Anaplasma phagocytophilum DNA amplified from lesional skin of seropositive dogs

Ticks and Tick-borne Diseases 5 (2014) 329–335

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Original article

Anaplasma phagocytophilum DNA amplified from lesional skin of seropositive dogs夽 Inese Berzina a,b,∗ , Christiane Krudewig b , Cornelia Silaghi c , Ilze Matise a , Renate Ranka d , Norbert Müller e , Monika Welle b a

Preclinical Institute, Faculty of Veterinary Medicine, Latvia University of Agriculture, Jelgava, Latvia Institute of Animal Pathology, Vetsuisse-Faculty, University of Bern, Bern, Switzerland c Institute for Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität München, Munich, Germany d Latvian Biomedical Research and Study Center, Riga, Latvia e Institute of Parasitology, Vetsuisse-Faculty, University of Bern, Bern, Switzerland b

a r t i c l e

i n f o

Article history: Received 1 March 2013 Received in revised form 19 December 2013 Accepted 24 December 2013 Available online 15 March 2014 Keywords: Persistent canine granulocytic anaplasmosis Skin lesions Histopathology A. phagocytophilum PCR

a b s t r a c t Canine granulocytic anaplasmosis (CGA) is caused by the rickettsial microorganism Anaplasma phagocytophilum. CGA is typically characterized by fever, thrombocytopenia, lethargy, anorexia, arthropy, and other nonspecific clinical signs. Skin lesions have been described in naturally infected lambs and humans. The pathophysiology of CGA is not entirely clear, and the persistence of the organism after the resolution of clinical signs has been described. The aim of the study was to investigate if A. phagocytophilum can be detected in canine lesional skin biopsies from A. phagocytophilum-seropositive dogs with etiologically unclear skin lesions that improved after the treatment with doxycycline. Paraffin-embedded lesional skin biopsies were allocated into separate groups: biopsies from A. phagocytophilum-seropositive dogs responsive to treatment with doxycycline (n = 12), biopsies from A. phagocytophilum-seronegative dogs (n = 2), and biopsies in which skin lesions histopathologically resembled a tick bite (n = 10). The serological status of the latter group was unknown. Histology of the seropositive and seronegative dog skin lesions did not indicate an etiology. DNA was extracted, and a conventional PCR for partial 16S rRNA gene was performed. Anaplasma phagocytophilum DNA was amplified from 4/12 seropositive dogs’ skin biopsies. All sequences were 100% identical to the prototype A. phagocytophilum human strain (GenBank accession number U02521). Anaplasma phagocytophilum was not amplified from the 2 seronegative and 10 suspected tick bite dogs. Serum antibody titers of the PCR-positive dogs ranged from 1:200 to 1:2048. Histopathologically, a mild-to-moderate perivascular to interstitial dermatitis composed of a mixed cellular infiltrate and mild-to-moderate edema was seen in all seropositive dogs. In 8/12 seropositive dogs, vascular changes as vasculopathy, fibrinoid necrosis of the vessel walls, and leukocytoclastic changes were observed. In summary, our results support the hypothesis that the persistence of A. phagocytophilum in the skin may be causative for otherwise unexplained skin lesions in seropositive dogs. © 2014 Elsevier GmbH. All rights reserved.

Introduction Canine granulocytic anaplasmosis (CGA) is caused by the obligatory intracellular rickettsial organism Anaplasma phagocytophilum (Dumler et al., 2001; Carrade et al., 2009).

夽 An abstract was presented at the International Society for Animal Clinical Pathology Conference, 3–7 July 2012, Ljubljana, Slovenia. ∗ Corresponding author at: Latvia University of Agriculture, Faculty of Veterinary Medicine, Preclinical Institute, Pathology Department, Kr. Helmana Street 8, Jelgava LV-3004, Latvia. Tel.: +371 26324105. E-mail address: [email protected] (I. Berzina). http://dx.doi.org/10.1016/j.ttbdis.2013.12.010 1877-959X/© 2014 Elsevier GmbH. All rights reserved.

Anaplasma phagocytophilum (A. phagocytophilum) is transmitted by several species of ixodid ticks in North America, Europe, and Asia. It infects dogs, cats, horses, small and large ruminants while various small mammals serve as reservoir hosts. The reported seroprevalence varies depending on the dog population tested and the methodology that was used (Carrade et al., 2009). In North America, the reported seroprevalence varies from 0 to 55% in South Ontario, Quebec, and Minnesota, respectively (Carrade et al., 2009). Seroprevalence varies also in Europe where 7.5% of dogs tested positive in Switzerland (Pusterla et al., 1998), while 50.1% were tested positive in Germany (Barutzki et al., 2006). Multiple epidemiological and molecular studies as well as case reports of CGA have been published in the recent years (Poitout

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et al., 2005; Carrade et al., 2009; Couto et al., 2010). The majority of naturally infected dogs do not show any clinical signs, and disease appears to be self-limiting (Carrade et al., 2009). CGA typically manifests as a febrile disease with short (4–14 days) bacteremia, lethargy, inappetence, lameness, anemia, and thrombocytopenia (Egenvall et al., 2000; Poitout et al., 2005; Carrade et al., 2009). Furthermore, A. phagocytophilum is capable of modifying several of neutrophil key functions such as phagocytosis and motility, therefore CGA is associated with impaired immunity (Woldehiwet, 2010; Rikihisa, 2011; Day, 2011). In cases of coinfection with other tickborne or vector-borne diseases, the clinical signs and the diagnosis might be more complicated (Beall et al., 2008; De Tommassi et al., 2013). Persistent A. phagocytophilum infection has been described in dogs, sheep, and horses (Egenvall et al., 2000; Stuen and Bergström, 2001; Franzén et al., 2009). The diagnosis of persistent granulocytic anaplasmosis is based on intermittently positive PCR results, reoccurrence of morulae in peripheral blood smears, and fluctuating antibody titers (Egenvall et al., 2000; Franzén et al., 2009; Scorpio et al., 2011). Since clinical signs are absent or vague and serial PCR and serological testing are frequently difficult to obtain, the persistent granulocytic anaplasmosis might be underdiagnosed in a clinical setting (Stuen and Bergström, 2001; Woldehiwet, 2010). Although granulocytic anaplasmosis has historically been frequently diagnosed in animals, ruminants, and sheep in particular, the research interest in this infection was boosted after this infection was recognized in humans (Bakken and Dumler, 2006). Despite the active research, there are several key mechanisms still not clear, such as the site of the persistence of the organism. To date, A. phagocytophilum DNA has been amplified from several sites in different animal species: circulating granulocytes in sheep and horses (Franzén et al., 2009; Stuen et al., 2008), poorly vascularized connective tissue in horses (Chang et al., 1998), and skin in sheep (Granquist et al., 2010). The above-mentioned sites have also been listed as possible sites of persistence of the organism in animals without typical clinical signs of granulocytic anaplasmosis. Up to now to the best of our knowledge, A. phagocytophilum DNA has been amplified only from peripheral blood in persistently infected dogs (Egenvall et al., 2000). The accumulation of unexplained skin lesions in biopsies from dogs seropositive for A. phagocytophilum together with the information that resolution of the lesions was reached after the treatment with doxycycline triggered our interest, and the question was raised if the skin lesions could be associated with an A. phagocytophilum infection. Skin lesions in dogs with CGA have been described only in 2 A. phagocytophilum-seropositive dogs from Slovenia. These dogs were reported to be pruritic, and the skin was edematous. The presence of A. phagocytophilum in the skin was not investigated in these 2 cases (Ravnik et al., 2011; Dr. N. Tozon, pers. communication). In humans, skin rash has been reported in up to 11% and 7% granulocytic anaplasmosis patients in USA and Europe, respectively (Dumler and Walker, 2001; Blanco and Oteo, 2002; Dumler et al., 2005). Furthermore, a case report of human granulocytic anaplasmosis and Sweet’s syndrome (acute febrile neutrophilic dermatosis) has been published in the USA (Halasz et al., 2005). In the described human case, A. phagocytophilum DNA was amplified from the lesional skin biopsy, but immunohistochemistry was negative, and the agent was not observed in the skin (Halasz et al., 2005). The aim of this retrospective study was to investigate if A. phagocytophilum can be detected by molecular and immunohistochemical means in canine lesional skin biopsies from A. phagocytophilum-seropositive dogs with etiologically unclear skin lesions that improved after the treatment with doxycycline.

Materials and methods Skin biopsy collection Biopsies were retrieved from the archive of the Institute of Veterinary Pathology, Vetsuisse-Faculty, University of Bern, Switzerland. All biopsies were preserved in 10% neutral buffered formalin and stored as paraffin blocks. Our criteria for the case selection in the test group were: (i) Histopathological presentation of biopsies from lesional skin was unusual, and the cause of the skin lesion remained unclear after examination; (ii) dog was seropositive against A. phagocytophilum; (iii) the skin lesions resolved after treatment with doxycycline. Based on our selection criteria, we obtained 52 lesional skin biopsies from 12 dogs, in the following referred to as seropositive biopsies and seropositive dogs, respectively. In addition to the skin biopsies, kidney, bone marrow, and spleen were tested from one dog (taken during necropsy). Biopsies were taken from the years 2006–2011. Two dogs were biopsied twice in one year, 2 dogs were biopsied twice in 2 consecutive years. In addition we opted to include 2 control groups: - Control group A consisted of dogs which met the following inclusion criteria: (i) Histopathological presentation of biopsies from lesional skin was unusual, and the cause of the skin lesion remained unclear after examination; (ii) dog was seronegative against A. phagocytophilum; (iii) the skin lesions resolved after treatment with doxycycline. Based on these inclusion criteria, we obtained 2 skin biopsies from 2 dogs, in the following referred to as seronegative biopsies and seronegative dogs, respectively. Biopsies were taken in 2009 and 2011. - Control group B consisted of dogs whose lesional skin biopsies were histologically consistent with a tick bite, and the serological status of the dogs was unknown to us. Based on these inclusion criteria, we obtained 11 biopsies from 10 dogs in the following referred to as tick bite biopsies. Biopsies were sampled in the years 2000–2012. Tick infestation in these cases has not been confirmed by the owner, but the described histopathology was suggestive for this entity (Szabo and Bechara, 1999). Serological examination in our test group and in the control group A was ordered in different laboratories by the clinics where the dogs had presented for their skin lesions. Therefore, the serological methods to identify specific antibodies against A. phagocytophilum differed. In 9 dogs, serological tests were performed in commercial laboratories, where an indirect fluorescent antibody test (IFAT) was performed. In 3 dogs, only a qualitative serology test was performed, and the exact titer is not known (Table 1). Signalment, breed, location, and macroscopical description of the skin lesions were available for all dogs included in the study, for seropositive dogs this information is included in Table 1. Information on the serological status of the dogs and the clinical history of the development of the skin lesions was either submitted together with the skin biopsies or was collected retrospectively upon request at the beginning of this study. All skin biopsies from test group dogs were analyzed by PCR to exclude leishmaniasis (Müller et al., 2003). Additional information on the exclusion of other tick-borne diseases, on treatment, and outcome was provided by the dogs’ veterinarians. PCR For DNA extraction, 3–5 tissue sections from each paraffinembedded tissue block (9–15 ␮m thick) were placed into a 1.5-ml Eppendorf tube. DNA extraction was performed with the QIAamp DNA FFPE Tissue Kit (Qiagen, Switzerland) following the

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Table 1 Detailed information on dogs seropositive for A. phagocytophilum and responsive to treatment with doxycycline. Dog no.

Age in years/sex/breed

Skin lesions

Location

Type

Serology titer

PCR

IHC

Morphological diagnoses

Vasculitis leukocytoclastic, diffuse, severe. Artherosclerosis multifocal, moderate. Folliculitis, luminal and furunculosis, deep, multifocal, moderate.

1

10/female neutered/Mongrel

Pinnae

Erythema, crusts

1:100

Negative

NT

2

7/female/German shepherd

Flank

Seropositive

Negative

NT

3

2/female neutered/Dalmatian

Teats, tarsi

Erythema, crusts, papules, alopecia Erythema, swelling

Seropositive

Negative

NT

4

5/female/Hovawart

Generalized

Erythema, pustules, nodules

1:2048

Positive

Negative

5

10/male castrated/German shepherd

Generalized

Seropositive

Negative

NT

6

2/male/German shepherd

Nose, tongue, metatarsus, elbow

1:1200

Negative

NT

7

7/female neutered/Rhodesian ridgebacka 8/male/German shepherd

Front legs

Pustules, ulceration, crusts, squames, alopecia Vesicles, ulcerations, crusts, lichenification Subcutaneous edema

1:800

Positive

Negative

Hair cycle arrest with follicular atrophy; dermal edema, moderate. Vasculopathy with deep dermal edema and focal hemorrhage. Dermatitis, neutrophilic, plasmacellular, mastocytic and focal eosinophils, perivascular to interstitial, superficial, moderate. Vasculitis, leukocytoclastic with septal panniculitis, neutrophilic, mild.

8

9

10

2/female neutered/Bernese mountain dog 6/male castrated/Australian shepherd

Generalized

Erythema, crusts

1:320

Positive

Negative

Generalized

Erythematous papules

1:40

Negative

NT

Pawpad, inner thigh, nasal planum, lip

Erythema, vesicles, pustules, nodules, crusts, erosions, ulcerations Erythema, papules

1:320

Negative

NT

1:224

Negative

NT

1:200

Positive

NT

11

4/female/Flatcoated retriever

Head, neck, axillary, inguinal

12

5/female/Flatcoated retriever

Generalized

Erythema, macules

Vasculitis, fibrinoid with thrombosis and severe dermal edema. Dermatitis, neutrophilic, deep, diffuse, moderate. Vasculopathy, cell-poor with associated multifocal dermal and epidermal necrosis. Dermatitis, neutrophilic, lymphocytic and plasma cellular, superficial, perivascular to interstitial, mild. Vasculitis, leukocytoclastic, neutrophilic and eosinophilic, diffuse, moderate. Dermatitis, eosinophilic, neutrophilic and mastocytic, superficial, perivascular, mild. Dermatitis, neutrophilic, eosinophilic and mastocytic, perivascular to interstitial, deep, moderate.

Vasculitis, leukocytoclastic, deep with edema, severe. Dermatitis, plasmacellular, perivascular to interstitial, superficial, moderate. Dermatitis, pyogranulomatous, multifocal to coalescing superficial, moderate. Vasculopathy, cell-poor with dermal edema and follicular atrophy, marked. Dermatitis, neutrophilic and mastocytic, superficial, interstitial, mild. Dermatitis, neutrophilic, eosinophilic and lymphocytic, perivascular to interstitial, moderate with severe leukocytostasis

NT, not tested with immunohistochemistry. Seropositive, quantitative serology titer not available. a Dog was also treated with prednisolone and euthanized due to splenic lymphoma.

manufacturer’s instructions with the following minor modifications: Histo-Clear (National Diagnostics, Atlanta, USA) was used for the dissolution of paraffin, and the amount of proteinase K was doubled to achieve better tissue lysis. DNA was eluted in 50 ␮l buffer. Conventional PCR (cPCR) was aimed to amplify partial A. phagocytophilum 16S rRNA gene with the anticipated amplicon size of 270 base pairs. We used commercially available HotStarTaq Master Mix Kit (Qiagen, Switzerland) with the A. phagocytophilumspecific primers Apl16SF (5 -CTCAGAACGAACGCTGGCGGCAA-3 ) and Apl16SR (5 -TCCTCACTCACGCGGCATAGC-3 ) (Mycrosynth, Switzerland). The Techne TC-312 thermal cycler (Techne,

Staffordshire, UK) was programmed to perform initial denaturation at 95 ◦ C for 15 min, denaturation at 95 ◦ C for 30 s, annealing at 63 ◦ C for 30 s, extension at 72 ◦ C for 45 s, for a total of 40 cycles with the final extension for 7 min at 72 ◦ C (Canelas Domingos et al., 2011). The final volume of the sample was 50 ␮l containing 25 ␮l Master Mix, 10 pmol of each primer, 1 ␮l DNA template, 0.25 ␮l UDG, 1 ␮l UTP, and 20.75 ␮l water. To exclude carry-over contamination from previous PCR reactions, we incubated the above-described mix of reagents for 1 h at room temperature with uracil DNA glycosylase (UDG). Amplified DNA together with Loading Dye, 6× (Promega, USA) was resolved in 2% agarose gel stained with RedSafe (iNtRON

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Biotechnology, Korea). After electrophoresis (Horizon 58, Life Technologies, Gibco BRL), DNA products were visualized under UV light (U:Genius, Syngene, Frederick, USA). Positive (A. phagocytophilum DNA from cell culture) and negative (distilled water) controls were run together with the samples. To minimize the risk of crosscontamination of the samples, additional precautions were taken such as performing all steps of the PCR in separate rooms and using filtered pipette tips. Amplicons were verified by sequencing as described previously (Ranka et al., 2004). The detection limit of our PCR was estimated to be equivalent to 1 in 1000 dilution of the control material which was DNA-extracted from A. phagocytophilum cultured in IDE8 tick cells at a time point when A. phagocytophilum started to be found free-floating in the culture medium (after approximately 4 weeks). We verified our conventional PCR method by testing known positive and negative sheep skin biopsies, respectively. Control skin biopsies were obtained from lambs naturally infected with A. phagocytophilum and were confirmed as positive or negative by PCR, sequencing of amplicons, immunohistochemistry (IHC), and IFAT. The paraffin-embedded tissue blocks were kindly provided by Dr. E. Granquist, Department of Production Animal Clinical Sciences, Section of Small Ruminant Research, Norwegian School of Veterinary Science, Sandnes, Norway. DNA extraction and cPCR protocols were performed like the ones described above.

Fig. 1. Skin lesions of an A. phagocytophilum-seropositive and PCR-negative dog (no. 1). Note the extensive crusts on the ear margins and the inner aspect of the pinna. In addition, the pinna is erythematous, hypotrichotic, and some papules are seen.

Histopathology and immunohistochemistry Four ␮m sections were cut from all biopsy blocks included in the study. Tissue was placed on glass slides (Thermo Scientific, USA) and stained with hematoxylin and eosin for microscopical evaluation. Slides were reviewed independently by 2 board-certified pathologists (CK and MW). Skin biopsies that were PCR-positive were subjected to immunohistochemical examination and therefore placed on positively charged slides (ColorFrost Plus, Thermo Scientific, USA). Deparaffinization and rehydration were performed in series of xylol and graded alcohol, respectively. Endogenous peroxidase inhibition was performed in 1% hydrogen peroxidase in methanol for 15 min. Antigen retrieval was carried out in citrate buffer (pH 6.0) and heated in a microwave to 95 ◦ C for 20 min. Incubation with normal goat serum diluted 1:50 in 5% BSA/PBST was carried out for 20 min. Primary antibodies were provided by Prof. S. Dumler (Division of Medical Microbiology Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, USA). Monoclonal (mouse anti-MSP2) and polyclonal (rabbit antiA. phagocytophilum) antibodies were used in dilutions 1:400 and 1:600, respectively, in 1% BSA/PBST overnight at 4 ◦ C (Granquist et al., 2010). A species- and isotype-specific negative control in a similar dilution was used. Thorough washing with PBST was followed by incubation with a species-specific secondary antibody bound with Streptavidin biotin for 30 min. After washing with PBST, the sections were treated with 3-amino-9-ethyl carbazole substrate (AEC) for 3–5 min, rinsed in tap water, counterstained with hematoxylin, and coversliped (Aquatex). Sheep skin biopsies were used as positive (naturally infected lamb skin tissue) and negative controls (tissue kindly provided by Dr. E. Granquist).

of the dogs displayed typical clinical signs of acute CGA (lethargy, fever). Seronegative and tick-bite dog groups included both male and female dogs from various countries and various breeds. Skin lesions from the seropositive dogs were not uniform with respect to location or type (Table 1). The majority of dogs presented with erythema, plaques, and erosions or ulcerations covered with crusts (Figs. 1–4). One of the seronegative dogs had papules, and erosions or ulcerations covered with crusts on the lower abdomen, paws, and neck. The other seronegative dog presented with generalized squames, lichenification, and crusts. In dogs from the tick bite biopsy group, lesions were mostly restricted to head, ventral neck, ears, and ventral abdomen. Gross lesions in this group were characterized as nodules, crusts, or ulcers.

Results Signalment, clinical presentation, and serology Detailed information on the signalment, location of the lesions, and serology results of the seropositive dogs are included in Table 1. The main complaints in these dogs were associated with the skin lesions. Anemia and thrombocytopenia were noted in 2 dogs within this group. Six dogs were negative for Leishmania by PCR, and none

Fig. 2. Representative skin lesions on the thorax of dog no. 8 which tested seropositive for A. phagocytophilum, and partial 16S rRNA gene of A. phagocytophilum was amplified by PCR from skin biopsies. Note the erythema associated with crusts and hyperpigmentation.

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Fig. 3. Skin lesions in the inguinal region of dog no. 8 which was also presented in Fig. 2. Note the moderate erythema, the hyperpigmentation, and the peripheral crusts.

PCR Anaplasma phagocytophilum DNA was amplified from 8 skin biopsies of 4 seropositive dogs further referred to as PCR-positive dogs; one dog had 5 positive biopsies, 3 dogs each had one positive biopsy (Table 1). In PCR-positive dogs, not all biopsied sites tested positive with PCR, and occasionally the paraffin blocks contained biopsies from more than one site. The results of sequencing in 3 cases revealed 100% identity to a human prototype strain of A. phagocytophilum (U02521) when blasted in GenBank. The sequences obtained in this study were deposited in GenBank under the following accession numbers: KC119573 (dog 8, 208 base pairs), KC119574 (dog 7, 253 base pairs), and KC119575 (dog 4, 266 base pairs). The PCR product from dog 12 was not sequenced. A. phagocytophilum DNA was not detected by PCR from biopsies of the 2 seronegative dogs and the 10 dogs with tick-bite lesions.

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Fig. 5. Histological appearance of a skin biopsy from dog no. 3, which tested seropositive and PCR-negative for A. phagocytophilum. Note the fibrinoid degeneration of the dermal blood vessels (black arrows) which are also partially occluded with fibrin (blue arrows/first and third from top) and lined by activated endothelial cells. In addition, there is severe dermal edema and an interstitial infiltrate with neutrophils which are multifocally accentuated. Hematoxylin and eosin, magnification 200×. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Histopathological presentation was unusual and did not identify a specific cause or diagnosis in the seropositive and seronegative dogs. Severity of histopathology among seropositive biopsies

varied among dogs and biopsy sites. The morphological diagnoses that summarize the findings in the investigated seropositive biopsies are depicted in Table 1. Vascular changes were seen in 8 of the 12 dogs (best seen in Fig. 5). They ranged from thickened, hyalinized wall of small caliber vessels with loss of nuclei (vasculopathy) to fibrinoid necrosis of the vessel walls, or leukocytoclastic vasculitis of small vessels. The vascular lesions were associated with moderate to severe edema and extravasation of erythrocytes (Fig. 6). In all dogs, a perivascular to interstitial infiltrate of the superficial and the deep dermis was noted (Figs. 5–7). Perivascular to interstitial neutrophils were very common, but folliculitis, suggesting classical pyoderma, was seen in only one case. Secondary lesions such as erosions, ulcerations, and crusts were common. The most commonly noted changes in the PCR-positive biopsies were variable cellular infiltrate (3/4), edema (2/4), and vasculopathy (2/4). Infectious agents were not visualized upon histological examination in hematoxylin and eosin and special stains for bacteria and fungi.

Fig. 4. Skin lesions of dog no. 12, which was seropositive for A. phagocytophilum and positive on PCR for the partial 16S rRNA gene of A. phagocytophilum. Note the abundant confluent erythematous papules.

Fig. 6. Histological appearance of a skin biopsy from dog no. 8, which tested seropositive and PCR-positive for A. phagocytophilum. Note the severe edema in the dermis and the moderate interstitial infiltrate with mostly neutrophils and lower amounts of eosinophils. Multifocally moderate numbers of extravasated erythrocytes are seen. Hematoxylin and eosin, magnification 100×.

Histopathological changes in skin biopsies

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Fig. 7. Histological appearance of a skin biopsy from dog no. 12, which was seropositive and PCR-positive for A. phagocytophilum. Note the moderate perivascular inflammatory infiltrate in the dermis which is composed of plasma cells and lymphocytes with fewer amounts of eosinophils, mast cells, and rare neutrophils. Blood vessels (arrow) are lined by activated endothelial cells and filled with numerous neutrophils. Moderate dermal edema is also present. Hematoxylin and eosin, magnification 200×.

Histopathological changes noted in the biopsies from seronegative dogs were similar to those noted in seropositive dogs. Histopathological changes in the tick-bite biopsies were similar to previously described lesions associated with tick bites (Szabo and Bechara, 1999) and did not resemble the histopathological findings in the biopsies from the seropositive and seronegative dogs. In short, the tick-bite lesions were characterized by dense, sharply demarcated cellular aggregates that were arranged in a nodular pattern. The dermal infiltrate was composed predominantly of lymphocytes, with fewer macrophages, mast cells, eosinophils, and plasma cells. Occasionally, epidermal ulceration was observed above the infiltrate.

Discussion To the authors’ knowledge, this is the first report which demonstrates the presence of A. phagocytophilum DNA in lesional skin biopsies from seropositive dogs that responded to the treatment with doxycycline. Currently, the cause for this positivity is not clear. Based on the information provided to us, our PCR-positive dogs lacked the typical clinical signs of acute CGA, such as lethargy, fever, thrombocytopenia (Jensen et al., 2007). Only one of the seropositive dogs had thrombocytopenia, the hallmark sign of acute CGA. Experimental studies have shown that thrombocytopenia is less frequently encountered in persistently infected dogs (Egenvall et al., 2000; Greig et al., 1996; Granick et al., 2009). Based on their studies of infected sheep, Granquist et al. (2010) hypothesized that the skin may be a site of persistence of this organism. Persistence could explain the PCR-positivity in the skin samples of our seropositive dogs which did not present with the classical signs of the acute CGA. Experimentally infected dogs with persistent CGA had intermittently positive PCR results from peripheral blood and positive serological titers (Egenvall et al., 2000; Eberts et al., 2011). Since our study was performed retrospectively, the peripheral blood was not tested by PCR, and serology was not performed repetitively. Another option for the PCR positivity in the skin biopsies could be that the amplified A. phagocytophilum DNA is associated with a previous tick bite. However, none of the PCR-positive biopsies had histopathological changes suggestive of an arthropod bite (acute or

chronic), and all of our control tick-bite biopsies were PCR-negative. Therefore, we assume this option to be unlikely. In our study, clinical and histopathological findings of the lesions varied in severity and appearance. Nevertheless erythema, papules, and plaques were a common clinical presentation. Gross lesions described in a female patient with human granulocytic anaplasmosis and Sweet’s syndrome were also erythematous papules, plaques, and nodules. These were histopathologically characterized by a dense dermal perivascular and interstitial, predominantly neutrophilic infiltrate (Halasz et al., 2005). In naturally infected lambs, clinical skin lesions from which A. phagocytophilum was amplified were focal as those described in a human case. Differences were seen upon histopathological examination, since the cellular infiltrate in lambs was characterized as mixed and not as predominantly neutrophilic like in the human case (Halasz et al., 2005; Granquist et al., 2010). A mixed cellular infiltrate, which was in most cases predominantly neutrophilic was also seen in our dogs (Table 1, dogs 4, 8, and 12). Vascular changes ranged from vasculopathy to fibrinoid necrosis of the vessel walls and were seen in 8/12 seropositive dogs in our study. Vasculitis was also described in A. phagocytophilum-infected lambs, but it was not a frequent finding (Granquist et al., 2010). However, it has to be mentioned that mild vascular changes such as vasculopathy can be easily overlooked by a pathologist not experienced in reading skin biopsies. In vitro and in vivo studies show that degranulation of A. phagocytophiluminfected neutrophils and the subsequent release of several potent chemotactic agents leads to an increased blood vessel permeability and helps to recruit other inflammatory cells (Carrade et al., 2009; Granquist et al., 2010; Day, 2011). This mechanism could explain the clinically observed erythema and papules in our dogs as well as the vascular changes we observed. Since the clinical and histological findings in the skin in our dogs are comparable to the reported human, dog, and lamb cases, these findings together with the positive PCR results indicate that the skin lesions in our dogs may be associated with a persistent A. phagocytophilum infection (Halasz et al., 2005; Egenvall et al., 2000; Granquist et al., 2010). In addition, negative leishmania PCR, lack of typical histopathological changes, and the failure to observe infectious organisms in any of the evaluated 65 biopsies decreases the possibility of other known infectious agents as the potential cause of these skin lesions. In our study, all PCR-positive canine skin biopsies were IHCnegative. A similar lack of a positive IHC result in the skin was found in a human granulocytic anaplasmosis and Sweet’s syndrome case (Halasz et al., 2005). But A. phagocytophilum morulae were detected by IHC in skin biopsies from acutely infected lamb skin biopsies (Granquist et al., 2010). Failure to observe A. phagocytophilum morulae in the skin biopsies can be explained by a lower amount of tissue examined by IHC as compared to PCR and the fact that the morulae could be unequally distributed within the sample (Franzén et al., 2009). Extensive IHC evaluation in our study was also limited by the amount of remaining tissue after the PCR testing was performed. We acknowledge that this retrospective study has the following limitations: lack of repeated serological and PCR testing, in order to clearly prove persistent CGA and unknown serological status of the tick-bite dogs. In summary, our results support the hypothesis that the persistence of A. phagocytophilum in the skin may be causative for otherwise unexplained skin lesions in seropositive dogs. However, further investigations in a larger cohort of dogs are warranted for a final proof. This is of importance since CGA is frequently diagnosed throughout the world, and further studies to gain information on skin lesions and A. phagoctyophilum pathology in persistently infected dogs are warranted (Pusterla et al., 1998; Tozon et al., 2003; Jensen et al., 2007; Carrade et al., 2009).

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