A Case of Prototheca Zopfii Genotype 1 Infection in a Dog (Canis Lupus

Mycopathologia https://doi.org/10.1007/s11046-018-0274-5

CASE REPORT

A Case of Prototheca zopﬁi Genotype 1 Infection in a Dog (Canis lupus familiaris)

Caroline S. Silveira . Deborah Cesar . M. Kelly Keating . Marlene DeLeon-Carnes . Anı´bal G. Armie´n . Martı´n Luhers . Franklin Riet-Correa . Federico Giannitti

Received: 15 January 2018 / Accepted: 28 May 2018 Ó Springer Science+Business Media B.V., part of Springer Nature 2018

Abstract Protothecosis is a rare disease caused by veterinary clinic in Montevideo, Uruguay, with mul- environmental algae of the genus Prototheca. These tiple skin nodules, one of which was excised by are saprophytic, non-photosynthetic, aerobic, color- surgical biopsy. The sample was examined histolog- less algae that belong to the Chlorellaceae family. ically and processed by PCR, DNA sequencing, and Seven different species have been described. Pro- restriction fragments length polymorphisms for the totheca zopfii genotype 2 and P. wickerhamii are most detection and genotyping of P. zopfii. In addition, commonly involved in pathogenic infections in transmission electron microscopy and scanning elec- humans and animals. The objective of this work is to tron microscopy were performed. Histology showed describe, for the first time, a case of protothecosis severe ulcerative granulomatous dermatitis and pan- caused by P. zopfii genotype 1 in a dog. The dog, a niculitis with myriads of pleomorphic algae. Algal 4-year-old mix bred male, was presented to a cells were 4–17 lm in size, with an amphophilic, 2–4- lm-thick wall frequently surrounded by a clear halo, contained flocculant material and a deeply basophilic Handling Editor: Rui Kano. nucleus, and internal septae with daughter cells (endospores) consistent with endosporulation. C. S. Silveira Á F. Riet-Correa Á F. Giannitti (&) Ultrastructurally, algal cells/endospores at different ´ Instituto Nacional de Investigacion Agropecuaria (INIA), stages of development were found within para- Ruta No. 50, km 11, La Estanzuela, Colonia 70006, Uruguay sitophorous vacuoles in macrophages. Prototheca e-mail: [email protected] zopfii genotype 1 was identified by molecular testing, confirming the etiologic diagnosis of protothecosis. D. Cesar Private Practice, Montevideo, Uruguay Keywords Algal diseases Á Algal infection Á Dog Á M. K. Keating Á M. DeLeon-Carnes Genotypic characterization Á Protothecosis Á South Centers for Disease Control and Prevention, Atlanta, GA, America USA

A. G. Armie´n Á F. Giannitti Veterinary Population Medicine Department, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN, USA

M. Luhers Universidad de la Repu´blica, Montevideo, Uruguay 123 Mycopathologia

Introduction and responsive and had good body condition and muscle tone, adequate hydration state, normal capil- Protothecosis is a rare disease caused by environmen- lary refill time, and adequate coloration of the mucous tal algae of the genus Prototheca, saprophytes of membranes. Differential diagnoses for the multiple worldwide distribution that can infect mammals and cutaneous nodules on clinical grounds included gran- result in either localized or systemic, life-threatening ulomatous or pyogranulomatous dermatitis and pan- disease, particularly in immunocompromised individ- niculitis caused by a bacterial or fungal infection, or a uals [1, 2]. Prototheca spp. is a non-photosynthetic, multinodular/multicentric skin malignancy (neo- achlorophyllic (colorless), aerobic alga in the family plasia). However, the owner refused ancillary labora- Chlorellaceae. Seven Prototheca species have been tory testing and the condition remained undiagnosed. described to date [3], of these P. zopfii and P. The dog was treated empirically and unsuccessfully wickerhamii have been more commonly implicated with enrofloxacin and prednisone. After 1 month of in pathogenic infections in humans and animals treatment, the animal was presented again to the clinic [2, 4, 7], while the novel species P. miyajii was with vomiting, diarrhea, and partial loss of vision (the isolated recently from a human patient with systemic patient would stumble into objects and would not blink protothecosis [3]. Within P. zopfii species, two differ- in response to visual stimuli). At that time, the larger ent genotypes (1 and 2) are currently recognized; mass, located in the left thoracic limb, was surgically genotype 2 is most commonly involved in different biopsied for histologic examination. Additional phys- clinical forms [4, 6]. ical examination and laboratory analyses were not Prototheca spp. have been associated with various performed on the basis of owner’s decision. syndromes in different species. The most commonly The skin biopsy was fixed in 10% neutral-buffered recognized forms of human protothecosis are dissem- formalin, routinely processed histologically, embed- inated, cutaneous, and olecranon bursitis [5]. In cattle, ded in paraffin, microtome-sectioned at 4 lm, stained mastitis is the more common manifestation [6], while with hematoxylin and eosin (HE), Gomori’s methe- rhinitis and sinusitis have been described in horses [8]. namine silver (GMS), and periodic acid–Schiff (PAS), In dogs, protothecosis is usually associated with colitis and examined under optic microscope (Axio Sco- and/or disseminated disease, sometimes with ocular pe.A1, Carl Zeiss, Germany). The lesion was charac- [9], or central nervous system involvement [10, 11]. terized by severe granulomatous and ulcerative The infection restricted only to the skin is rare in this dermatitis and panniculitis, with occasional multinu- species [12], although it appears to be the most cleate giant cells and myriads of algal organisms. The common form in the cat [13]. The cutaneous form in single microorganisms were spherical to ovoid, varied the dog is usually associated with P. wickerhamii in size between 4 and 17 lm, had a thin amphophilic infection [2, 7, 14], although it has recently been cell wall that ranged from 2 to 4 lm, and were described in a case of P. zopfii genotype 2 infection frequently surrounded by a clear halo. Algal cells [4]. Here we describe a case of protothecosis caused by contained a distinct deeply basophilic nucleus and P. zopfii genotype 1 in a dog from Uruguay, which to flocculent material. Some organisms contained a the best of our knowledge represents the first report of refractile capsule or displayed internal septation P. zopfii genotype 1 infection in dogs. typical of endosporulation, with daughter cells (endospores) filling the sporangia (Fig. 1a). The organisms were positive with GMS and PAS (Fig. 1b). Case Report Transmission electron microscopy (TEM) was performed on formalin-fixed paraffin-embedded A 4-year-old, intact male, medium-sized (* 15 kg of (FFPE) skin following a procedure previously body weight), mix breed dog was presented to the described [15]. Briefly, the sample was deparaffinized veterinary clinic in Montevideo, Uruguay, on June and post-fixed in 0.166 M cacodylate-buffered 3% 2015. The dog had multiple (5) skin nodules in the glutaraldehyde with 1% tannic acid (Electron Micro- right knee, thoracic region, dorsal aspect of the nose, scopy Sciences, Hatfield, Pennsylvania), followed by and right and left thoracic limbs. Upon initial exam- a second post-fixation treatment in 1% osmium ination, the patient was normothermic, bright, alert, tetroxide (Electron Microscopy Sciences, Hatfield, 123 Mycopathologia

Fig. 1 Lesions and morphology of P. zopfii genotype 1 in the a have strongly PAS-positive cell wall and septae (arrow). PAS skin of a dog. a Severe granulomatous dermatitis. Extracellu- stain, original magnification 9400. c Degenerated algae within larly and within infiltrating macrophages (arrowhead) there are parasitophorous vacuoles of macrophages. Alga walls (arrows) numerous pleomorphic algal cells, that range from 4 to 17 lmin are collapsed, and internal structures are lacking. M macrophage size, have a 2–4-lm-thick wall occasionally surrounded by a nucleus. Transmission electron microscopy. Bar = 5 lm. d Al- clear halo, and contained internal septae consistent with gae within parasitophorous vacuoles of macrophages (arrows). endosporulation (arrow). HE stain, original magnification Scanning electron microscopy. Bar = 50 lm 9400. b Severe granulomatous dermatitis. Algae depicted in

Pennsylvania). Using a graded series of ethanol, tissue commercial software (iTEM, Olympus SIS, Munster, blocks were dehydrated and embedded in resin Germany). (EMbed, Electron Microscopy Sciences, Hatfield, For scanning electron microscopy (SEM), sections Pennsylvania) and sectioned on a ultramicrotome were post-fixed in 2.5% glutaraldehyde in 0.1 M (UC6, Leica Microsystems, Vienna, Austria). Thin sodium cacodylate buffer overnight. The sample was sections of 60–90 nm were collected on 100-mesh washed three times in 0.1 M sodium cacodylate buffer copper grids (Electron Microscopy Sciences, Hatfield, and post-fixed in 1% osmium tetroxide in 0.1 M Pennsylvania) and stained with 5% uranyl acetate for sodium cacodylate buffer. The sample was rinsed 20 min and Satos’ lead citrate for 6 min. Stained three times in deionized water and passed through a sections were examined under a transmission electron series of ethanol dehydration steps in the following microscope (JEOL Ltd., Tokyo, Japan). The dimen- order: 25, 50, 70, 95, and 100%. After dehydration, sions of the microorganisms were obtained using samples were placed in a critical point dryer and dehydrated further, followed by coating in the sputter

123 Mycopathologia coater. Microorganisms were examined on a Hitachi cutaneous entrance through skin trauma or penetration S3500N scanning electron microscope (Tokyo, through mucosal surfaces has also been documented Japan). [1, 2, 10, 11]. Given that the skin nodules developed At TEM and SEM, microorganisms at different before the onset of other systemic clinical signs, it is stages of development and degeneration were found possible that the skin was the point of entry. However, within the parasitophorous vacuoles of macrophages. ingestion as a possible route of transmission should Algal cells in early stages of degeneration had not be ruled out. prominent central nuclei and large nucleoli. Algal cell Once the diagnosis of protothecosis was reached, walls were thick, dense, and irregular, with fragments treatment with amphotericin B intravenously of cell wall material extending into the macrophage (12.5 mg weekly) and itraconazole orally (100 mg cytoplasm (Fig. 1c, d). daily) was initiated (data not shown). After 2 months For molecular confirmation and speciation, a poly- of treatment, the dog improved clinically and the merase chain reaction (PCR) restriction fragments cutaneous masses reduced in size significantly. Unfor- length polymorphism (RFLP) method [16], previously tunately, the long-term outcome remained unknown, validated against positive culture controls at the since the patient was not accessioned into the clinic Centers for Disease Control and Prevention (Atlanta, again for follow-up examination. It is important to Georgia), was adopted. DNA extracted from skin note that there can be some variation in the in vitro obtained from the FFPE blocks was amplified using susceptibility of P. zopfii genotypes 1 and 2 to primers targeting the 18S rRNA gene (forward: Proto amphotericin B, gentamicin, and kanamycin [17], so 18-2f: 50-CGCGCAAATTACCCAATCC-30; reverse: culture, drug susceptibility testing, and genotyping of Proto 18-2r: 50-AACGGGACGGCCAGAGT-30). The clinical isolates should be implemented when possible sequenced amplicon showed 100% homology to P. to aid in the decision making in face of specific zopfii. Further typing was performed using RFLP therapy. analysis, as previously described [16]. Briefly, the The most common clinical manifestation of pro- 450-bp fragment was cut by the P. zopfii genotype tothecosis in dogs is hemorrhagic colitis, progressing 1-specific restriction enzyme Kpn21, and not by to systemic disease [2]. The cutaneous/tegumentary the P. zopfii genotype 2-specific restriction enzy- presentation is more frequently observed in cats [13]. me SmaI, revealing the presence of P. zopfii genotype In dogs, this form is rare [12] and is characterized by 1. multiple ulcerated skin nodules, mainly in the extrem- ities of the limbs. These lesions have been predom- inantly associated with P. wickerhamii infection [2, 7]. Discussion Because P. zopfii can be associated with other clinical syndromes in various animal species, includ- The diagnostic investigation in this case allowed for ing bovine mastitis [6, 18] and rhinitis in horses [8], unequivocal confirmation of a P. zopfii genotype 1 veterinary practitioners and laboratory diagnosticians infection in a dog, by a combination of morphological in Uruguay should be aware of the spectrum of and molecular methods. Due to the clinical progres- diseases potentially associated with protothecal infec- sion and presence of concomitant signs of vomiting, tion to increase the chances of reaching an etiologic diarrhea, and loss of vision, it can be speculated that diagnosis, in clinical cases or specimens submitted to the infection was not restricted to the skin, but that the diagnostic laboratories, respectively. dog may have been suffering from disseminated Although canine protothecosis has been described protothecosis. A similar presentation of disseminated sporadically in neighboring countries such as Brazil protothecosis with skin involvement has been [19, 20], and recently in Argentina [21], the condition described recently in a dog infected with P. zopfii is unprecedented in Uruguay. Reports on genotypic genotype 2 [4]. characterization of P. zopfii strains infecting dogs in The pathogenesis of protothecosis in dogs is not yet South America are so far limited to one description of fully elucidated. Ingestion has been regarded as the the genotype 2 in Brazil [19], which has also been most likely route of transmission in most cases of implicated in cases of bovine mastitis in this country disseminated protothecosis by P. zopfii [2]; however, [22]. This genotype has also been detected in diseased 123 Mycopathologia dogs in Italy [4] and the USA [23]. Analysis of 350 References clinical Prototheca isolates, including 342 bovine strains from around the world, 6 canine strains from 1. Pal M, Abraha A, Rahman MT, Dave P. Protothecosis: an Germany, Brazil, and USA, and 2 human strains from emerging algal disease of humans and animals. Int J Life Sci Biotechnol Pharm Res. 2014;3:1–13. Austria and China, revealed that 90.6% of the bovine 2. 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