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Atypical Cowpox Virus Infection in a Series of Cats
J. Comp. Path. 2018, Vol. 158, 71e76
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
INFECTIOUS DISEASE
Atypical Cowpox Virus Infection in a Series of Cats N. Jungwirth*,{, C. Puff*, K. K€ oster†, R. Mischke†, H. Meyer‡, A. Starkx, x ‡ B. Thoma , G. Z€ oller , F. Seehusen*, M. Hewicker-Trautwein*, A. Beineke*,{, W. Baumg€ a rtner*,{ and P. Wohlsein* * Department of Pathology, University of Veterinary Medicine Hannover, † Small Animal Clinic, University of Veterinary Medicine Hannover, Hannover, ‡ Department of Viruses and Intracellular Agents, Bundeswehr Institute of Microbiology, x Central Diagnostic Laboratory Division, Bundeswehr Institute of Microbiology, Munich and { Centre for Systems Neuroscience, Hannover, Germany
Summary Within 4 weeks, five cats with skin lesions affecting the hindlimbs and mainly consisting of oedema, hyperaemia and plaque-like alterations were presented to the same veterinary clinic. The cats were suffering from lameness, trauma, renal insufficiency or complicated tail amputation. Although the lesions seemed unusual for a poxvirus infection, microscopical examination of biopsy samples or specimens taken during necropsy examination revealed ballooning degeneration of keratinocytes with eosinophilic, cytoplasmic inclusion bodies indicative of an orthopoxvirus infection. Cowpox virus infection was verified using immunohistochemistry and virus isolation. Molecular analysis revealed identical haemagglutinin gene sequences in four cases and spatiotemporal circumstances in some cases pointed to hospital-acquired transmission. Unusual manifestations of feline cowpox may have an unexpected risk for human infection. Ó 2017 Elsevier Ltd. All rights reserved. Keywords: cat; cowpox; orthopox; unusual presentation
Cowpox virus belongs to the genus Orthopoxvirus and is an endemic pathogen in Europe and Northern and Central Asia, with a zoonotic potential for man resulting in localized skin lesions or non-fatal or fatal systemic disease (Scarff and Clough, 1990; Pfeiff et al., 1991; Bennett and Baxby, 1996; Chantrey et al., 1999; Essbauer et al., 2010; Hemmer et al., 2010). Natural infections are seen in cattle, cats, dogs, rodents, non-human primates and various zoo animals (M€atz-Rensing et al., 2006; Kurth et al., 2008; Essbauer et al., 2010; Von Bomhard et al., 2011; J€ager et al., 2016). About 50% of human cowpox infections are caused by stray cats that had been infected by hunting rodents (Lawn, 2010; Herder et al., 2011; Appl et al., 2013). The highest seasonal incidence of Correspondence to: P. Wohlsein (e-mail: peter.wohlsein@tiho-hannover. de). 0021-9975/$ - see front matter https://doi.org/10.1016/j.jcpa.2017.12.003
feline cowpox is from late summer to autumn, corresponding to the peak size of rodent populations, especially of bank voles (Clethrionomys glareolus) or wood mice (Apodemus sylvaticus), which are regarded as reservoir hosts (Bennett and Baxby, 1996; Chantrey et al., 1999; Appl et al., 2013; Hnilica and Patterson, 2016). Typically, lesions in infected cats are located on the head, neck or forelimbs and consist of erythematous macules and papules extending into nodules of up to 1 cm over a period of 3e5 days (Hnilica and Patterson, 2016; Mauldin and Peters-Kennedy, 2016). These nodules tend to ulcerate, forming central necrosis, craters and crusts. During the first week of viraemia, secondary bacterial infection or systemic illness may be observed (Hnilica and Patterson, 2016; Mauldin and Peters-Kennedy, 2016). Additionally, individual cases develop fatal necrotizing pneumonia during the viraemic period, Ó 2017 Elsevier Ltd. All rights reserved.
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without typical skin lesions (Godfrey et al., 2004; Sch€ oniger et al., 2007; Mauldin and PetersKennedy, 2016). Fatal outcomes due to concurrent immunosuppression caused by feline leukaemia virus, feline immunodeficiency virus (FIV) or feline panleukopenia virus are described (Bennett et al., 1986; Schaudien et al., 2007). Most cats infected with cowpox recover completely (Sch€ oniger et al., 2007; Appl et al., 2013; Mauldin and Peters-Kennedy, 2016). In this report, five cats originating from the Greater Hannover area, infected with cowpox virus and presented to the same veterinary clinic over a 4-week period in 2015, are described in order to draw attention to both an unusual clinical presentation and an atypical morphological manifestation. A 7-year-old, neutered male European shorthair cat (case 1) with access to the outside was presented in late August with pain in the left hindlimb that had been present for the previous 5 days. Clinically, there was normal general behaviour despite an elevated body temperature (40.0 C). Multiple nodular skin lesions with crusts (<0.8 cm) were present on the head (lower lid, ear and mandible), right forelimb and trunk. The left hindlimb was diffusely and severely swollen and painful with mild exudation. Despite antibiotic treatment and wound dressing, no recovery was achieved and lesions progressed within 3 days to dermal necrosis on the dorsal part of the paw of the left hindlimb. Therefore, surgical debridement was performed 2 weeks after initial presentation (Fig. 1). The follow-up showed almost complete healing of the skin lesions within 3 weeks and after 4 months the cat was able to go outside again. A 1-year-old, neutered male, European shorthair cat (case 2) with access to the outside was referred to the same clinic 1 day after case 1 with a history of a car accident. The cat had severe abrasions on the left hindpaw and a severely swollen right forepaw with lameness. Wound debridement and amputation of the fifth toe of the left hindpaw were performed. Within 9 days after surgery, even with antibiotic treatment, severe necrosis of the left hindlimb developed and a complete amputation was performed. Twelve days after initial presentation the cat had developed multiple cutaneous red nodules (w0.5e1 cm) extending from the forelimbs to the trunk with central necrosis. Multiple macules were observed on the left flank and right hindlimb, which also showed diffuse, moderate, subcutaneous oedema. Due to its poor general condition and inappetence the cat was humanely destroyed 22 days after initial presentation. A 6-year-old, neutered male European shorthair cat (case 3) had been hospitalized in the same
Fig. 1. Cutaneous poxvirus infection, case 1. Dorsoplantar lesion of the left hindpaw showing focal extensive ulceration with diffuse, moderate oedema and diffuse mild hyperaemia.
clinic twice, in July and late August, due to chronic renal insufficiency, anaemia and thrombocytopaenia. The cat was seropositive for FIV. In midSeptember, the cat had developed severe swelling of the right hindlimb with lameness and poor general condition. Multifocal to coalescing areas of alopecia with mild hyperaemia were present on the right hindlimb, extending from the hip to the paws. Additionally, multifocal serocellular crusts and moderate, diffuse subcutaneous oedema were present at this location. The cat died spontaneously 1 day after hospitalization. A 5-year-old, neutered male European shorthair cat (case 4) with access to the outside had a tail amputation in early September after unknown trauma. Several days post surgery the cat developed poor general condition and diffuse, severe swelling with lameness affecting the left hindlimb that had started 5 days post surgery, followed by similar changes in the right hindlimb 12 days after surgery. This cat was also referred to the same clinic in mid-September and displayed diffuse, severe dermal hyperaemia of the left hindlimb and a brown, raised, dermal plaque (w1 cm) proximal to the foot pad (Fig. 2). Subsequently, the cat developed fever with deterioration of health status and was humanely destroyed. A 4-month-old, male cat (case 5) was presented in late September to the same clinic with a 2-day history
Atypical Cowpox Infection in Cats
Fig. 2. Cutaneous poxvirus infection, case 4. Ventroplantar lesion of the left hindpaw with diffuse, severe dermal hyperaemia and a focal raised, brown, plaque-like lesion (arrow).
of lameness. There was severe diffuse swelling of the left inguinal region and the knee and paw of the left hindlimb. A multifocal moderate erosive dermatitis was present on the left hindlimb and papules (<0.5 cm) were present at different locations including the left chin and shoulder. After a 3-day period of treatment with antibiotic, non-steroidal anti-inflammatory and immunomodulatory agents, the cat showed an improved health status and 14 days after presentation the skin lesions were completely resolved. Microscopical examination was performed on skin biopsy samples taken from different locations in cases 1, 2, 4 and 5. Following excisional skin biopsy in cases 2 and 4, complete necropsy examinations were performed on these animals as well as on case 3. In these necropsy cases, tissue samples from various organs including the skin were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections were stained with haematoxylin and eosin (HE). For immunohistochemistry (IHC), a monoclonal murine orthopoxvirus-specific antibody (clone 5B4) and the avidinebiotineperoxidase complex (ABC) method (Vector Laboratories, Burlingame, California, USA) were applied as previously described (Schaudien et al., 2007). A real-time polymerase chain reaction (PCR) with the RealStar OrthopoxÔ PCR kit targeting sequences of the fusion protein gene (Altona, Hamburg, Germany; Olson et al., 2004) was performed. A standard PCR spanning the entire open reading frame of the haemagglutinin (HA) gene (Damaso et al., 2000), followed by sequencing, resulted in detection of cowpox virus. An alignment was created using ClustalW v.1.83 and sequences of the National Center for Biotechnology Information (NCBI) database. Tissue homogenates from cases 2 and 4 were inoculated on MA104 (African green monkey kidney cells) and the identity of the cytopathogenic agent was confirmed
73
as cowpox virus by standard HA-gene PCR followed by sequencing (Schaudien et al., 2007). Histopathological examination confirmed typical poxvirus lesions in all five cases, consisting of moderate to severe ulcerative to necrosuppurative dermatitis with epidermal hyperplasia, ballooning degeneration of keratinocytes and large, cytoplasmic, eosinophilic inclusion bodies within epidermal and follicular keratinocytes (Fig. 3). Case 3 also had multicentric lymphoma affecting the lung, pulmonary lymph node, heart and kidneys. IHC revealed intralesional orthopoxvirus antigen in affected epidermal and hair follicle cells of all five cats. Nucleotide sequencing of the HA gene verified the presence of cowpox virus in all five cases with identical sequences of 939 nucleotides in cases 1 to 4 (Fig. 4). The same HA gene sequence had already been reported from cowpox cases in elephants (Hannover 1980; Hameln 1980), cats (Hannover 2004 and UK 2000) and man (Detmold 2001 and Paderborn 2007). The sequence of case 5 differed (924 nucleotides) and was not identical to any published sequence present within the NCBI GenBank database. In general, cowpox infections are considered as uncommon. with only 46 cats reported in a retrospective study between 2004 and 2010 in Germany (Appl et al., 2013). Typically, lesions are located on the head, neck or forelimbs and consist of ulcerated nodules with crusts followed by macules and papules (Appl et al., 2013; Hnilica and Patterson, 2016). In the present report, five cats from the Greater Hannover area with a cowpox virus infection were presented to the same veterinary clinic within a period of 4 weeks. These cats displayed unusual clinical manifestations with lameness and skin lesions affecting the
Fig. 3. Skin biopsy sample from case 2. Epidermal hyperplasia with ballooning degeneration of keratinocytes and multifocal large, cytoplasmic, eosinophilic inclusion bodies within keratinocytes (arrows). HE. Bar, 50 mm.
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Fig. 4. Evolutionary relationships of five cases of feline cowpox that occurred in Hannover, 2015, with a further 17 cowpox virus isolates. The evolutionary history was reconstructed by using the maximum-likelihood method and sequences of the haemagglutinin gene. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis revealed 12 identical sequences (GenBank accession number in parenthesis) including cases 1 to 4 Hannover 2015 (this paper; labelled in yellow), EPHannover 1980 (AY902292), cat Hannover S2216/2004, CPXV K780 UK 2000 (AY902281), CPXV K779 UK 2000 (AY902282), HumPad07/1 Paderborn 2007 (KC813496), EP4 Hameln 1980 (AY902292), cpv-922-99 Germany 1999 (AF377885) and CPXV 75/01 Detmold 2001 (AY902293). Additional sequences used for the analysis comprised HumBer07/1 Berlin 2007 (KC813509), HumMag07/1 Magdeburg 2007 (KC813495), case 5 Hannover 2015 (this paper, labelled in green), RatHei09/1 Heidelberg 2009 (KC813504), HumLan08/1 Landau 2009 (KC813492), CPXV Brighton UK 1927 (AF482758), Ratpox09 Marl 2009 (LN864565), RatKre08/2 Krefeld 2008 (KC813505), RatAac09/1 Aachen 2009 (KC813501) and RatGer09/1 Germering 2009 (KC813503). Scale bar shows nucleotide substitutions per site.
hindlimbs consisting of severe oedematous, hyperaemic swellings often with plaque-like lesions or erosions. Two (cases 1 and 5) of the five cats had sparse nodular lesions on the head and forelimbs, but lacked further characteristic skin changes. Initially, there was no clinical suspicion of a cowpox infection because the cats suffered from lameness, sequelae of a car accident or chronic renal insufficiency. Skin lesions were initially regarded as incidental findings and misinterpreted as cellulitis or pyoderma. In four cases (cases 1 to 4) an identical HA gene sequence was shown (Fig. 4). Identical HA gene sequences had already been reported in eight previous cases of cowpox (Schaudien et al., 2007; Carroll
et al., 2011; Dabrowski et al., 2013) since 1980 in the Greater Hannover area and also in 2000 in the UK. This probably indicates a permanent circulation of the virus in the reservoir populations. In this case series, case 1 may represent the index case with few nodular skin lesions at initial presentation. At the time of presentation a possible cowpox virus infection was not considered. However, the most consistent scenario would be indirect transmission of the virus, because within a narrow time frame, case 2 with a history of a car accident, case 3, which was treated for renal insufficiency, and case 4 with lameness after tail amputation were presented at the same clinic. Case 3 was the only animal known to be immunodeficient due to a FIV infection. The
Atypical Cowpox Infection in Cats
epidemiological circumstances of the cowpox virus infection in case 5 remain obscure. This cat already had some skin lesions at the time of initial presentation, indicating an infectious source outside the clinic, which is also supported by the different HA gene sequence. In conclusion, all five cats demonstrated an unusual clinical manifestation of a cowpox virus infection characterized by lameness and dermal changes affecting the hindlimbs. Typical pox lesions of the forelimbs or head were lacking or sparse and had been misinterpreted as incidental findings. The origin of the infection was not determined, but all cats had the opportunity for contact with free-living rodents. The identical HA gene sequence in cases 1 to 4 may indicate a common source of infection, either from the natural reservoir or from possible hospitalassociated virus transmission, which is supported by the presentation at the same clinic within a narrow time frame. Cowpox virus infection should be considered as a differential diagnosis even in cats with an unusual clinical presentation bearing in mind the potential zoonotic risk.
Acknowledgments The authors thank B. Buck, P. Gr€ unig, C. Sch€ utz, C. Namneck and C. Hermann for excellent technical assistance. The current address for B. Thoma is Medizinische Laboratorien D€ usseldorf, D€ usseldorf, Germany. This study received part funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 643476.
Conflicts of interest The authors declare no conflict of interest with respect to the publication of this manuscript.
References Appl C, von Bomhard W, Hanczaruk M, Meyer H, Bettenay S et al. (2013) Feline cowpoxvirus infections in Germany: clinical and epidemiological aspects. Berliner und M€unchener Tier€arztliche Wochenschrift, 126, 55e61. Bennett M, Baxby D (1996) Cowpox. Journal of Medical Microbiology, 45, 157e158. Bennett M, Gaskell CJ, Gaskell RM, Baxby D, GruffyddJones TJ (1986) Poxvirus infection in the domestic cat: some clinical and epidemiological observations. Veterinary Record, 118, 387e390. Carroll DS, Emerson GL, Li Y, Sammons S, Olson V et al. (2011) Chasing Jenner’s vaccine: revisiting cowpox virus classification. PLoS One, 6, e23086.
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Chantrey J, Meyer H, Baxby D, Begon M, Bown KJ et al. (1999) Cowpox: reservoir hosts and geographic range. Epidemiology and Infection, 122, 455e460. Dabrowski PW, Radonic A, Kurth A, Nitsche A (2013) Genome-wide comparison of cowpox viruses reveals a new clade related to variola virus. PLoS One, 8, e79953. Damaso CR, Esposito JJ, Condit RC, Moussatche N (2000) An emergent poxvirus from humans and cattle in Rio de Janeiro State: Cantagalo virus may derive from Brazilian smallpox vaccine. Virology, 277, 439e449. Essbauer S, Pfeffer M, Meyer H (2010) Zoonotic poxviruses. Veterinary Microbiology, 140, 229e236. Godfrey DR, Blundell CJ, Essbauer S, Pfeffer M, Shearer DH et al. (2004) Unusual presentations of cowpox infection in cats. Journal of Small Animal Practice, 45, 202e205. Hemmer CJ, Littmann M, L€ obermann M, Meyer H, Petschaelis A et al. (2010) Human cowpox virus infection acquired from a circus elephant in Germany. International Journal of Infectious Diseases, 14(Suppl. 3), e338ee340. Herder V, Wohlsein P, Grunwald D, Janssen H, Meyer H et al. (2011) Poxvirus infection in a cat with presumptive human transmission. Veterinary Dermatology, 22, 220e224. Hnilica K, Patterson A (2016) Viral, rickettsial, and protozoal skin diseases. In: Small Animal Dermatology, 4th edit., KA Hnilica, AP Patterson, Eds., Elsevier, St. Louis, p. 181. J€ ager K, Steinborn P, Weider K, Wohlsein P (2016) Kutane Infektion mit Orthopoxvirus bovis bei einem Wachtelhund. Tier€arztliche Praxis Kleintiere, 44, 273e277. Kurth A, Wibbelt G, Gerber HP, Petschaelis A, Pauli G et al. (2008) Rat-to-elephant-to-human transmission of cowpox virus. Emerging Infectious Diseases, 14, 670e671. Lawn R (2010) Risk of cowpox to small animal practitioners. Veterinary Record, 166, 631. M€ atz-Rensing K, Ellerbrok H, Ehlers B, Pauli G, Floto A et al. (2006) Fatal poxvirus outbreak in a colony of New World monkeys. Veterinary Pathology, 43, 212e218. Mauldin AE, Peters-Kennedy J (2016) Integumentary system. In: Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals, 6th edit., Vol. 1, GM Maxie, Ed., Elsevier, London, pp. 619e620. Olson VA, Laue T, Laker MT, Babkin IV, Drosten C et al. (2004) Real-time PCR system for detection of orthopoxviruses and simultaneous identification of smallpox virus. Journal of Clinical Microbiology, 42, 1940e1946. ubinger AM, Gerritzen A, Pfeiff B, Pullmann H, Eis-H€ Schneweis KE et al. (1991) Letale TierpockenInfektion bei einem Atopiker unter dem Bild einer Variola vera (Lethal animal pox virus infection in an atopic patient reminiscent of variola vera). Hautarzt, 42, 293e297. Scarff D, Clough C (1990) Fatal cowpox infection in a human. Veterinary Record, 127, 459.
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Schaudien D, Meyer H, Grunwald D, Janssen H, Wohlsein P (2007) Concurrent infection of a cat with cowpox virus and feline parvovirus. Journal of Comparative Pathology, 137, 151e154. Sch€oniger S, Chan DL, Hollinshead M, Humm K, Smith GL et al. (2007) Cowpox virus pneumonia in a domestic cat in Great Britain. Veterinary Record, 160, 522e523. Von Bomhard W, Mauldin EA, Breuer W, Pfleghaar S, Nitsche A (2011) Localized cowpox infection in a 5-
month-old 111e114.
rottweiler.
Veterinary
Dermatology,
July 28th, 2017 ½ Received, Accepted, December 8th, 2017
22,
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
INFECTIOUS DISEASE
Atypical Cowpox Virus Infection in a Series of Cats N. Jungwirth*,{, C. Puff*, K. K€ oster†, R. Mischke†, H. Meyer‡, A. Starkx, x ‡ B. Thoma , G. Z€ oller , F. Seehusen*, M. Hewicker-Trautwein*, A. Beineke*,{, W. Baumg€ a rtner*,{ and P. Wohlsein* * Department of Pathology, University of Veterinary Medicine Hannover, † Small Animal Clinic, University of Veterinary Medicine Hannover, Hannover, ‡ Department of Viruses and Intracellular Agents, Bundeswehr Institute of Microbiology, x Central Diagnostic Laboratory Division, Bundeswehr Institute of Microbiology, Munich and { Centre for Systems Neuroscience, Hannover, Germany
Summary Within 4 weeks, five cats with skin lesions affecting the hindlimbs and mainly consisting of oedema, hyperaemia and plaque-like alterations were presented to the same veterinary clinic. The cats were suffering from lameness, trauma, renal insufficiency or complicated tail amputation. Although the lesions seemed unusual for a poxvirus infection, microscopical examination of biopsy samples or specimens taken during necropsy examination revealed ballooning degeneration of keratinocytes with eosinophilic, cytoplasmic inclusion bodies indicative of an orthopoxvirus infection. Cowpox virus infection was verified using immunohistochemistry and virus isolation. Molecular analysis revealed identical haemagglutinin gene sequences in four cases and spatiotemporal circumstances in some cases pointed to hospital-acquired transmission. Unusual manifestations of feline cowpox may have an unexpected risk for human infection. Ó 2017 Elsevier Ltd. All rights reserved. Keywords: cat; cowpox; orthopox; unusual presentation
Cowpox virus belongs to the genus Orthopoxvirus and is an endemic pathogen in Europe and Northern and Central Asia, with a zoonotic potential for man resulting in localized skin lesions or non-fatal or fatal systemic disease (Scarff and Clough, 1990; Pfeiff et al., 1991; Bennett and Baxby, 1996; Chantrey et al., 1999; Essbauer et al., 2010; Hemmer et al., 2010). Natural infections are seen in cattle, cats, dogs, rodents, non-human primates and various zoo animals (M€atz-Rensing et al., 2006; Kurth et al., 2008; Essbauer et al., 2010; Von Bomhard et al., 2011; J€ager et al., 2016). About 50% of human cowpox infections are caused by stray cats that had been infected by hunting rodents (Lawn, 2010; Herder et al., 2011; Appl et al., 2013). The highest seasonal incidence of Correspondence to: P. Wohlsein (e-mail: peter.wohlsein@tiho-hannover. de). 0021-9975/$ - see front matter https://doi.org/10.1016/j.jcpa.2017.12.003
feline cowpox is from late summer to autumn, corresponding to the peak size of rodent populations, especially of bank voles (Clethrionomys glareolus) or wood mice (Apodemus sylvaticus), which are regarded as reservoir hosts (Bennett and Baxby, 1996; Chantrey et al., 1999; Appl et al., 2013; Hnilica and Patterson, 2016). Typically, lesions in infected cats are located on the head, neck or forelimbs and consist of erythematous macules and papules extending into nodules of up to 1 cm over a period of 3e5 days (Hnilica and Patterson, 2016; Mauldin and Peters-Kennedy, 2016). These nodules tend to ulcerate, forming central necrosis, craters and crusts. During the first week of viraemia, secondary bacterial infection or systemic illness may be observed (Hnilica and Patterson, 2016; Mauldin and Peters-Kennedy, 2016). Additionally, individual cases develop fatal necrotizing pneumonia during the viraemic period, Ó 2017 Elsevier Ltd. All rights reserved.
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N. Jungwirth et al.
without typical skin lesions (Godfrey et al., 2004; Sch€ oniger et al., 2007; Mauldin and PetersKennedy, 2016). Fatal outcomes due to concurrent immunosuppression caused by feline leukaemia virus, feline immunodeficiency virus (FIV) or feline panleukopenia virus are described (Bennett et al., 1986; Schaudien et al., 2007). Most cats infected with cowpox recover completely (Sch€ oniger et al., 2007; Appl et al., 2013; Mauldin and Peters-Kennedy, 2016). In this report, five cats originating from the Greater Hannover area, infected with cowpox virus and presented to the same veterinary clinic over a 4-week period in 2015, are described in order to draw attention to both an unusual clinical presentation and an atypical morphological manifestation. A 7-year-old, neutered male European shorthair cat (case 1) with access to the outside was presented in late August with pain in the left hindlimb that had been present for the previous 5 days. Clinically, there was normal general behaviour despite an elevated body temperature (40.0 C). Multiple nodular skin lesions with crusts (<0.8 cm) were present on the head (lower lid, ear and mandible), right forelimb and trunk. The left hindlimb was diffusely and severely swollen and painful with mild exudation. Despite antibiotic treatment and wound dressing, no recovery was achieved and lesions progressed within 3 days to dermal necrosis on the dorsal part of the paw of the left hindlimb. Therefore, surgical debridement was performed 2 weeks after initial presentation (Fig. 1). The follow-up showed almost complete healing of the skin lesions within 3 weeks and after 4 months the cat was able to go outside again. A 1-year-old, neutered male, European shorthair cat (case 2) with access to the outside was referred to the same clinic 1 day after case 1 with a history of a car accident. The cat had severe abrasions on the left hindpaw and a severely swollen right forepaw with lameness. Wound debridement and amputation of the fifth toe of the left hindpaw were performed. Within 9 days after surgery, even with antibiotic treatment, severe necrosis of the left hindlimb developed and a complete amputation was performed. Twelve days after initial presentation the cat had developed multiple cutaneous red nodules (w0.5e1 cm) extending from the forelimbs to the trunk with central necrosis. Multiple macules were observed on the left flank and right hindlimb, which also showed diffuse, moderate, subcutaneous oedema. Due to its poor general condition and inappetence the cat was humanely destroyed 22 days after initial presentation. A 6-year-old, neutered male European shorthair cat (case 3) had been hospitalized in the same
Fig. 1. Cutaneous poxvirus infection, case 1. Dorsoplantar lesion of the left hindpaw showing focal extensive ulceration with diffuse, moderate oedema and diffuse mild hyperaemia.
clinic twice, in July and late August, due to chronic renal insufficiency, anaemia and thrombocytopaenia. The cat was seropositive for FIV. In midSeptember, the cat had developed severe swelling of the right hindlimb with lameness and poor general condition. Multifocal to coalescing areas of alopecia with mild hyperaemia were present on the right hindlimb, extending from the hip to the paws. Additionally, multifocal serocellular crusts and moderate, diffuse subcutaneous oedema were present at this location. The cat died spontaneously 1 day after hospitalization. A 5-year-old, neutered male European shorthair cat (case 4) with access to the outside had a tail amputation in early September after unknown trauma. Several days post surgery the cat developed poor general condition and diffuse, severe swelling with lameness affecting the left hindlimb that had started 5 days post surgery, followed by similar changes in the right hindlimb 12 days after surgery. This cat was also referred to the same clinic in mid-September and displayed diffuse, severe dermal hyperaemia of the left hindlimb and a brown, raised, dermal plaque (w1 cm) proximal to the foot pad (Fig. 2). Subsequently, the cat developed fever with deterioration of health status and was humanely destroyed. A 4-month-old, male cat (case 5) was presented in late September to the same clinic with a 2-day history
Atypical Cowpox Infection in Cats
Fig. 2. Cutaneous poxvirus infection, case 4. Ventroplantar lesion of the left hindpaw with diffuse, severe dermal hyperaemia and a focal raised, brown, plaque-like lesion (arrow).
of lameness. There was severe diffuse swelling of the left inguinal region and the knee and paw of the left hindlimb. A multifocal moderate erosive dermatitis was present on the left hindlimb and papules (<0.5 cm) were present at different locations including the left chin and shoulder. After a 3-day period of treatment with antibiotic, non-steroidal anti-inflammatory and immunomodulatory agents, the cat showed an improved health status and 14 days after presentation the skin lesions were completely resolved. Microscopical examination was performed on skin biopsy samples taken from different locations in cases 1, 2, 4 and 5. Following excisional skin biopsy in cases 2 and 4, complete necropsy examinations were performed on these animals as well as on case 3. In these necropsy cases, tissue samples from various organs including the skin were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections were stained with haematoxylin and eosin (HE). For immunohistochemistry (IHC), a monoclonal murine orthopoxvirus-specific antibody (clone 5B4) and the avidinebiotineperoxidase complex (ABC) method (Vector Laboratories, Burlingame, California, USA) were applied as previously described (Schaudien et al., 2007). A real-time polymerase chain reaction (PCR) with the RealStar OrthopoxÔ PCR kit targeting sequences of the fusion protein gene (Altona, Hamburg, Germany; Olson et al., 2004) was performed. A standard PCR spanning the entire open reading frame of the haemagglutinin (HA) gene (Damaso et al., 2000), followed by sequencing, resulted in detection of cowpox virus. An alignment was created using ClustalW v.1.83 and sequences of the National Center for Biotechnology Information (NCBI) database. Tissue homogenates from cases 2 and 4 were inoculated on MA104 (African green monkey kidney cells) and the identity of the cytopathogenic agent was confirmed
73
as cowpox virus by standard HA-gene PCR followed by sequencing (Schaudien et al., 2007). Histopathological examination confirmed typical poxvirus lesions in all five cases, consisting of moderate to severe ulcerative to necrosuppurative dermatitis with epidermal hyperplasia, ballooning degeneration of keratinocytes and large, cytoplasmic, eosinophilic inclusion bodies within epidermal and follicular keratinocytes (Fig. 3). Case 3 also had multicentric lymphoma affecting the lung, pulmonary lymph node, heart and kidneys. IHC revealed intralesional orthopoxvirus antigen in affected epidermal and hair follicle cells of all five cats. Nucleotide sequencing of the HA gene verified the presence of cowpox virus in all five cases with identical sequences of 939 nucleotides in cases 1 to 4 (Fig. 4). The same HA gene sequence had already been reported from cowpox cases in elephants (Hannover 1980; Hameln 1980), cats (Hannover 2004 and UK 2000) and man (Detmold 2001 and Paderborn 2007). The sequence of case 5 differed (924 nucleotides) and was not identical to any published sequence present within the NCBI GenBank database. In general, cowpox infections are considered as uncommon. with only 46 cats reported in a retrospective study between 2004 and 2010 in Germany (Appl et al., 2013). Typically, lesions are located on the head, neck or forelimbs and consist of ulcerated nodules with crusts followed by macules and papules (Appl et al., 2013; Hnilica and Patterson, 2016). In the present report, five cats from the Greater Hannover area with a cowpox virus infection were presented to the same veterinary clinic within a period of 4 weeks. These cats displayed unusual clinical manifestations with lameness and skin lesions affecting the
Fig. 3. Skin biopsy sample from case 2. Epidermal hyperplasia with ballooning degeneration of keratinocytes and multifocal large, cytoplasmic, eosinophilic inclusion bodies within keratinocytes (arrows). HE. Bar, 50 mm.
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Fig. 4. Evolutionary relationships of five cases of feline cowpox that occurred in Hannover, 2015, with a further 17 cowpox virus isolates. The evolutionary history was reconstructed by using the maximum-likelihood method and sequences of the haemagglutinin gene. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis revealed 12 identical sequences (GenBank accession number in parenthesis) including cases 1 to 4 Hannover 2015 (this paper; labelled in yellow), EPHannover 1980 (AY902292), cat Hannover S2216/2004, CPXV K780 UK 2000 (AY902281), CPXV K779 UK 2000 (AY902282), HumPad07/1 Paderborn 2007 (KC813496), EP4 Hameln 1980 (AY902292), cpv-922-99 Germany 1999 (AF377885) and CPXV 75/01 Detmold 2001 (AY902293). Additional sequences used for the analysis comprised HumBer07/1 Berlin 2007 (KC813509), HumMag07/1 Magdeburg 2007 (KC813495), case 5 Hannover 2015 (this paper, labelled in green), RatHei09/1 Heidelberg 2009 (KC813504), HumLan08/1 Landau 2009 (KC813492), CPXV Brighton UK 1927 (AF482758), Ratpox09 Marl 2009 (LN864565), RatKre08/2 Krefeld 2008 (KC813505), RatAac09/1 Aachen 2009 (KC813501) and RatGer09/1 Germering 2009 (KC813503). Scale bar shows nucleotide substitutions per site.
hindlimbs consisting of severe oedematous, hyperaemic swellings often with plaque-like lesions or erosions. Two (cases 1 and 5) of the five cats had sparse nodular lesions on the head and forelimbs, but lacked further characteristic skin changes. Initially, there was no clinical suspicion of a cowpox infection because the cats suffered from lameness, sequelae of a car accident or chronic renal insufficiency. Skin lesions were initially regarded as incidental findings and misinterpreted as cellulitis or pyoderma. In four cases (cases 1 to 4) an identical HA gene sequence was shown (Fig. 4). Identical HA gene sequences had already been reported in eight previous cases of cowpox (Schaudien et al., 2007; Carroll
et al., 2011; Dabrowski et al., 2013) since 1980 in the Greater Hannover area and also in 2000 in the UK. This probably indicates a permanent circulation of the virus in the reservoir populations. In this case series, case 1 may represent the index case with few nodular skin lesions at initial presentation. At the time of presentation a possible cowpox virus infection was not considered. However, the most consistent scenario would be indirect transmission of the virus, because within a narrow time frame, case 2 with a history of a car accident, case 3, which was treated for renal insufficiency, and case 4 with lameness after tail amputation were presented at the same clinic. Case 3 was the only animal known to be immunodeficient due to a FIV infection. The
Atypical Cowpox Infection in Cats
epidemiological circumstances of the cowpox virus infection in case 5 remain obscure. This cat already had some skin lesions at the time of initial presentation, indicating an infectious source outside the clinic, which is also supported by the different HA gene sequence. In conclusion, all five cats demonstrated an unusual clinical manifestation of a cowpox virus infection characterized by lameness and dermal changes affecting the hindlimbs. Typical pox lesions of the forelimbs or head were lacking or sparse and had been misinterpreted as incidental findings. The origin of the infection was not determined, but all cats had the opportunity for contact with free-living rodents. The identical HA gene sequence in cases 1 to 4 may indicate a common source of infection, either from the natural reservoir or from possible hospitalassociated virus transmission, which is supported by the presentation at the same clinic within a narrow time frame. Cowpox virus infection should be considered as a differential diagnosis even in cats with an unusual clinical presentation bearing in mind the potential zoonotic risk.
Acknowledgments The authors thank B. Buck, P. Gr€ unig, C. Sch€ utz, C. Namneck and C. Hermann for excellent technical assistance. The current address for B. Thoma is Medizinische Laboratorien D€ usseldorf, D€ usseldorf, Germany. This study received part funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 643476.
Conflicts of interest The authors declare no conflict of interest with respect to the publication of this manuscript.
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July 28th, 2017 ½ Received, Accepted, December 8th, 2017
22,