Surto de leishmaniose causada por Leishmania enriettii em cobaias (Cavia porcellus)

pvb Pesquisa Veterinária Brasileira Pesq. Vet. Bras. 0100-736X 1678-5150 Colégio Brasileiro de Patologia Animal - CBPA RESUMO: Descrevemos um surto de leishmaniose em sete cobaias (Cavia porcellus), com lesões cutâneas nodulares ulceradas de tamanhos variados observadas na cavidade nasal, lábio superior, pavilhões auriculares, vulva e região periarticular dos membros. No exame citológico foram encontrados macrófagos contendo vacúolos parasitóforos no citoplasma de aproximadamente 4.0μm em diâmetro com morfologia sugestiva de Leishmania sp. Apesar de regressão espontânea das lesões cutâneas terem ocorrido em duas das sete cobaias, apenas um sobreviveu. Seis dos sete animais afetados morreram e foram necropsiados. Macroscopicamente, todos os animais apresentaram lesões cutâneas nodulares, crostosas e sanguinolentas. Uma das cobaias tinha pulmões vermelho-escuros, distendidos e firmes. A histopatologia das lesões cutâneas revelou dermatite acantótica intersticial histiocítica associada a miríades de organismos de Leishmania no citoplasma de macrófagos. Nos pulmões as lesões eram características de pneumonia bronco-intersticial com infiltrado focal de neutrófilos, eosinófilos, macrófagos epitelioides e células gigantes multinucleadas contendo amastigotas basofílicos de 2µm com morfologia compatível com Leishmania spp. Lesões granulomatosas focais associadas ao agente no pulmão são um achado inédito na leishmaniose causada por L. enriettii em cobaias. A técnica de reação em cadeia da polimerase (PCR) com primer mini-exon realizada em amostras de lesões de duas cobaias afetadas foi positiva, identificando Leishmania enriettii. Os aspectos macroscópicos, citológicos, e histológicos associados à técnica da (PCR), permitiram o diagnóstico da leishmaniose e a identificação da espécie L. enriettii. Introduction Leishmaniases are diseases caused by protozoans from the genus Leishmania (Kinetoplastida: Trypanosomatidae). Clinically and epidemiologically, there are three forms of leishmaniasis, each species specific: visceral, cutaneous, and mucocutaneous (Ecco et al. 2000, Fighera et al. 2003, McAdam et al. 2015). The protozoan is transmitted by sandfly vectors (Diptera: Phlebotominae) when females take a blood meal from the vertebrate host (Paranaiba et al. 2017). Mucocutaneous leishmaniasis affects several animal species, including humans (McAdam et al. 2015). Guinea pigs (Cavia porcellus) are affected by a non-zoonotic mucocutaneous presentation of leishmaniasis caused by the protozoan Leishmania enriettii (Medina 1946, Muniz & Medina 1948, Lainson 1997). This disease was described 75 years ago in southern Brazil (Medina 1946). Although a recent review paper on leishmaniasis caused by L. enriettii was published (Paranaiba et al. 2017), the disease is still poorly understood and clinically mistaken by other conditions, such as neoplastic lesions (Fighera et al. 2003). This study aims to evaluate the clinical, epidemiological, pathological, and molecular aspects of an outbreak of leishmaniasis in guinea pigs caused by L. enriettii. Materials and Methods Seven guinea pigs were affected, five males and two females, aged between eight months and three years (Table 1). Clinical data came from on-site visits done by the authors to the household where the outbreak occurred and by the owners, whom the authors asked to monitor the guinea pigs. All the guinea pigs that died did so spontaneously. Table 1. Outbreak of leishmaniose by Leishmania enriettii. Data on seven affected guinea pigs and distribution of mucocutaneosus lesions ID Sex Age Distribution of lesions 1 M 10 months Mucocutaneous junction of the muzzle and nasal cavity, and both forelimbs 2a M 1 year Mucocutaneous junction of the muzzle, both pinnae, and the four limbs 3b M 1 year Right pinna 4c,d M 2 years Mucocutaneous junction of the muzzle and nasal cavity, both pinnae, and the four limbs 5c M 3 years Mucocutaneous junction of the muzzle and nasal cavity, the upper lip, and the four limbs 6 F 8 months Mucocutaneous junction of the muzzle and nasal cavity, both pinnae, the four limbs, and vulva 7e F 2 years ID = identification of guinea pig, M = male, F = female, a macroscopic and histopathological lesions in the lung, b survived, c material from lesions sampled for polymerase chain reaction, d material from lesions sampled for cytology, e regression of lesions, no lesions at necropsy. We performed cytology on material sampled by fine-needle aspiration from ulcerated skin lesions of the pinna and limbs from Guinea pig 4 in slides stained by a commercial Romanowsky stain (Panótico Rápido, LB, Laborclin, Brazil). Fragments of several organs from necropsied Guinea pigs 1, 2, 4-7 were harvested, fixed in 10% buffered formalin, routinely processed for histopathology, and stained with hematoxylin and eosin (HE). Tissue samples from five guinea pigs (1,4-7) revealed unfit for histological examination due to advanced postmortem changes. Punch biopsy for the polymerase chain reaction (PCR) for identification of the protozoan taken from the lesions of the pinna and muzzle of Guinea pigs 4 and 5 and processed according to a technique already described (Marfurt et al. 2003). Results We found leishmaniasis in five male and two female guinea pigs with ages ranging between eight months and three years (Table 1, raised as pets in the same household, located in the municipality of Viamão (30°4′51″ S, 51°1′22″ W), Rio Grande do Sul, Southern Brazil. The guinea pigs were outdoors in a space bounded by fences, which limited their contact with other animals, such as dogs, cats, rabbits, and domestic chickens that also lived or had access to the household. None of the other animals in this home were affected. Guinea pigs fed at will on fruits, vegetables, and legumes leftovers, commercial pelleted feed for rabbits, and water. The owner observed the appearance of lesions two months before submission and noted they evolved quickly and in a similar manner across all animals. During clinical examination, the affected guinea pigs had identical skin lesions that consisted of ulcerated, bloody, and crusty nodules, being grossly similar in all animals, with variations only in the number (focal or multifocal lesions), size, and in the affected anatomical region (Table 1). The most frequent sites of injuries were the mucocutaneous junction of the snout and the nasal cavity (Fig.1) and the pinnae (Fig.2), and the limbs (Fig.3). Aside from mucocutaneous lesions, guinea pigs didn’t present any other clinical signs and remained clinically stable. All animals died spontaneously and quickly. The number and aspect of lesions were unrelated to the age or sex of the guinea pigs. Guinea pigs 3 and 7 showed spontaneous regression of the lesions, and one of them (Guinea pig 3) remains alive so far with only a small lesion in the dorsal aspect of the right pinna; over five months, this lesion significantly regressed and is approximately half of the initial lesion. Lesions of Guinea pig 7 regressed utterly, and it survived for six months, then died unexpectedly. Fig.1. Crusted and ulcerated nodule on the muzzle and upper lip. The nodule also affecting the mucocutaneous junction and the anterior mucosa of the nasal cavity. Leishmaniasis (Leishmania enriettii), Cavia porcellus 5. Fig.2. A crusted, ulcerated and bleeding mass partially destroys the right pinna. Leishmaniasis (Leishmania enriettii), Cavia porcellus 4. Fig.3. An ulcerated and bleeding nodule partially in the distal portion of the thoracic limb. Leishmaniasis (Leishmania enriettii), Cavia porcellus 6. Cytology displayed numerous macrophages with abundant pink foamy cytoplasm filled with oval parasitophorous vacuoles containing amastigotes approximately 3-4µm in diameter and round to oval nuclei (Fig.4). Fig.4. Foamy macrophages contain numerous oval amastigotes approximately 4.0μm in diameter in their cytoplasm from ulcerated skin lesions of the pinna and limbs. Cytology. Leishmaniasis (Leishmania enriettii), Cavia porcellus 4. Quick Panoptic, obj.40x. DNA amplification for Leishmania isolated in guinea pigs and biopsy samples was similar to the reference strains of L. enriettii with approximately 350 base pairs (bp) in length. For L. infantum, the band was 435 bp, and for L. amazonensis and L. braziliensis were 283 and 226 bp, respectively. In the he polymerase chain reaction (PCR) technique with mini-exon primer, both samples were positive and equal to the reference strain, identifying L. enriettii. The necropsy lesions were similar in five of the six necropsied guinea pigs, except for Guinea pig 7, where cutaneous lesions had regressed. The nodules were of varying sizes. The lesions have thick crusts consisting of coagulated blood and adhere to the subepidermal tissue. The cut surface of the nodules was white and firm. The most extensive lesion (3.5x2.5cm) was in the muzzle. In Guinea pig 2, the lungs were dark-red, non-collapsing, and firm (Fig.5). Fig.5. The lungs were dark-red, non-collapsing, and firm. Gross lesions in the lung. Leishmaniasis (Leishmania enriettii), Cavia porcellus 2. The superficial dermis of Guinea pig 2 had moderate e focally extensive chronic dermatitis, fibrosis, and acanthosis. There was marked diffuse lamellar orthokeratotic hyperkereratosis. Pulmonary histology of Guinea pig 2 showed neutrophilic, eosinophilic, histiocytic, diffuse, and moderate broncho-interstitial pneumonia. The alveolar septa were thickened with mild neutrophil and macrophage infiltrate; there was type II pneumocyte hyperplasia. Abundant neutrophils and foamy macrophages occasionally filled the alveolar lumen. In multifocal areas, there were granulomatous nodules with epithelioid macrophages, which sometimes formed multinucleated giant cells (MGC). Macrophages and MGC contained intracytoplasmic basophilic amastigotes approximately 2µm in diameter with morphology compatible with Leishmania sp. (Fig.6). Amastigotes were also in focal areas with marked eosinophilic infiltrate. Fig.6. The alveolar septa are thickened by mononuclear inflammatory infiltrate; epithelioid macrophages, sometimes arranged as multinucleated giant cells, contain intracytoplasmic basophilic organisms (arrow) approximately 2µm with morphology compatible with Leishmania sp. Leishmaniasis (Leishmania enriettii), histopathology of the lung, Cavia porcellus 2. HE, obj.40x. Discussion The epidemiological data, the clinical picture, the macroscopic lesions, and the histopathology observed in the guinea pigs of the present outbreak bear a close similarity to those described by several authors for the mucocutaneous leishmaniasis by Leishmania enriettii (Medina 1946, Muniz & Medina 1948, Paraense 1952, 1953, Bray et al. 1969, Bryceson et al. 1974, Kanan 1975a, 1975b, Machado et al. 1994, Lainson 1997, Ecco et al. 2000, Fighera et al. 2003, Paranaiba et al. 2017). Also, the agent was confirmed as L. enriettii by PCR. In the mucocutaneous cases of leishmaniasis by L. enriettii in guinea pigs previously reported, the anatomical sites that most frequently presented lesions were the pinnae, muzzle, and mucocutaneous junction of the nasal cavity and periocular region (Paraense 1953, Kanan 1975b, Thomaz-Soccol et al. 1996). In the outbreak reported here, the pinnae, muzzle, and mucocutaneous junction of the nasal cavity were the most affected sites. The evolution and gross aspect of the mucocutaneous lesions did not seem to be correlated to the degree of infection, as seen by Animal 2, which had discrete lesions while showing marked pulmonary changes associated with L. enrietti amastigotes. Our findings led us to avoid the denomination “mucocutaneous” that is used in the literature to designate the condition (Kanan 1975a, 1975b, Ecco et al. 2000, Fighera et al. 2003), since a visceral lesion co-existed. In animal leishmaniasis, the separation of the condition into visceral, cutaneous, and mucocutaneous forms, seems sometimes arbitrary, and perhaps the use of these denominations should be replaced by the more comprehensive and uncompromising term “leishmaniasis”. The finding of a granulomatous focal lesion in association with the causative agent in the lung of one guinea pig is a novelty in reported cases of L. enriettii caused by cases of leishmaniasis in guinea pigs. L. enriettii inoculated intravenously into guinea pigs induces, within five days, nodules in the nostrils, nasal septum, scrotum, vulva, and limbs (Kanan et al. 1975b). The findings indicate a hematogenic distribution of the infection, and immunosuppression is a known risk factor for visceral leishmaniasis. The inoculation of L. enriettii in wild guinea pigs (Cavia aperea) intravenously produces similar lesions (Lainson 1997). This may also be the mechanism of dissemination of the agent to the lung. Previous reports note severe pneumonia during necropsy; however, the etiological agent is not described (Kanan et al. 1975b, Fighera et al. 2003). In the current outbreak, pneumonia was present during necropsy, and none of the animals showed clinical respiratory signs. The first documented mucocutaneous case of leishmaniasis in guinea pigs caused by L. enriettii is from southern Brazil (Medina 1946). The causative agent demonstrates 100% infectivity to guinea pigs and no zoonotic potential (Medina 1946, Muniz & Medina 1948, Machado et al. 1994, Lainson 1997). After almost 70 years since this documentation, cases of L. enrietti in captive red kangaroos (Macropus rufus) and humans were reported from Australia and Africa, respectively. Phylogenetic analysis of the protozoa allotted them as new members of the L. enrietti complex, a new subgenus of the Leishmania spp. Parasites (Dougall et al. 2011, Pothirat et al. 2014, Kwakye-Nuako et al. 2015). These studies demonstrate the vast geographic spread of the L. enrietti complex and its capability to affect different vertebrate hosts, including humans. The mucocutaneous lesions described in kangaroos and humans had a similar gross presentation of ulceration and crusts as those seen in the Cavia porcellus of this report. Conclusion The cytological, gross, and histological findings associated with the PCR results allowed for the leishmaniasis diagnosis in Cavia porcellus, with Leishmania enriettii being identified as the agent. Due to the presence of both cutaneous and pulmonary lesions, the best denomination for the disease is leishmaniasis in guinea pigs and should be considered as a differential diagnosis for pneumonia in this species. Acknowledgments The authors are grateful to the professionals of the Axys Análises laboratory in Porto Alegre/RS, who processed the material for histopathology. References Bray R.S., Ellis D.S. & Bird R.G. 1969. 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