【病毒外文文獻(xiàn)】2010 Detection of Coronavirus in Capybaras (Hydrochoeris hydrochaeris) by Transmission Electron Microscopy in S_o Paulo,

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549 Int J Morphol 28 2 549 555 2010 Detection of Coronavirus in Capybaras Hydrochoeris hydrochaeris by Transmission Electron Microscopy in S o Paulo Brazil Detecci n de Coronavirus in Capybaras Hydrochoeris hydrochaeris por Microscop a Electr nica de Transmisi n en S o Paulo Brasil Catroxo M H B Miranda L B Lavorenti A Petrella S Melo N A Martins A M C P R F CATROXO M H B MIRANDA L B LAVORENTI A PETRELLA S MELO N A MARTINS A M C P R F Detection of coronavirus in capybaras Hydrochoeris hydrochaeris by transmission electron microscopy in S o Paulo Brazil Int J Morphol 28 2 549 555 2010 SUMMARY Coronaviruses are enveloped positive stranded RNA belongs to the Nidovirales order Coronaviridae family and are classified into three groups Groups 3a and 3c comprising the avian coronaviruses They are worldwide in distribution and can cause respiratory enteric and in some cases hepatitis and neurological diseases in a wide variety of animals resulting in economic losses to breeders and breeding In this study we describe an outbreak of diarrhea in a herd of capybaras found in the city of Piracicaba S o Paulo Brazil The animals had sudden symptoms of anorexia profuse and watery diarrhea yellowish or greenish dehydration weight loss sharp and twisting of limbs leading to death in less than a week mostly weaned and with an age of three months A total of 59 stool samples and 10 fragments of small intestine were processed for negative staining technique rapid preparation By transmission electron microscope a large number of particles with morphology similar to coronavirus pleomorphic rounded or elongated features with radial projections form a corona and measuring 80 140 nm in diameter was visualized in all samples analyzed The technique was extremely useful for rapid viral diagnosis in the affected animals KEYWORDS Coronavirus Capybaras Transmission electron microscopy INTRODUCTION Coronaviruses are worldwide in distribution highly infectious and extremely difficult to control They can cau se respiratory enteric and in some cases hepatic and neurological diseases in a wide variety of animals Gorbalenya et al 2004 resulting in economic losses to breeders and breeding and dairy cattle industry Boileau Tsunemitsu et al 1995 Catroxo et al 1999 2007 Brand o et al 2002 equine Cappellaro et al 1988 swine Cappellaro et al 1998 dog Hagiwara et al 1989 Catroxo et al 1998 monkey Catroxo et al 1997 and bird Catroxo et al 2000 The enteric coronaviruses is characterized by ano rexia prostration dehydration dyspnea and death of animals of all ages mainly young and those submitted to a confinement system Fenner et al 1992 The virus replicates itself in the villi of small intes tinal absorptive cells and differentiated cells in the crypts of the colon resulting in replacement by immature cuboidal cells desquamation and villus shortening decreasing the absorption surface of the intestine causing an imbalance and mal absorptive diarrhea that progresses to a state of dehydration which leads in most cases the death of animals Dea et al 1981 Heckert et al 1990 Cappellaro et al 1998 Laboratory of Electron Microscopy Biological Institute of S o Paulo SP Brazil Departamento de Medicina Veterin ria Escola de Veterin ria Universidade Federal de Goi nia UFG Goi nia GO Brazil Departamento de Patologia de N o Ruminates Escola Superior de Agricultura Luiz de Queiroz Esalq Piracicaba SP Brazil Aldolfo Lutz Institute S o Paulo SP Brazil 550 The incubation period ranges from 2 to 8 days and the morbidity reaches 100 by the fourth day Guard Woo et al 2006 The capybara Hydrochoerus hydrochaeris is a large herbivore rodent mammal semi aquatic habit found throughout the national territory Its captive breeding has been increasing in Brazil due to the marketing of beef in the national and international market whose protein content is high and fat low Reproduction is more efficient in these animals than in the bovine species and diet of low cost are factors that economically incentive breeding Moreover the leather is appreciated in the international market due to the impermeability lightness and resistance being used in the ma nufacture of shoes gloves and clothing The oil extracted from subcutaneous fat can be used for medicinal purposes and fur in the manufacture of brushes and upholstery Silva 1986 Arouca et al 2000 Oda 2002 In spite of several factors encouraging breeding little has been discussed about the diseases that affect this species and the importance of these animals as reservoirs of infectious agents Sarkis 2002 Direct electron microscopy technique is the most perfect method to detect the presence of enteric viruses in stool samples Arcangeletti et al 2005 It is generally used for the diagno sis of enteric coronaviral infections Dea Tsunemitsu et al 1995 Cappellaro et al 1998 Catroxo et al 2007 Considering the efficiency and speed of this technique this study aimed to detect viral agents in fecal and intestine samples during an outbreak of diarrhea occurred in a herd of cabybaras in the city of Piracicaba Brazil MATERIAL AND METHOD Description of the outbreak In the period from June to December 1995 an outbreak of diarrhea occurred in a herd of capybaras Hydrochoerus hydrochaeris located in Piracicaba SP Brazil where the animals were kept in indivi dual sectors maternity nursery picket and reproduction for experimental studies to determine levels of energy and protein for growth and weight gain For verification of weight gain the capybaras were captured every two weeks and submitted to conditions of intense stress Suddenly animals showed symptoms of anorexia profuse and watery diarrhea yellowish or greenish dehydration severe weight loss and twisting of anterior limbs leading many animals to death in less than a week mostly weaned and at three months of age Stool samples from 59 capybaras 22 maternity sector 10 pickets 9 nursery 17 of reproduction and one free living capybara and fragments of small intestines of 10 capybaras of the maternity sector were collected kept under refrigeration and sent to the Electron Microscopy Laboratory of the Biology Institute of S o Paulo to investigate viral agents Feces and small intestine fragments were processed for transmission electron microscopy using negative staining rapid preparation technique Negative staining technique rapid preparation In the negative staining process feces and small intestine fragments were suspended in phosphate buffer 0 1 M pH 7 0 Drops of the obtained suspension were placed in contact with metallic copper grids with carbon stabilized supporting film of 0 5 collodium in amyl acetate Next the grids were drained with filter paper and negatively stained at 2 ammonium molybdate pH 5 0 Brenner Hayat Madeley 1997 Observations were made in a Philips EM 208 electron microscope at 80 kV RESULTS Necropsia Gross lesions confined to the gastro intestinal tract and a stomach distended and congested were observed during the necropsy in 10 100 capybaras The small intestine was also distended with yellow foamy fluid The intestinal wall was thin and translucent due to severe atrophy of intestinal villi Negative staining technique rapid preparation Under the transmission electron microscopy in 49 83 of 59 feces samples and in the 10 100 samples of intestinal suspension coronavirus particles were visualized in a great number when negatively contrasted with ammonium molibdate 2 and pH5 0 Figs 1 2 The viral particles showed pleomorphism marked rounded or elongated shaped Fig 2 with characteristic radial projections forming a corona Figs 3 4 and mostly presenting a central electron dense corion Fig 3 and measuring 80 140 nm in diameter The club shaped surface projections measured approximately 20 nm long CATROXO M H B MIRANDA L B LAVORENTI A PETRELLA S MELO N A MARTINS A M C P R F Detection of coronavirus in capybaras Hydrochoeris hydrochaeris by transmission electron microscopy in S o Paulo Brazil Int J Morphol 28 2 549 555 2010 551 Fig 1 Negatively stained showing coronaviruses particles grouping and external envelope arrow Bar 80 nm Fig 2 Negatively stained coronaviruses particles showing pleomorphism marked rounded minor arrow or elongated shaped big arrow Bar 90 nm Fig 3 Negatively stained coronavirus particle showing a central electron dense corion minor arrow and characteristic radial projections forming a corona big arrow Fig 4 Negatively stained coronavirus particle exhibiting thin wispy and widely spaced spikes Bar 100nm DISCUSSION In this study we observed by transmission electron microscopy the presence of coronavirus in feces of capybaras during an outbreak of diarrhea in a herd Clinical signs observed in the affected animals were anorexia prostration dehydration dyspnea and death accompanied by yellowish or greenish diarrhea with liquid to pasty consistency recurrent type represented by intermittency of symptoms with periods of improvement followed by diarrhea episodes Similar outbreaks have been reported in cattle Brand o et al 2007 Barry et al 2009 swine Cappellaro 1998 equine Cappellaro et al 1988 dog Hagiwara et al 1989 Dezengrini et al 2007 Decaro et al 2010 sheep Pass et al 1982 feline Pedersen et al 2008 and avian Circella et al 2007 and among wild animals of zootecnical breeding such as collared peccary Catroxo et al 1995 rhea Catroxo et al 1996 a white lipped peccary Catroxo et al 1996 b ostrich Els MIRANDA L B LAVORENTI A PETRELLA S MELO N A MARTINS A M C P R F Detection of coronavirus in capybaras Hydrochoeris hydrochaeris by transmission electron microscopy in S o Paulo Brazil Int J Morphol 28 2 549 555 2010 552 Studies indicate that among animals there is a large reservoir for coronavirus not yet been recognized such as for influenza virus Holmes 2009 The coronavirus has a high frequency of recombination and high mutation rate Lai Catroxo et al 1996a Circella et al 2007 dog Catroxo et al 1998 Hagiwara et al cattle Benfield Dea Tsunemitsu et al Catroxo et al 1999 Barrera Valle et al 2005 Jeong et al 2005 Catroxo et al 2007 monkey Catroxo et al 1997 swine Capellaro et al 1998 According to Fenner et al the virus more severely affects young animals spreading very quickly and producing severe economic losses even before the esta blishment of control measures In our study the most affected animals were the weaned at three months During the experimental study in the herd the capybaras were constantly submitted to intense stress situation since they were captured every two weeks for check weighing In cattle other risk factors such as closed confinement improper ventilation inadequate handling of food the constant presence of visitors or the introduction of a new animal on the property may lead to the onset of the outbreak Boileau Nogueira Roman 1999 In suspected viral infection the use of the method of transmission electron microscopy for detection of viral particles in stool samples is required when there is a need of a rapid epidemiological clinical diagnosis and in cases of unsatisfactory diagnoses Arcangeletti et al and particularly valuable for following outbreaks of naturally acquired or viral gastroenteritis Brandt et al 1981 In this study the use of negative contrast technique for transmission electron microscopy which is essential to detect coronavirus in capybaras allowed a rapid prophylactic and control measures in the herd of capybaras Although the enteric coronaviruses is often reported in several animal species this is the first occurrence in capybaras Hydrochoerus hydrochaeris CATROXO M H B MIRANDA L B LAVORENTI A PETRELLA S MELO N A MARTINS A M C P R F Detection of coronavirus in capybaras Hydrochoeris hydrochaeris by transmission electron microscopy in S o Paulo Brazil Int J Morphol 28 2 549 555 2010 RESUMEN Coronavirus est n envueltos en una cadena positiva de ARN orden Nidovirales familia Coronaviridae y se clasifican en tres grupos Grupos que integran la tercera 3c y los coronavirus aviario Se distribuyen por todo el mundo y pueden causar enfermedades respiratorias ent ricas y en algunos casos hepatitis y enfermedades neurol gicas en una amplia variedad de animales resultando en p rdidas econ micas a la ganader a y los criadores En este trabajo se describe un brote de diarrea en un reba o de capibaras en la ciudad de Piracicaba S o Paulo Brasil Los animales presentaron s bitamente los s ntomas de anorexia diarrea profusa y acuosa de color amarillento o verdoso deshidrataci n p rdida de peso agudo torsi n de los miembros y la muerte en menos de una semana en su mayor a con tres meses de edad Un total de 59 muestras de heces y 10 trozos de intestino delgado se procesaron por la t cnica de contraste negativo preparaci n r pida En todas las muestras analizadas al microscopio electr nico de transmisi n se visualiz un gran n mero de part culas con morfolog a similar a los coronavirus pleom rficas con caracter sticas redondeadas o alargadas con proyecciones radiales en forma de corona midiendo de 80 140 nm de di metro La t cnica es sumamente til para el diagn stico r pido de virus en animales afectados PALABRAS CLAVE Coronavirus Capybara Microscop a electronic de transmisi n CATROXO M H B MIRANDA L B LAVORENTI A PETRELLA S MELO N A MARTINS A M C P R F Detection of coronavirus in capybaras Hydrochoeris hydrochaeris by transmission electron microscopy in S o Paulo Brazil Int J Morphol 28 2 549 555 2010 553 REFERENCES Arcangeletti M C De Conto F Pinardi F Medici M C Valcavi P Ferraglia F Motta F Covan S Calderaro A Chezzi C Miranda L B Lopes R S Takahira R K Kohayagawa A Ciarlini P C Landa H D A Beiras A M A Por tal S C Batista E R Redondo A V R Urquiaga Varela R U Alfieri A F Stipp D T Catroxo M H B Villalobos E M C Birgel Jr E H Gregori F Rosales C A R Ruiz V L A Villarreal L Y B de Souza S L P L J Richtzenhain L J Kim H W Rodriguez W J Thomas L Yolken R H Arrobio J O Kapikian A Z Parrott R H Catroxo M H B Suga O Mueller S B K Oliveira J M Carrascosa A A T A Bersano J G Catroxo 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Detection of coronavirus in capybaras Hydrochoeris hydrochaeris by transmission electron microscopy in S o Paulo Brazil Int J Morphol 28 2 549 555 2010 556