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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-7 July- 2017 Page | 7 Presence of herpesvírus in diseased fishes Fabíola de Souza 1 Marcia H. B. Catroxo 2 Ana Maria Cristina R. P. F. Martins 3 Rodrigo Barbosa de Souza 4 Christiane Alves de Oliveira 5 Marcio Hipólito 6 125 Electron Microscopy Laboratory Research and Development Center in Animal Health Biological Institute São Paulo SP Brazil 36 Interinstitutional Laboratory of Aquaculture Health Research and Development Center in Animal Health Biological Institute São Paulo SP Brazil 4 Federal University of São Paulo State - UNIFESP São Paulo SP Brazil Abstract — Herpesviruses that infect fishes belong to the Herpesvirales order and Alloherpesvirus family. In these species the different types of herpesvirus can cause tumors adenocarcinoma and skin lesions. This study aims detect to presence of herpesvirus in fishes from commercial recreation or experimental creations of the States of São Paulo and Minas Gerais Brazil. Organ fragments and lesions of 53 fish species coming of mortality cases were forwarded at Biological Institute for examination by transmission electron microscopy by research of etiological agent. By transmission electron microscopy through negative staining technique were observed herpes virus-like particles in 46 fishes and through embedding resin technique in ultrathin sections were visualized herpes virus immature particles measuring 90-110nm in diameter located in the nuclei and complete particles measuring 160nm. In the histopathology technique lesions associated with the virus as corpuscles inclusion papillomas and dermal lesions and in the gills were observed in 27 fishes. The evaluated techniques of TEM and the histopathology were effective for the rapid detection of herpesvirus in the examined samples. Keywords — Disease histopathology transmission electron microscopy herpesvirus. I. INTRODUCTION Aquaculture in full development is becoming more important as livestock activity although it is still considered by many as an appendage of the fishing industry. Practiced in all Brazilian states aquaculture mainly covers the following creations of fish shrimp frogs shellfish oysters and mussels and other aquatic crops such as growing algae are practiced on a smaller scale 1. According to Toranzo et al. 2004 2 infectious diseases caused by viruses can cause damage to the economic viability of aquaculture activities. These diseases occur mostly confined animals and can develop serious infectious processes affecting the development of animals and even cause death. Herpesviruses belong to Herpesvirales order divided into three families Alloherpesvirus Herpesviridae and Malacoherpesvirus encompassing viruses that occur in several animal species such as molluscs fish amphibians reptiles birds and mammals 3. The Herpesviridae family keeps viruses of mammals birds and reptiles the Alloherpesviridae family includes fish and frogs viruses and Malacoherpesviridae family contains the virus from bivalves 4. The virus mainly affecting the early stages of fish life and therefore eggs larvae and young fish are more susceptible. The infected eggs can produce larvae newly hatched with clinical signs of illness while the infected adult fish may or may not present any symptoms 5. Within the family of herpesviruses can highlight herpesvirus papilloma of cyprinids 6 channel catfish herpesvirus and herpesvirus salmon 7. The need for a more detailed and thorough knowledge of the damage caused by herpesviruses in aquaculture especially in fish farming has led us to this research. The pathological relationship between etiologic agents involving these animals is not well established since in Brazil there is no survey of the pisces herpesviroses and the reports are sporadic and opportunistic depending on the shipment of samples to a diagnostic center. In this way all studies in the area are important because the virus can remain latent in healthy animals or decimate entire flocks in a breeding unfeasible a production.

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-7 July- 2017 Page | 8 This study aimed to detect the presence of herpes virus in organs fragments of fishes from the states of São Paulo and Minas Gerais using transmission electron microscopy techniques negative staining and resin embedding and associating viral presence with indicative lesions observed by optical microscopy using histological technique hematoxylin-eosin HE. II. MATERIAL AND METHOD 2.1 Ethics statement This study was approved by Animal Experimentation Research Ethics Committee Instituto Biológico protocol 137/14. 2.2 Experimental design The collection of material was performed from samples received for examination by the Laboratório Interinstitucional de Sanidade em Aquicultura and the Laboratório de Microscopia Eletrônica Instituto Biológico São Paulo SP Brazil. The samples were from comercial recreation or experimental creations of the States of São Paulo and Minas Gerais Brazil. Were utilized 202 organ fragments such as liver kidney spleen intestine gills heart brain and skin lesions of 53 fish. The examined species were 3 catfish Bagre sp 2 african sharptooth catfish Clarias gariepinus 3 cobia Rachycentron canadum 14 carp Ciprinus carpio 1 grouper Epinephelus marginatus 1 lenticulata pike cichlid Crenicichlalenticulata 3 pacu Piaractus mesopotamicus 1 mida cichlid Cichlassoma citrinellum 1 red piranha Serrasalmus nateteri 1 snookfish Centropomus parallelus 1 black pacu Colossoma macropomum and 22 tilapia Oreochromis niloticus from cases of diseases and mortalities. 2.3 Negative staining technique rapid preparation In the negative staining the fragments of organs and skin lesions 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 8910. 2.4 Resin embedding technique Thin slices the fragments of organs and skin lesions were fixed in 2.5 glutaraldehyde in 0.1M pH7.0 phosphate buffer and pos-fixed in 1 osmium tetroxide in the same buffer. After dehydration in cetonic series the fragments were embedded in Spurr resin 1112. Ultrathin sections were cut on the LKB ultratome and mounted on copper grids. The sections were stained by combination of uranyl acetate and lead citrate 1314. 2.5 Routine histological technique Fragments of organs and skin lesions of 27 fish were fixed in 10 buffered formalin dehydrated diaphonized and embedded in paraffin. 4 µm thick sections were performed and stained with hematoxylin Harris and eosin technique 15. III. RESULTS 3.1 Negative staining technique rapid preparation On transmission electron microscopy Philips EM 208 using the negative staining technique rapid preparation it was found enveloped figure 1 and non-enveloped figure 2 isometric pleomorphic herpesvirus-like particles measuring 120-200 nm of diameter in 46 86.8 out of the 53 examined fish from 202 samples of organs fragments. The positive species were 3 catfish Bagre sp 2 african sharptooth catfish Clarias gariepinus 3 cobia Rachycentron canadum 14 carp Ciprinus carpio 1 grouper Epinephelus marginatus 1 lenticulata pike cichlid Crenicichla lenticulata 3 pacu Piaractus mesopotamicus 1 midas cichlid Cichlassoma citrinellum 1 red piranha Serrasalmus nateteri 1 snookfish Centropomus parallelus 1 black pacu Colossoma macropomum e 15 tilapias Oreochromis niloticus. A total of 7animals 13.21 tilapias – O. niloticus were negative for herpesvirus.

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-7 July- 2017 Page | 9 FIG. 1 – ELECTRON MICROGRAPH OF ENVELOPED HERPESVIRUS PARTICLE ARROW IN CARP LIVER FRAGMENT SUSPENSION. BAR: 140 nm. FIG. 2 – ELECTRON MICROGRAPH SHOWING VIRAL NUCLEOCAPSIDS ARROW IN CARP LIVER SUSPENSION. BAR: 120 nm. 3.2 Resin embedding technique All positive samples from liver brain and skin by negative staining technique were subjected the resin embedding technique. In ultrathin sections was observed immature herpesvirus particles figure 3 measuring 90-110 nm in diameter located in the nucleus and complete particles measuring 160 nm in diameter. Extensive proliferation of the nuclear membrane producing complex finger-like extensions and amorphous inclusions located near the nucleus were visualized figure 4. FIG. 3 – ELECTRON MICROGRAPH OF ULTRATHIN SECTIONS OF SNOOKFISHS BRAIN SHOWING THE PRESENCE OF INCOMPLETE HERPESVIRUS PARTICLE LOWER ARROW AND EXTENSIVE PROLIFERATION OF NUCLEAR MEMBRANE PRODUCING FINGER-LIKE EXTENSIONS LARGE ARROW. BAR: 800 nm. FIG. 4 – ELECTRON MICROGRAPH OF ULTRATHIN SECTIONS OF SNOOKFISH BRAIN INDICATING THE PRESENCE OF INCOMPLETE HERPESVIRUS PARTICLE MINOR ARROW AND AMORPHOUS INCLUSIONS LOCATED NEAR THE NUCLEUS BIG ARROW. BAR: 800 nm. 3.3 Routine histological technique Positive samples by negative staining technique of 27 fish were examined by routine histological technique HE. The examined species were 13 carps 8 tilapia 2 african sharptooth catfish 1 tambaqui 1 red piranha 1grouper and 1 midas cichlid. The observed lesions associated with the viral presence figure5 are shown in Table 1.

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-7 July- 2017 Page | 10 FIG. 5 - PHOTOMICROGRAPH OF HISTOLOGICAL SECTION OF AFRICAN SHARPTOOTH CATFISH LIVER INDICATING HERPESVIRUS BASOPHILIC NUCLEAR INCLUSION ARROW. HE STAINING. BAR: 20 µm. TABLE 1 MAIN HISTOPATHOLOGICAL LESIONS OBSERVED IN FISH AND NUMBER OF AFFECTED ANIMALS / ANALYZED ANIMALS. Live Kidney Spleen Hepatopancreas Gill Pancreas Skin CNS Foamy areas 0/16 0/15 0/17 0/9 0/16 6/9 0/7 0/3 Dystrophic calcification 0/16 7/15 0/17 0/9 0/16 0/9 0/7 0/3 Corpuscles inclusion 3/16 1/15 0/17 2/9 0/16 0/9 3/7 1/3 Fibrin exudates 0/16 6/15 4/17 0/9 0/16 0/9 0/7 1/3 Secondary fusion of primary lamellae 0/16 0/15 0/17 0/9 6/16 0/9 0/7 0/3 Bleeding 2/16 8/15 15/17 0/9 0/16 0/9 2/7 1/3 Hyperplasia mononuclear cells 0/16 0/15 10/17 0/9 0/16 0/9 0/7 0/3 Epithelial hyperplasia 0/16 0/15 0/17 0/9 0/16 0/9 4/7 0/3 Lesions suggestive of papilloma 0/16 0/15 0/17 0/9 0/16 0/9 3/7 0/3 Melanomacrophages 3/16 6/15 13/17 0/9 0/16 7/9 3/7 1/3 Necrosis 2/16 4/15 2/17 2/9 1/16 1/9 4/7 1/3 Nephrosis 0/16 3/15 0/17 0/9 0/16 0/9 0/7 0/3 Cytoplasmic rarefaction 12/16 0/15 0/17 5/9 0/16 0/9 0/7 0/3 Inflammatory reaction 2/16 9/15 0/17 3/9 3/16 6/9 4/7 1/3 Congestion of blood vessels 8/16 0/15 0/17 0/9 7/16 3/9 0/7 3/3 IV. DISCUSSION In this study 202 samples of organ fragments from 53 fish were processed by the negative staining technique for transmission electron microscopy. Of these 53 fish 46 86.8 were positive for herpes virus.

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-7 July- 2017 Page | 11 Studies around the world show a mortality rate of 80 to 100 in creations of carps infected by both CyHV-2 and CyHV-3 1617181920 as well by IcHV-1 that infects channel catfish 21 the 3 types of fish herpesvirus most frequent. Not yet know here in Brazil which are the types of herpes viruses that are present in fish. Particles with morphology similar to herpesvirus pleomorphic isometric some enveloped measuring between 120 to 200 nm in diameter were visualized in all samples of organ fragments from the 46 positive fish of our study. These morphological features are also described by other authors in studies with herpesvirus made in tilapia larvae 22 in Osmerus eperlanus 23 and in Ictalurus nebulosu 24. In ultrathin sections of brain liver and skin of the positive fish of our study were observed immature and complete herpesvirus particles located in the nucleus aspects also seen in tumor cells of Osmerus eperlanus where herpesvirus particles were viewed in the cytoplasm 23. The resin embedding technique has also been applied to study ultrastructural aspects of herpesvirus into cells infected by CyHV-3 1725262728 of the Koi herpesvirus KHV infecting carp Cyprinus carpio 29 and of the CCV channel catfish virus in a kind of catfish Ictalurus nebulosus. 4 From this technique herpesvirus particles were visualized in all stages of development in the nucleus granular bodies containing virus particles lamellar structures and altered nuclear membrane and numerous viral particles in the cytoplasm 24. This technique applied in the spleen and kidney lesions carried out in goldfish by Lovy and Friend 2014 30 confirmed the presence of the herpesvirus in the nucleus and cytoplasm of necrotic cells. The authors also observed that the nucleus had marginalized chromatin. We found the incomplete particles measured between 90 and 110 nm and the complete particles measured on average 160 nm in diameter. These findings were consistent with other ultrastructural studies that reported who the nucleocapsids of the CyHV-1 CyHV- 2 and CyHV-3 measured 100 a 113 nm and the mature enveloped particles measured 190 nm to 230 nm in diameter 3132333435 located in the cytoplasm and extracellular space 33. In fish of our study the clinical signs and symptoms most commonly observed were incoordination lethargy sudden death skin lesions fin and mouth liver nodules and liver and intestinal disorders. These symptoms and signs have also been reported in fish infected by CyHV-2 CyHV-3 and IcHV such as Carassius auratus 3319 Cyprinus carpio 1736 Ictalurus punctatus 3724 and Oreochromis aureus 22. We find lesions like-papillomas in skin of 3 tilapia. Lesions petechial or papillomatous around the body mouth cornea kidneys and jaw have been reported in fish infected with herpesvirus Salmon such as Oncorhynchus keta 3839 Cyprinus carpio 40 Oncorhynchus kisutch Oncorhynchus keta Oncorhynchus masou and Salmo gairdneri Yoshimizu et al. 1987 41. Papilloma like lesions were also observed in CyHV-1 infection in the fish species 424331. In Cyprinus carpio infected by CyHV-3 the most commonly encountered lesions were located in the gills 1744454629. Regarding histopathology in 5 carp and in 1 african sharptooth catfish of our study we found gill lesions such as fusion the primary and secondary blades. Histopathological examinations performed by Lovy and Friend 2014 30 in goldfish Carassius auratus also showed the presence of fusion of gill filaments in all fish positive for herpesvirus examined in their study. This finding was attributed to epithelial hyperplasia the infiltration of eosinophilic cells and occasionally diffuse necrosis. Pikarsky et al. 2004 46 also included as branchial arch lesions suggestive of herpesvirus subepithelial increase inflammation and congestion of blood vessels in carp. These lesions also were found by us in grouper carp and tilapia. In kidney of catfish carp and tilapia were observed focuses with hemorrhage and the light of many tubules was visualized fibrinoid and hyaline material necrotic areas and the presence of melanomacrophages. It was observed particulary marked mononuclear glomerulonephritis as well as dystrophic calcification calcium deposition. Plumb et al. 1974 47 and Wolf et al. 1972 48 observed in your studies conducted in catfish Ictalurus punctatus the presence of extensive edema inflammation and necrosis of the renal tubules. Pikarsky et al. 2004 46 found blood vessels

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-7 July- 2017 Page | 12 congested in carp infected by herpesvirus while Lovy and Friend 2014 30 verified the presence of necrosis and dilated blood vessels in goldfish. In tilapia O. niloticus and catfish Bagre sp hepatic tissue in general showed cytoplasmic rarefaction mineral protein deficiency numerous melanomacrophages hepatitis focal monolimphocytary and necrosis. Plumb et al. 1974 47 and Wolf et al. 1972 48 cite the focal necrosis hemorrhage and edema as the main lesions found in the liver of animals infected with herpesvirus of Ictaluridae IcHV1. In the spleen of catfish carp and tilapia the most significant lesions found were hemorrhagic areas presence of melanomacrophages hyperplasia of mononuclear cells and necrosis. These findings were corroborated by Lovy and Friend 2014 30 in goldfish. Moreover Plumb et al. 1974 47 and Wolf et al. 1972 48 found in their studies apart of changes observed in our work congested blood vessels. In our study we found the presence of inclusion bodies in skin of 2 tilapia and of 1 catfish in hepatopancreas of 1 tilapia and of 1 carp in liver of 1 catfish and 2 carps in kidney of 1 tilapia and in the SNC of 1 midas cichlid. Gibson-Kueh et al. 2012 49 also found these inclusion bodies in the spleen kidney liver and heart in their studies in giant perch Lates calcarifer infected with herpesvirus. According to Yamamoto et al. 1983 50 and Elias et al. 2004 51 the presence of inclusion bodies basophilic is the major histopathologic alteration characteristic of the presence of herpesvirus. V. CONCLUSION From these findings it was concluded that electron microscopy techniques used in this study were effective in diagnosing the presence of herpesvirus allowing the visualization of agent and should be spread widely. Routine histopathological technique was also effective in the visualization of lesions suggestive of the viral agent as the presence of inclusion bodies. Moreover enabled to perform the analysis of the animals overall condition indicating that the quality of creation can make it susceptible to infection by herpesvirus. Histopathology and electron microscopy tests must be complementary. We cannot here say that the disease followed by mortality are directly caused by herpesvirus however serious conditions predisposing to poor water quality unsuitable managements high density and poor nutritional quality must have contributed to viral replication. The viral presence of course contributed to the worsening of the picture intensifying clinical symptoms followed of mortality. Studies to assess what are the fish herpesvirus subtypes that are circulating in our country and its real impact on mortality should be conducted. ACKNOWLEDGEMENTS The research was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior CAPES. REFERENCES 1 Scorvo-Filho J.D. Panorama da aquicultura nacional. In: Textos técnicos - Instituto de Pesca. Disponível em: ftp://ftp.sp.gov.br/ftppesca/panorama_aquicultura.pdf Acesso em: 22 de agosto de 2013. 2 Toranzo AE Barja JL Dopazo CP and Romalde JL. 2004. Enfermidades bacterianas y víricas de peces marinos. In: Ranazani-Paiva MJT Takemoto RM Lizama MLA. 2004. Sanidade de Organismos Aquáticos. Editora Varela São Paulo. p.3-49. 3 International Committee on Taxonomy of Viruses ICTV. 2013. Virology Division – IUMS. Disponível em: http://ictvonline.org/virusTaxonomy.asp. Acesso em: 12 de dezembro de 2013. 4 Davison AJ Eberle R Ehlers B Hayward GS Mcgeoch DJ Minson AC Pellett PE Roizman B Studdert MJ and Thiry E. 2009. The order Herpesvirales. Arch. Virol. v.154 n. 1 p. 171-177. 5 Martins AMCRPF Hipolito M and Catroxo M.B. 2011. A importância da piscicultura e algumas doenças virais e bacterianas písceas. Comunicado técnico Instituto Biológico Centro de pesquisa e desenvolvimento sanidade animal n.156. Disponível em: http://www.biologico.sp.gov.br/artigos_ok.phpid_artigo156. Acesso em: 03 de fevereiro de 2014. 6 Schubert GH. 1966. The infective agent in carp pox. Bulletin Office of International Epizootics p.1011-1022. 7 Wolf K. 1983. Biology and properties of fish and reptilian herpesviruses. In: The Herpersviruses. v.2 Roizman b. ed.. Plenum Press: New York. p.319-366.

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