RESUMO

Abstract Contagious Ecthyma (CE) is a severe exanthematous dermatitis caused by the Orf virus (ORFV) that mainly affects domestic small ruminants such as sheep and goats. It is a worldwide-distributed occupational zoonosis, particularly infecting those in close contact with animals or animal products such as shepherds, farmers and veterinarians, among others. In the present work, we report the first human CE case confirmed in Argentina. A phylogenetic analysis based on four gene sequences of the isolated strain responsible for the disease showed that this isolate grouped with other ORFV sequences that caused reported CE cases in sheep from the same Argentine province. We also sequenced a sample from a Chilean human case reported in 2017, whose phylogenetic analysis showed that it groups together with other Argentine isolates from locations close to the border with Chile. Keywords: Contagious Ecthyma; Dermatitis; Human Orf; Zoonosis; Molecular characterization.

Resumen El ectima contagioso (EC) es una dermatitis exantemática grave causada por el virus Orf (ORFV), que afecta mayormente a pequeños rumiantes domésticos, como ovinos y caprinos. Es una zoonosis ocupacional con distribución mundial, infecta a humanos en estrecho contacto con animales o sus productos, como granjeros, esquiladores y veterinarios, entre otros. En este trabajo se informa el primer caso humano de EC confirmado en Argentina. Un análisis filogenético basado en cuatro genes de la cepa responsable de este caso mostró que el aislamiento agrupa con otras secuencias de ORFV que causaron casos en ovinos en la misma provincia argentina. También se secuenció una muestra del caso de ectima humano reportado en Chile en 2017 y el análisis filogenético mostró que dicho aislamiento forma un grupo con otros aislamientos argentinos de localidades cercanas a la frontera con Chile. Palabras clave: Ectima contagioso; Dermatitis; Orf en humanos; Zoonosis; Caracterización molecular.

RESUMO

Contagious Ecthyma (CE) is a severe exanthematous dermatitis caused by the Orf virus (ORFV) that mainly affects domestic small ruminants such as sheep and goats. It is a worldwide-distributed occupational zoonosis, particularly infecting those in close contact with animals or animal products such as shepherds, farmers and veterinarians, among others. In the present work, we report the first human CE case confirmed in Argentina. A phylogenetic analysis based on four gene sequences of the isolated strain responsible for the disease showed that this isolate grouped with other ORFV sequences that caused reported CE cases in sheep from the same Argentine province. We also sequenced a sample from a Chilean human case reported in 2017, whose phylogenetic analysis showed that it groups together with other Argentine isolates from locations close to the border with Chile.

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El Ectima gangrenoso es un trastorno infeccioso infrecuente, clásicamente relacionado a bacteriemia, descrito principalmente en poblaciones inmunodeprimidas. Se asocia con la sepsis y bacteriemia por P. aeruginosa en pacientes inmunodeprimidos, incluidos aquellos con neutropenia o inmunodeficiencias, también se han descrito casos por hongos filamentosos y levaduras. Se ha observado en aproximadamente un 1,3-3 % de los casos de bacteriemia por P. Aeruginosa1. Se ha descrito ectima en pacientes con déficit en su inmunidad2, como en el caso que se describe a continuación. El Ectima puede presentarse como lesión única o múltiple, caracterizadas por máculas eritematosas que progresan a vesículas, bullas o pústula, evolucionando hasta la necrosis central con halo eritematoso. Se presenta el caso de un paciente con Ectima Gangrenoso, refractario inicialmente a tratamiento, que evolucionó con empeoramiento progresivo, presentándose en el hemograma hiperleucocitosis asociado a anemia y trombopenia, con diagnóstico ulterior de Leucemia Mieloide Aguda.

Ecthyma gangrenosum is an uncommon infectious disorder classically associated with bacteremia and found mainly in immunocompromised populations. It is associated with sepsis and Pseudomonas aeruginosa bacteremia in immunocompromised patients, including those with neutropenia or immune deficiencies. There have also been cases caused by filamentous fungi and yeasts. It has been seen in approximately 1.3 ­ 3% of Pseudomonas aeruginosa bacteremias. Ecthyma has been reported in immune-deficient patients, as in the case described below. Ecthyma can present with a single or multiple lesions with erythematous macules progressing to vesicles, bullas or pustules, which develop central necrosis with an erythematous halo. We present the case of a patient, later diagnosed with Acute Myeloid Leukemia, with ecthyma gangrenosum, initially refractory to treatment, which worsened progressively, presenting hyperleukocytosis associated with anemia and thrombocytopenia in the whole blood count.

RESUMO

El ectima gangrenoso es un trastorno infeccioso infrecuente clásicamente relacionado a bacteriemia, descrito principalmente en poblaciones inmunodeprimidas. El agente más comúnmente relacionado es Pseudomonas aeruginosa, sin embargo, se han descrito otras etiologías bacterianas, hongos filamentosos y levaduras. Su patogénesis está dada por la invasión de la pared de los vasos sanguíneos, causando trombosis arterial y venosa, desencadenando necrosis de epidermis y tejidos subdérmicos. Clínicamente, se manifiesta como máculas, vesículas o pústulas hemorrágicas que evolucionan a úlceras de superficie necrótica rodeadas por un halo eritematoso característico, habitualmente en un contexto clínico de sepsis. El diagnóstico de este cuadro es clínico, sin embargo, el estudio microbiológico es clave en la identificación del agente etiológico y un posterior tratamiento dirigido. En su manejo es esencial una alta sospecha clínica y el inicio de tratamiento antibiótico en forma precoz. La duración del tratamiento es variable y en algunos casos debe asociarse a debridación quirúrgica. El pronóstico es variable dependiendo de múltiples factores: estado inmunológico, agente etiológico, presencia de sepsis y el tiempo de inicio de tratamiento.(AU)

Ecthyma gangrenosum is an infrequent infectious disorder classically related to bacteremia, described mainly in immunosuppressed populations. The most related agent is Pseudomonas aeruginosa, however other bacterial etiologies, filamentous fungi and yeasts have been described. Its pathogenesis is given by the invasion of the blood vessel wall, causing arterial and venous thrombosis, triggering necrosis of epidermis and subdermal tissues. Clinically, it manifests as hemorrhagic macules, vesicles or pustules that evolve into ulcers with a necrotic surface surrounded by a characteristic erythematous halo, usually in a clinical setting of sepsis. The diagnosis of this condition is clinical, however the microbiological study is key in the identification of the etiological agent and a subsequent directed treatment. In its management, a high clinical suspicion and an early start of antibiotic treatment are essential. The duration of treatment is variable and in some cases it must be associated with surgical debridement. The prognosis is variable depending on multiple factors: immunological status, etiological agent, presence of sepsis and time of initiation of treatment.(AU)

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Las infecciones cutáneas producidas por microorganismos afectan directamente a la piel, partes blandas y tejidos, donde proliferan y ocasionan graves alteraciones. Estas infecciones constituyen un problema de salud pública, ya que muchos microorganismos muestran resistencia a antimicrobianos comunes y no comunes, lo cual incide directamente en la aplicación del tratamiento adecuado al paciente. El objetivo de este estudio fue identificar los microorganismos más frecuentes en infecciones cutáneas, su sensibilidad y resistencia a los antibióticos en los pacientes con infección que acudieron al Hospital Provincial General "Ambato" en el período mayo 2017 ­ junio 2018. La metodología empleada en esta investigación se basa en un estudio descriptivo, de corte transversal y enfoque cuali-cuantitativo, empleando la técnica documental y el reporte de resultados como instrumento. La información se tabuló y analizó mediante el paquete operativo Microsoft 2010. Se encontró que en el 29% de las muestras procesadas se aisló Staphylococcus aureus como el más frecuente en este tipo de infecciones, con mayor sensibilidad a Clindamicina, Doxacilina y Linezolid (100%) y resistencia a Penicilinas y Oxacilina (47,82%). Es importante destacar que el 47,83 % de las cepas de S. aureus aisladas expresaron fenotípicamente el gen mecA. La entidad clínica más frecuente asociada a este tipo de infección fue el ectima con un 55%. En conclusión, se comprobó la resistencia de cepas a diversos antibióticos presentando las más relevantes como meticilino resistentes, vancomicina resistentes y Klebsiella pneumoniae carbapenemasas

The skin infections caused by microorganisms directly alter the skin, soft tissues and tissues cause serious damage and the proliferation of them, which is a problem for health centers and hospitals, since these microorganisms are often resistant to antibacterial which are not very common is a problem for the treatment of the patient. The objective of this study was to identify the most frequent microorganisms in cutaneous infections, their sensitivity and resistance to antibiotics in the patients with infection who when to the General Provincial Hospital "Ambato", in the period May 2017 - June 2018 the methodology used in this investigation is based on a descriptive cutting study cross-section and qualitative-quantitative approach, using the documentary technique and the report of results as an instrument. The information was tabulated and analyzed using the Microsoft 2010 operating package. It was found that 29% of the samples processed were isolated Staphylococcus aureus as the most frequent in this type of infections, with greater sensitivity to Clindamycin, Doxacillin and Linezolid (100%) and resistance to Penicillins and Oxacillin (47,82%). It is important to note that 47,82% of strains of S. aureus were expressly phenotypically expressed in the mecA gene. The most frequent clinical entity associated with this type of infection was efficacy with 55%. In conclusion, the resistance of strains in several antibiotics was proved, presenting the most relevant ones as methicillin resistant, vancomycin resistant and Klebsiella pneumoniae carbapenemasas.

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El ectima gangrenoso es una manifestación dermatológica característica de una infección severa causada casi siempre por Pseudomonas aeruginosa (P. aeruginosa), con o sin bacteriaemia. Las infecciones severas por P. aeruginosa se presentan habitualmente en pacientes con inmunodepresión u hospitalizados, en los pacientes sanos existe el antecedente de foliculitis y forunculosis. El diagnóstico es evidentemente clínico, con confirmación en cultivos. Se presenta el caso de una paciente joven inmunocompetente que presentó lesiones características de ectima gangrenoso por P. aeruginosa acompañado de cuadro gastrointestinal.

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Resumen La infección por el virus orf, también conocida como ectima contagioso, es reconocida una zoonosis ocupacional. Se diagnostica por lesiones cutáneas que evolu cionan rápidamente desde máculas a pápulas, vesículas y pústulas. Se presenta el caso clínico de una estudiante de medicina veterinaria que había tenido contacto con caprinos, clínicamente sanos y sin lesiones aparentes, hacía 19 días. Presentó dos lesiones vesiculares que coalescieron hasta formar una lesión de mayor tamaño rodeada por un halo eritematoso. Las lesiones fueron compatibles con la presentación clásica de las producidas por el virus orf en humanos. Se confirmó la presencia del virus orf mediante una RPC anidada del tejido de biopsia. Es uno de los primeros casos confirmados mediante técnicas moleculares en seres humanos en Chile.

Infection with the orf virus, also known as contagious ecthyma, is recognized as an occupational zoonosis worldwide. It is diagnosed by cutaneous lesions that progress rapidly from macules to papules, vesicles and pustules. The clinical case of a student of veterinary medicine who had had contact with goats, clinically healthy and without apparent lesions, which occured 19 days ago, is reported. She presented two vesicular lesions that coalesced to form a larger lesion surrounded by an erythematous halo. The lesions were compatible with the classical presentation of those produced by the orf virus in humans. The presence of the orf virus was confirmed by a nested PCR from biopsy tissue. It is one of the first cases confirmed by molecular techniques in humans in Chile.

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El ectima gangrenoso es una infección poco frecuente de la piel generalmente causada por Pseudomona aeruginosa, usualmente ocurre en pacientes inmunocomprometidos pero ocasionalmente afecta a individuos sanos. Debido a las complicaciones que el paciente puede presentar en el transcurso de la enfermedad, la mortalidad por la misma es alta, sobre todo en los pacientes con bacteriemia. Se describió el caso de un niño de 1 año, atendido por varias lesiones en piel y toma del estado general hasta la gravedad. Se realizó tratamiento multidisciplinario y se logró la evolución satisfactoria al egreso(AU)

Ecthymagangrenosum is a rare infection of the skin usually caused by Pseudomoma aeruginosa, usually occurs in immunocompromised patients but occasionally affects healthy individuals. Due to the complications that the patient may present during the course of the disease, the mortality is high, especially in patients with bacteremia. It was described a case of 1-year-old child, treated for several skin lesions and general conditions to the point of gravity that were taking into account. A multidisciplinary treatment was carried out and satisfactory evolution was achieved at discharge(AU)

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Se efectúa una breve revisión del tratamiento del ectima gangrenoso. El ceftazimide demostró menor morbi-mortalidad, mientras que los amino glucósidos y el imipemen, mayor mortalidad y resistencia respectivamente.

A brief review about the best treatment of the echthyma gangrenosum are reported. Ceftazidime showed less morbi-mortality while aminoglycosides and imipenem higher mortality and resistance.

RESUMO

El ectima gangrenoso es una vasculitis necrosante poco frecuente, en la mayoría de los casos secundaria a sepsis por Pseudomonas aeruginosa en pacientes inmunocomprometidos. Sin embargo, existen reportes de ectima gangrenoso secundarios a otras etiologías infecciosas. Presentamos un caso de ectima gangrenoso asociado a una infección por Staphylococcus aureus resistente a meticilina en una paciente sin los factores de riesgo clásicos de inmunosupresión que se describen en la literatura médica.

Ecthyma gangrenosum is an uncommon necrotizing vasculitis, in most cases secondary to sepsis by Pseudo-mona aeruginosa in immunocompromised patients. However, there have been several reports of ecthyma gangre-nosum caused by other infectious etiologies. We report an unusual case of ecthyma gangrenosum associated with methicillin-resistant Staphylococcus aureus infection in a patient without the classic immunological risk factors described in the literature.

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La otitis externa maligna es considerada una infección invasiva del conducto auditivo externo, infrecuente y sumamente agresiva, que llega, en ocasiones, a comprometer la base del cráneo. Se presenta habitualmente en inmunocomprometidos, incluidos aquellos con infección por VIH. El ectima gangrenoso es una infección cutánea localizada, inusual, causada, en la mayoría de los casos por Pseudomonas aeruginosa, con compromiso multisistémico y evolución tórpida. La asociación entre ambas entidades es aún más rara. A continuación, presentamos el caso de una mujer de 37 años, con el antecedente principal de infección por VIH desde hace 10 años, adherente al tratamiento antirretroviral y, a pesar de mantener una adecuada respuesta inmunológica y virológica, se presenta a la urgencia de nuestro hospital en shock séptico por otitis externa maligna, con ectima gangrenoso y posible síndrome hemofagocítico como complicación, aislándose Pseudomonas aeruginosa como agente causal.

Malignant otitis externa is an aggressive, infrequent and invasive infection of the external auditory canal. In some cases it leads to skull base compromise, particularly in immunocompromised individuals, including HIV-positive patients. Ecthyma gangrenosum is an unusual, localized cutaneous infection, caused mostly by Pseudomonas aeruginosa, with systemic compromise and a torpid evolution. An association between these 2 entities is even more rare. We present the case of a 37-year old woman with a history of 10 years’ HIV infection and good adherence to antiretroviral therapy. Although she had an adequate immunological and virological response, she presented at our emergency department in septic shock due to malignant otitis externa with ecthyma gangrenosum and possible haemophagocytic syndrome as a complication, with Pseudomonas aeruginosa determined to be the causative agent.

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Background: Contagious ecthyma is a viral disease caused by a Parapoxvirus, which affects primarily sheep and goats. The disease has a worldwide distribution and is characterized by cutaneous pustules and crusts mainly in the muzzle and lips. Although the disease has a worldwide distribution, there are few reports in the literature of contagious ecthyma outbreaks in Brazil. Moreover, this is an important disease, as well as causing huge economic losses due to high morbidity rates, is also a zoonosis occupational character. This report describes the epidemiological, clinical, and anatomopathological aspects of an outbreak of contagious echtyma in sheep in the State of Rio Grande do Sul, Brazil. Case: Cases were observed on January and February of 2016. Seventeen out of 45 Texel sheep were affected including five 4-6-month-old lambs, three 7-12-month-old male sheep and nine 2-year-old ewes. Before the outbreak, a Texel ram was introduced in the herd as replacement. Clinically, affected sheep had pustules, ulcers, and crusts in the lips, labial commissures, muzzle, and nasal bridge. They also presented dyspnea, submandibular and facial subcutaneous edema. One of the affected sheep was euthanized due to the poor prognosis. At necropsy, the lesions observed clinically were confirmed. Histopathology of the skin in the lips and muzzles showed marked acanthosis of the epidermis, [...]

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Background: Contagious ecthyma is a viral disease caused by a Parapoxvirus, which affects primarily sheep and goats. The disease has a worldwide distribution and is characterized by cutaneous pustules and crusts mainly in the muzzle and lips. Although the disease has a worldwide distribution, there are few reports in the literature of contagious ecthyma outbreaks in Brazil. Moreover, this is an important disease, as well as causing huge economic losses due to high morbidity rates, is also a zoonosis occupational character. This report describes the epidemiological, clinical, and anatomopathological aspects of an outbreak of contagious echtyma in sheep in the State of Rio Grande do Sul, Brazil. Case: Cases were observed on January and February of 2016. Seventeen out of 45 Texel sheep were affected including five 4-6-month-old lambs, three 7-12-month-old male sheep and nine 2-year-old ewes. Before the outbreak, a Texel ram was introduced in the herd as replacement. Clinically, affected sheep had pustules, ulcers, and crusts in the lips, labial commissures, muzzle, and nasal bridge. They also presented dyspnea, submandibular and facial subcutaneous edema. One of the affected sheep was euthanized due to the poor prognosis. At necropsy, the lesions observed clinically were confirmed. Histopathology of the skin in the lips and muzzles showed marked acanthosis of the epidermis, [...](AU)

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Resumen El ectima gangrenoso es una infección poco frecuente de la piel, conocida con una característica lesión causada principalmente por la Pseudomoma aeruginosa, pero que también se puede presentar por otro tipo de bacterias del tipo Staphylococcus aureus o el Streptococcus beta hemolítico del grupo A ( S. beta hemolítico del grupo A), entre otros. Esta entidad se asocia frecuentemente con inmunodeficiencias primarias y secundarias. Las lesiones, clínicamente, se presentan como máculas eritematovioláceas, descamativas que rápidamente evolucionan a papulovesículas, ampollas hemorrágicas y costrosas que dejan unas úlceras profundas en sacabocado con fondo necrótico. La mortalidad es alta, entre 40 y 75% de los individuos inmunológicamente comprometidos. Se describe el caso de una niña de 5 años que inicialmente presentó varicela y luego una sobreinfección por S. aureus ocasionando un cuadro clínico de un ectima gangrenoso; al diagnóstico se llega por la clínica, cultivos y biopsias de piel. Se realizó manejo con aciclovir, clindamicina y cefepime endovenoso, con buena y rápida respuesta de la paciente.

Abstract Ecthyma gangrenosum (EG) is a rare infection of the skin, known as a characteristic lesion caused by Pseudomoma aeruginosa , but also can occur from other types of bacteria like Staphylococcus aureus or beta hemolytic group, among others. This condition is often associated with primary and secondary immunodeficiencies. The lesions present clinically as scaly, erythematous violacious macules that rapidly evolve into papule-vesicles, bleeding and crusty blisters leaving a deep punch ulcer with a necrotic base. Mortality is high, between 40% and 75% of immunologically compromised individuals. We describe the case of a girl of 5 years of age who initially presented chickenpox and then superinfection with Staphylococcus aureus, causing a clinical picture of EG. The diagnosis was reached based on symptoms, cultures and skin biopsies. Management was achieved with acyclovir, intravenous clindamycin and cefepime, with a good and quick response by the patient.

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Las infecciones por pseudomonas aeruginosa se presentan habitualmente en individuos con factores predisponentes, siendo excepcional su ocurrencia en los previamente sanos. Se describe el caso de un lactante de 2 meses sin antecedentes patológicos a destacar que presentó una infección grave por pseudomonas aeruginosa, presentando al momentode la consulta en emergencia lesiones en piel características de ectima gangrenoso. El reconocimiento de estas lesiones permite adecuar el tratamiento antibiótico empírico a la susceptibilidad antimicrobiana de este germen. La presencia de una infección por esta bacteria obliga al clínico a la búsqueda de inmunodeficiencias no diagnosticadas previamente...

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Las infecciones por pseudomonas aeruginosa se presentan habitualmente en individuos con factores predisponentes, siendo excepcional su ocurrencia en los previamente sanos. Se describe el caso de un lactante de 2 meses sin antecedentes patológicos a destacar que presentó una infección grave por pseudomonas aeruginosa, presentando al momentode la consulta en emergencia lesiones en piel características de ectima gangrenoso. El reconocimiento de estas lesiones permite adecuar el tratamiento antibiótico empírico a la susceptibilidad antimicrobiana de este germen. La presencia de una infección por esta bacteria obliga al clínico a la búsqueda de inmunodeficiencias no diagnosticadas previamente.

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O Ectima contagioso, popularmente conhecido como Boqueira, é uma doença causada pelo vírus Orf, que induz lesões pustulares agudas na pele de ovinos e caprinos. No presente trabalho, é relatada pela primeira vez a identificação e confirmação laboratorial do vírus Orf em rebanhos caprinos da Bahia. A Agência Estadual de Defesa Agropecuária da Bahia (ADAB) relatou a ocorrência de focos de uma doença epitelial nos caprinos das raças Canindé e Alpina Britânica nas localidades de Barreira e Pedra Alta, no município de Araci. Foram coletadas as amostras das lesões de crostas labiais do rebanho da localidade de Barreira e das crostas dos tetos do rebanho de Pedra Alta. Os materiais coletados foram submetidos à extração de DNA e posterior reação em cadeia da polimerase (PCR), para amplificação dos genes do vírus Orf: ORFV011 (B2L, 1022 pares de bases [pb]) e ORFV 059 (F1L, 1062 pb). Todas as amostras foram positivas na reação de PCR, confirmando-se a presença do vírus Orf nas lesões observadas nos rebanhos caprinos das comunidades de Barreira e Pedra Alta.

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Ectima contagioso (EC) ou orf é uma doença infecto-contagiosa da pele causada pelo vírus do ectima contagioso (ECV) ou ORFV, que acomete principalmente ovinos e caprinos, e ocasionalmente o homem. Protótipo do gênero Parapoxvírus da família Poxviridae, está amplamente disseminado em todo mundo, inclusive no Brasil, especialmente na Região Nordeste, onde a caprinovinocultura é amplamente praticada para a produção de pele, carne e leite. Em Pernambuco, a doença ocorre de forma endêmica e pode ser confundida com enfermidades vesiculares, como a Febre Aftosa (FA), havendo a necessidade de diferenciação, sobretudo como suporte às ações do Programa Nacional de Erradicação da Febre Aftosa (PNEFA). Uma grande limitação no controle da doença é a dificuldade de replicação do vírus em cultivo celular, visando sua manipulação biotecnológica para a produção de vacinas. O objetivo deste trabalho é isolar e avaliar o comportamento de amostras de ECV em cultura primária de células de córnea fetal caprina, além de estabelecer um diagnóstico molecular eficiente para amostras de campo e avaliar o perfil filogenético das amostras de campo estudadas. Amostras de crostas de 22 ovinos e de sete caprinos que apresentavam sinais clínicos de EC, originários dos Estados de Pernambuco, Bahia, Sergipe e Paraíba, foram inoculadas em monocamadas de células epiteliais de córnea de feto caprino, durante sete passagens consecutivas, a intervalos semanais. Para realização do diagnóstico molecular foi utilizado para amplificação de um fragmento de 235pb do gene do envelope B2L o par de primers, PPP-3 e PPP-4 denominados pan-parapoxvirus. Observou-se em todas as passagens, a partir de 24 horas pós-infecção, efeito citopático (ECP) caracterizado pelo arredondamento celular, fusão com formação de pequenos sincícios, vacuolização e corpúsculos de inclusão citoplasmática, com intensidade de 25% a 100% de desprendimento da camada celular, que variou de acordo com a amostra viral. Conclui-se que as culturas de células primárias de córnea fetal caprina mostraram-se altamente permissíveis à replicação do ECV e que as amostras virais isoladas mostraram-se adaptadas ao cultivo utilizado, com pequena variação entre as amostras virais, e que, a identificação viral através da PCR é um método aplicável ao diagnóstico diferencial de enfermidades vesiculares, sobretudo como suporte às ações do PNEFA, no qual caprinos e ovinos são considerados animais sentinelas. A análise filogenética mostrou que as seqüências de ORFV do Nordeste do Brasil estão altamente relacionadas entre si, com seqüências idênticas acima de 99%. Além de apresentarem mais de 80% de identidade com seqüências de outros isolados de ORFV em todo mundo.

Contagious ecthyma is a severe and proliferative viral disease affecting ovine and caprine species, and eventually men, caused by the contagious ecthyma virus (ECV) of the Parapoxvirus genus. The disease is spread worldwide, including in Brazil, where in the state of Pernambuco, due to its endemicity, can be mistaken by other vesicular diseases such as the Foot and mouth Disease, which requires its diagnostic differentiation. Meanwhile, the control of the infection in endemic regions is limited due to the difficulties in replicating the virus in cell cultures, for the development of vaccines. The purpose of this work is to evaluate the behavior of ECV samples in primary cultures of fetal caprine cornea cells, and also to establish an efficient molecular diagnostic method for field samples, and to analyze the phylogenetic profile of the virus samples studied. Crust samples from twenty-two sheep and seven goats from the states of Pernambuco, Bahia, Sergipe and Paraiba presenting the clinical symptoms of EC were inoculated in cell monolayers, during seven consecutive passages at weekly intervals. During all passages, starting at 24 hours post-infection was observed a cytopathic effect (CE) characterized by cell rounding, cell fusion with the formation of small syncytia, cytoplasmic inclusion and vacuolization. The intensity of cell detachment from the layers ranged from 25% to 100% which varied according to the viral sample. We concluded that the primary cultures of the fetal caprine cornea cell appeared highly permissible to replication of ECV and the isolated samples of ECV seemed to adapt to the utilized culture with a slight variation among samples, and the identification by PCR showed to be an applicable method to the differential diagnosis of vesicular diseases, especially to support the actions of PNEFA, where goats and sheep are considered sentinel animals. The phylogenetic analysis showed that ORFV sequences from the northeastern of Brazil are highly correlated showing identical sequences up to 99%. In addition, they present more than 80% sequence identity with other isolates of ORFV worldwide.

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RESUMO

O ectima contagioso (também conhecido como orf), é uma doença debilitante de ovinos e caprinos causada pelo vírus do orf (ORFV). A vacinação tem sido usada com relativo sucesso no controle da doença. No entanto, as vacinas atuais contêm amostras virulentas do agente, são produzidas por escarificação cutânea de animais, e apresentam eficácia questionável. Assim, o presente trabalho teve como objetivo produzir e testar a eficácia de uma vacina experimental produzida em cultivo celular. A cepa IA-82 do ORFV foi submetida a 21 passagens em cultivo de células BHK-21 e usada para vacinar ovinos jovens (n=30), por escarificação cutânea na face interna da coxa. A vacinação produziu pústulas e crostas em 16 dos 30 ovinos vacinados, indicando imunização adequada. Noventa dias após a vacinação, ovinos vacinados (n=16) e controles (n=16) foram inoculados com uma cepa virulenta do ORFV (10(6,9)DICC50/mL) após escarificação na comissura labial. Todos os animais desenvolveram lesões típicas de ectima, incluindo hiperemia, vesículas, pústulas e crostas. No entanto, os animais vacinados desenvolveram lesões mais leves e passageiras do que os controles, e os escores clínicos foram estatisticamente diferentes (p<0,05) entre os dias 10 e 22 pós-desafio. Além disso, o tempo de duração da doença foi significativamente inferior (p<0,05) nos animais vacinados. Os animais vacinados também excretaram menor quantidade de vírus (p<0,05) e por um período significativamente mais curto do que os controles (13 dias versus 22 dias, p<0,001). Esses resultados demonstram a proteção parcial conferida pela vacina experimental e, dependendo da melhoria dos índices de imunização e proteção, são promissores no sentido da utilização de vacinas contra o ORFV produzidas em cultivo celular.(AU)

Contagious ecthyma, also known as orf, is a debilitating disease of sheep and goats caused by the parapoxvirus, orf virus (ORFV). Vaccination has been used with relative success to reduce the losses caused by the disease, yet the current vaccines contain virulent virus, are empirically produced through skin scarification of live lambs, and present questionable efficacy. Therefore, the present study aimed at developing and testing an experimental ORFV vaccine produced in tissue culture. The ORFV strain IA-82 was submitted to 21 passages in BHK-21 cells and then used to immunize lam bs (n=30) through skin scarification of the internal face of the hind limb. Vaccination produced localized pustules and scabs lesions in 16 out of 30 animals, indicating an adequate replication of the vaccine virus. Ninety days after vaccination, vaccinated (n=16) and control lambs (n=16) were inoculated with a virulent ORFV strain (10(6,9)TCID50/ml) in the labial commissure. Vaccinated and control lambs developed typical orf lesions, characterized by hyperemia, vesicles, pustules and scab formation. Nonetheless, vaccinated animals developed milder lesions compared to controls and the clinical scores were significantly lower (p<0.05) between days 10 and 22 post-challenge. In addition, the mean duration of clinical disease was significantly reduced in vaccinated animals (p<0.05). Furthermore, vaccinated animals excreted much less virus (p<0.05) and for a significantly shorter period of time than did the controls (13 days versus 22 days, p<0.001). These results demonstrate partial protection by the experimental vaccine and, upon improvement of immunization and protection indices, are promising towards the use of tissue culture-based ORFV vaccines.(AU)

Assuntos

RESUMO

O ectima contagioso (também conhecido como orf), é uma doença debilitante de ovinos e caprinos causada pelo vírus do orf (ORFV). A vacinação tem sido usada com relativo sucesso no controle da doença. No entanto, as vacinas atuais contêm amostras virulentas do agente, são produzidas por escarificação cutânea de animais, e apresentam eficácia questionável. Assim, o presente trabalho teve como objetivo produzir e testar a eficácia de uma vacina experimental produzida em cultivo celular. A cepa IA-82 do ORFV foi submetida a 21 passagens em cultivo de células BHK-21 e usada para vacinar ovinos jovens (n=30), por escarificação cutânea na face interna da coxa. A vacinação produziu pústulas e crostas em 16 dos 30 ovinos vacinados, indicando imunização adequada. Noventa dias após a vacinação, ovinos vacinados (n=16) e controles (n=16) foram inoculados com uma cepa virulenta do ORFV (10(6,9)DICC50/mL) após escarificação na comissura labial. Todos os animais desenvolveram lesões típicas de ectima, incluindo hiperemia, vesículas, pústulas e crostas. No entanto, os animais vacinados desenvolveram lesões mais leves e passageiras do que os controles, e os escores clínicos foram estatisticamente diferentes (p<0,05) entre os dias 10 e 22 pós-desafio. Além disso, o tempo de duração da doença foi significativamente inferior (p<0,05) nos animais vacinados. Os animais vacinados também excretaram menor quantidade de vírus (p<0,05) e por um período significativamente mais curto do que os controles (13 dias versus 22 dias, p<0,001). Esses resultados demonstram a proteção parcial conferida pela vacina experimental e, dependendo da melhoria dos índices de imunização e proteção, são promissores no sentido da utilização de vacinas contra o ORFV produzidas em cultivo celular.

Contagious ecthyma, also known as orf, is a debilitating disease of sheep and goats caused by the parapoxvirus, orf virus (ORFV). Vaccination has been used with relative success to reduce the losses caused by the disease, yet the current vaccines contain virulent virus, are empirically produced through skin scarification of live lambs, and present questionable efficacy. Therefore, the present study aimed at developing and testing an experimental ORFV vaccine produced in tissue culture. The ORFV strain IA-82 was submitted to 21 passages in BHK-21 cells and then used to immunize lam bs (n=30) through skin scarification of the internal face of the hind limb. Vaccination produced localized pustules and scabs lesions in 16 out of 30 animals, indicating an adequate replication of the vaccine virus. Ninety days after vaccination, vaccinated (n=16) and control lambs (n=16) were inoculated with a virulent ORFV strain (10(6,9)TCID50/ml) in the labial commissure. Vaccinated and control lambs developed typical orf lesions, characterized by hyperemia, vesicles, pustules and scab formation. Nonetheless, vaccinated animals developed milder lesions compared to controls and the clinical scores were significantly lower (p<0.05) between days 10 and 22 post-challenge. In addition, the mean duration of clinical disease was significantly reduced in vaccinated animals (p<0.05). Furthermore, vaccinated animals excreted much less virus (p<0.05) and for a significantly shorter period of time than did the controls (13 days versus 22 days, p<0.001). These results demonstrate partial protection by the experimental vaccine and, upon improvement of immunization and protection indices, are promising towards the use of tissue culture-based ORFV vaccines.

Assuntos