with Rickettsia rickettsii to generate sequentially timed blood specimens to facilitate optimization of Rocky Mountain Spotted Fever (RMSF) diagnosis in naturally infected dogs. When retrospectively tested, the R. rickettsii inoculum used for this study was PCR negative for Bartonella spp. DNA. As planned, infection resulted in a short duration illness, accompanied by self-limiting fever and thrombocytopenia, with spontaneous recovery beginning three days after R. rickettsii inoculation. All dogs in the study remained afebrile and clinically normal for the next six weeks, during which time sequential blood specimens were collected and R. rickettsii seroconversion was documented in all six dogs.

在适应环境后，6只犬皮内感染立氏立克次氏体，按感染后时间顺序采集血液标本，便于优化自然感染犬的落基山斑点热(RMSF)诊断。当进行回顾性检测时，本研究使用的立克次氏体接种物对巴尔通体属DNA呈PCR阴性。按计划，感染导致短期疾病，伴有自限性发热和血小板减少，在接种立克次氏体后3天开始自行恢复。研究中的所有犬在接下来的6周内保持无发热和临床表现正常，在此期间连续采集血样，并记录所有6只犬的立克次体血清转化情况。

By post-infection day (PID) 46, one of the six dogs developed moist dermatitis with haemorrhage along the right aural margin. Assuming a minor traumatic injury to the ear, treatment for seven days with a combination antibiotic, antifungal and anti-inflammatory topical ointment (Quadritop, Henry Schein Inc.; Melville, NY, USA) resulted in complete lesion resolution. Approximately one month later (PID 80) aural margin dermatitis involving both pinnae reoccurred (Figure 1). The lesions progressed despite treatment with a topical moisturizer spray once daily (Douxo Seborrhea, Ceva Animal Health; Lenexa, KS, USA). By PID 108, alopecia, scaling, mild crusting and hyperpigmentation involved both the inner and outer aural margins (Figure 1a and b). Hairs were easily epilated from the skin over the ears. The moisturizer spray was stopped. An in-house dermatophyte test was negative for fungal culture. At PID 128, the only abnormality in the complete blood count was a mild nonregenerative anaemia {haematocrit 36% [reference interval (RI) 39.2–55.9], absolute reticulocytes 51,800/lL (RI < 60,000)}, a decrease from a 47% haematocrit pre- infection with R. rickettsii and 49% on PID 39. A serum biochemical panel and thyroid (T4 and free T4 equilibrium dialysis) results were unremarkable (Antech Diagnostics; Southhaven, MS, USA).

感染后第46天（PID 46），6只犬中的1只出现湿性皮炎，且右耳沿耳缘出血。假设是耳部小外伤，联合使用抗生素、抗真菌和抗炎外用软膏治疗七天，病变完全消退。大约一个月后(PID 80)，双侧耳廓耳缘皮肤病复发(图1)。尽管每日一次使用外用保湿喷雾治疗(多可素)。到感染第108天，耳缘内侧和外侧均出现脱毛、皮屑、轻度结痂和色素沉着(图1a和b)。耳周皮肤易脱毛。后停用保湿喷雾。室内皮肤真菌培养试验为阴性。在感染的第128天, 唯一的异常是全血细胞计数下呈非再生性贫血 {红细胞压积为36% [参考区间 (RI) 39.2 –55.9], 绝对网织红细胞是51800 / lL (RI < 60000 )}, 红细胞压积比在感染立克次氏体前的47%和感染后第39天的49%有降低。血清生化和甲状腺(T4和游离T4平衡透析法)结果不显著。

Figure 1. Beagle dog with Bartonella henselae associated skin lesions.

(a) Scaling and alopecia of the outer pinna margins at 10 weeks post-Rickettsia rickettsii infection. (b) Scaling, mild crusting and alopecia of concave pinna margin, and (c) progressive alopecia and hyperpigmentation along pinna 12 weeks post-Rickettsia rickettsii infection. (d) Healed outer pinna after treatment 57 weeks post-R. rickettsii infection.

图 1. 感染汉氏巴尔通体的比格犬的皮肤病变。

(a)立克次体感染10周后，耳缘外侧出现皮屑和脱毛。(b)耳廓边缘凹面皮屑、轻度结痂和脱毛，(c)立克次体感染12周后，沿耳廓出现渐进性脱毛和色素沉着。(d)立克次体感染57周后 ，经治疗后耳廓外侧恢复。

As per the R. rickettsii study protocol, sequential blood and serum samples were collected during the study period (from pre-inoculation day 38 to PID 39). When one of five dogs developed cutaneous aural margin lesions after completion of the R. rickettsii study (one dog was adopted out after the completion of the R. rickettsii study and was lost to follow-up), additional blood and serum samples were collected on PID 113, 123, 201, 204 and 206. Due to lesion progression, biopsies were obtained from both aural margins and from normal-appearing ventral abdominal skin. Skin lesions were never observed in the other five dogs. Following general anaesthesia on PID 128, skin biopsies were obtained aseptically from each aural margin and abdominal skin.

根据立克次氏体研究方案，在研究期间（从接种前第38天到感染后的第39天）连续采集了血液和血清样本。当5只犬中的1只犬在完成立克次体研究后出现皮肤耳缘病变（1只犬在完成立克次体研究后被领养并失去随访），在感染后的第113、123、201、204和206天还额外采集了血液和血清样本。由于病变进展，均从耳缘和正常腹部皮肤进行活检采样。其他5只犬从未观察到皮肤病变。在感染的第128天进行全身麻醉后，从每侧的耳缘和腹部皮肤进行无菌皮肤活检。

Testing procedures used to assess exposure (serological investigation) and infection (PCR/DNA sequencing) for R. rickettsii, B. henselae and for other vector-borne organisms in the dog with aural margin vasculitis are described in Data S1.The dog seroconverted to R. rickettsii (IFA titre <1:16 pre-infection, peak titre 1:4,096 on PID 11) and remained R. rickettsii seroreactive with titres progressively decreasing (seroreversion) to 1:256 by PID 206. Based upon retrospective serological testing, B. henselae antibodies were not detected in the dog’s pre-R. rickettsii infection (four time points tested) sera, but were detectable (≥1:64) on PID 9, 113, 123, 201, 204 and 206. All EDTA blood specimens were Bartonella spp. and Rickettsia spp. PCR negative throughout the study. Bartonella spp. DNA was not amplified from BAPGM enrichment blood cultures processed between PID 113 and 206.

在资料S1中描述了在耳缘血管炎患犬身上评估立式立克次体、汉氏巴尔通体和其他媒介传播生物暴露(血清学调查)和感染(PCR/DNA测序)的测试程序。犬血清转化为立克次体（感染前 IFA 滴度 < 1:16，PID 11 时滴度峰值为 1:4,096），并保持立克次体血清反应性，PID 206 时滴度逐渐降低（血清回复）至 1:256。基于回顾性血清学检测，在犬的立克次体感染前（检测的4个时间点）血清中未检测到汉氏巴尔通体抗体，但在PID 9、113、123、201、204 和 206 时可检测到 (≥1:64)。所有EDTA血液标本在整个研究期间巴尔通体属和立克次体属的 PCR呈阴性。巴尔通体属在 PID 113和206之间处理的BAPGM 富集血培养物中未扩增出DNA。

By targeting the 16S–23S ITS region, B. henselae SA2 DNA was PCR amplified and successfully sequenced from each of the dog’s three tissue biopsies (100% similarity, 550 of 550 bp to GenBank accession # AF369529). The ITS result was further confirmed by PCR amplification and DNA sequencing of the partial ssrA gene from the left aural margin and abdominal biopsy (PCR negative for right aural margin DNA extraction).

通过靶向16S-23S ITS区，汉氏巴尔通体SA2 DNA进行了PCR 扩增，并从犬的3份组织活检标本中各成功测序（100%相似性，550/550 bp 至 GenBank 登录号 AF369529）。通过左耳缘部分ssrA 基因的PCR扩增和DNA测序及腹部活检（右耳缘DNA提取PCR阴性）进一步证实了ITS结果。

By laser confocal immunohistochemical evaluation, Bartonella organisms were visualized within dermal blood vessels in the dog’s aural lesions; illustrated in a z-stack projection of 83, 0.45 um optical sections (total thickness = 37 um) (Figure 2).

通过激光共聚焦免疫组织化学评价，在犬耳部病变的真皮血管内观察到巴尔通体微生物；如 83,0.45 μm光学切片的z-stack投影所示（总厚度 = 37 μm）（图 2）。

Figure 2. Beagle dog with Bartonella henselae associated skin lesions.

Laser scanning confocal projection of the left ear margin skin biopsy obtained 12 weeks post-Rickettsia rickettsii infection. Immunoreactive Bartonella spp. organisms (green) are predominantly associated with a vessel wall (red) which is immunoreactive to collagen type IV.

图 2. 感染汉氏巴尔通体的比格犬的皮肤病变。

立克次体感染12周后获得左耳缘皮肤活检的激光共聚焦投影。免疫反应性巴尔通体微生物（绿色）主要与血管壁（红色）相关，其对IV型胶原具有免疫反应。

With serial tissue sectioning, histopathological investigation revealed mild, multifocal necrotizing small vessel vasculitis (Figure 3) with degenerate neutrophils and fewer macrophages associated with small foci of lytic dermal necrosis. Mild perivascular infiltrates of lymphocytes and plasma cells were present in some areas. Warthin– Starry silver staining of three stepped histological sections of lesions was negative or equivocal for the presence of bacteria. Bartonella henselae IFA seroconversion (<1:16 to 1:128 on PID 113 and 1:1,024 on PID 123) was documented. Rickettsia rickettsii DNA was not amplified from the dog’s three cutaneous biopsies.

通过连续组织切片，组织病理学研究显示轻度的多灶性坏死性小血管炎（图3）伴有退行性中性粒细胞和小的溶解性真皮坏死灶伴有较少量的巨噬细胞。部分区域存在轻度血管周围淋巴细胞和浆细胞浸润。病变的三个阶梯式组织切片的 Warthin–Starry 银染色为阴性或不确定是否存在细菌。记录了汉氏巴尔通体IFA血清转化（PID113为< 1:16至1:128，PID123为1:1,024）。犬的3份皮肤活检均未扩增出立克次体DNA。

Figure 3. Beagle dog with Bartonella henselae associated ear margin skin lesions.

Histological evaluation revealed a necrotizing small vessel vasculitis. Small swollen pale vessels (arrowhead) in the dermis are obscured by lytic necrosis, mild haemorrhage and degenerate neutrophils (arrows). Haematoxylin and eosin; 9400.

图3. 感染汉氏巴尔通体的比格犬的皮肤病变。

组织学检查显示为坏死性小血管炎。真皮内的小而肿胀的苍白血管(箭头)被溶解性坏死、轻度出血和退行性中性粒细胞(箭号)所掩盖。苏木精和伊红;9400.

Based on B. henselae serological investigation and tissue PCR results, the dog was treated with oral doxycycline (10 mg/kg twice daily) and enrofloxacin (10 mg/kg once daily) for six weeks. While awaiting diagnostic test results and prior to administering antibiotics, the skin lesions improved with hair regrowth beginning along both pinnae. Following the course of antibiotics, both ears appeared normal. Bartonella seroreversion (B. henselae IFA titre decreased from 1:1,024 on PID 123 to 1:256 on PID 201) was documented, and the nonregenerative anaemia had resolved (haematocrit 47%). One year later, the dog remained healthy without developing additional cutaneous lesions.

根据汉氏巴通体血清学调查和组织样本PCR结果，该犬口服多西环素（10 mg/kg，每日2次）和恩诺沙星（10 mg/kg，每日1次）治疗6周。在等待诊断试验结果和提前服用抗生素，皮肤病变改善，两侧耳廓开始长毛。抗生素治疗后，双耳外观正常。记录了巴尔通体血清逆转（汉氏巴尔通体 IFA 滴度从PID123的1:1,024 降至PID 201的1:256），非再生性贫血恢复（红细胞压积 47%）。1年后，该犬保持健康，未发生其他皮肤病变。

Discussion

讨论

We describe a dog that developed aural margin vasculitis, accompanied by alopecia, scaling, crusting and hyperpigmentation. Bartonella henselae infection was confirmed by seroconversion, PCR amplification / DNA sequencing targeting two B. henselae genes, visualization of organisms using immunohistochemical investigation and seroreversion in conjunction with antibiotic administration. Vasculitis secondary to B. henselae infection has been associated with dermal lesions, cerebral infarction and splenic infarction in dogs and humans.In dogs, cutaneous vasculitis can be caused by infectious agents, drug reactions, autoimmune diseases or remain idiopathic.Diagnosis is based on biopsy and histological examination; therapy is directed at the underlying cause. In this case, the aural dermatitis resolved in conjunction with antibiotic administration with no recurrence within a 12 month follow-up period, supporting our suspicions for an infectious aetiology. Although case reports cannot prove causation, the findings in this dog may be useful to clinicians evaluating similar dermatological aural lesions. It is of interest that the initial right aural lesion resolved with a short course of topical medications, only to have more severe aural lesions reoccur bilaterally approximately one month later. Whether these aural lesions would have resolved spontaneously or additional reoccurrences would have developed if the dog were not treated with antibiotics is unknown.

我们描述了一只犬出现耳缘血管炎，并伴有脱毛、皮屑、结痂和色素沉着。通过血清转化、靶向两个汉氏巴尔通体基因的PCR扩增/DNA测序、使用免疫组化和血清研究观察到微生物确诊了汉氏巴尔通体感染，联合抗生素治疗实现了血清转化。继发于汉氏巴通体感染的血管炎，与犬和人的真皮病变、脑梗死和脾梗死有关。在犬中，皮肤血管炎的形成与感染病原、药物反应、自体免疫性疾病或先天性等因素有关。诊断基于活检和组织学检查，针对潜在病因进行治疗。在该病例中，耳部皮肤病在给予抗生素后恢复，在12个月随访期内无复发，支持我们对感染性病因的怀疑。尽管病例报告不能证明因果关系，但该犬的结果可能对临床医生评估相似的耳部皮肤病变有用。有趣的是，最初的右耳病变通过短期外用药恢复，仅在约1个月后双侧再次发生更严重的耳部病变。尚不清楚如果犬未接受抗生素治疗，这些耳病变是否会自发消退或会再次发生。

Several arthropod vectors, including fleas and ticks, can transmit Bartonella species to dogs and humans. The bacterium is most often transmitted through the contamination of a bite or scratch with Bartonella spp. contained within the vectors faeces. The dog in this case report lived in a barrier facility with no access to vectors or the outside environment, but did interact with five other dogs and human caretakers. Thus, access to vectors or vector faeces was unlikely. The exact mode of infection for this dog was not determined, but one other dog in the R. rickettsii study group was B. henselae IFA and Western immunoblot  seroreactive prior to infection with R. rickettsii. That seroreactive dog was clinically healthy with no skin lesions, but may have been a source for horizontal transmission of B. henselae. Bartonella spp. DNA has been amplified from dog saliva, but a potential role for salivary transmission is unknown. Once infected, the oozing aural lesions may have posed a risk for bacterial transmission to other dogs or humans.Bartonella spp. have been transmitted to humans through dog bites and by needle stick,1,2 but it is unknown if direct contact with B. henselae infected skin lesions could result in zoonotic bacterial transmission. We conclude that additional research is needed to further define modes of transmission and the potential role of Bartonella spp. as a cause of vasculitis, thromboembolism and cutaneous aural lesions in dogs.

几种节肢动物，包括跳蚤和蜱虫，可以将巴尔通体传播给犬和人类。该细菌最常通过叮咬和抓伤而传播，也会因巴尔通体感染的病媒的粪便污染传播。本病例报告中的犬居住在隔离设施中，无法接触病媒或外部环境，但确实可以与另外5只犬和管理人员互动接触。因此，不太可能接触病媒或病媒粪便。确切的感染方式尚未确定，但立式立克次体研究组中的另外1只犬在感染立式立克次体前为汉氏巴尔通体IFA 和 Western 免疫印迹血清阳性。该血清阳性犬在临床上是健康的，无皮肤病变，但可能是汉氏巴尔通体水平传播的来源。已从犬唾液中扩增出巴尔通体属DNA，但唾液传播的潜在作用尚不清楚。一旦感染，耳部病变渗出可能是细菌传播给其他犬或人的风险。巴尔通体属已通过犬咬伤和针刺传播给人类，但尚不清楚直接接触汉氏巴通体感染的皮肤病变是否会导致人畜共患细菌传播。我们的结论是，需要更多的研究来进一步确定巴尔通体的传播方式及作为犬血管炎、血栓栓塞和皮肤耳部病变的病因的潜在作用。

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