The persistence of infectious pancreatic necrosis virus (IPNV) in Atlantic salmon was examined with particular emphasis on the association of IPNV with leucocytes. Atlantic salmon were infected with IPNV via the water and via the feed. Despite relatively low mortalities, a high prevalence of IPNV was demonstrated particularly when the virus was introduced via the water. No virus-specific pathology or reduction in the growth performance of infected populations was evident. The conditions for the stimulation of Atlantic salmon leucocytes with the mitogen, phytohemagglutinin (PHA) were optimised and utilized in a co-stimulation assay. In this assay, leucocytes were infected with IPNV and simultaneously stimulated with PHA. It was demonstrated that the ability of IPNV to infect and replicate in leucocytes was enhanced when the cells were stimulated with mitogen. DNA synthesis was inhibited in the infected leucocytes. The inhibition was dependent upon the presence of infectious virus; inactivated virus failed to inhibit DNA synthesis. Three groups of salmon were examined to investigate the <i>in vivo</i> relationship of IPNV with leucocytes. Group A were the control group. IPNV was not isolated from these fish and the leucocytes responded to stimulation with PHA. Group B were experimentally infected IPNV carriers; virus was isolated from 6% of the fish using standard diagnostic methods but was not isolated from the supernatants of leucocyte cultures. The leucocytes of most fish responded to PHA stimulation. Neutralising antibody titres were variable and did not correlate with virus isolation. Group C were also IPNV carriers; virus could not be isolated using standard diagnostic methods but was isolated from the supernatants of stimulated leucocyte cultures of 44% of the fish. A significant inhibition of DNA synthesis in response to PHA stimulation was observed. The persistence of IPNV in Atlantic salmon is discussed in the light of the data presented here and existing knowledge of the persistence of mammalian viruses.

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-06-19T11:33:37Z No. of bitstreams: 1 resumo.pdf: 14552 bytes, checksum: c7feb5483902e97430fd0ecfd37fc98b (MD5) / Made available in DSpace on 2017-06-19T11:33:37Z (GMT). No. of bitstreams: 1 resumo.pdf: 14552 bytes, checksum: c7feb5483902e97430fd0ecfd37fc98b (MD5) Previous issue date: 2005-03-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O vírus da doença infecciosa bursal é um importante agente patogênico de aves e desde 1962 tem estado relacionado com grandes prejuízos econômicos. Ainda hoje não foi estabelecido um protocolo de vacinação realmente eficiente e falhas na imunização das aves são geralmente descritas. Os motivos dessas falhas vem sendo pesquisados e as causas alegadas são desde o surgimento de cepas patogênicas por mutações na região do gene que codifica para a VP2, uso mal assessorado de vacinas pouco atenuadas e a reversão vacinal. Este trabalho teve como objetivo pesquisar e comparar a patogenicidade de isolados de campo com a de cepas vacinais. Foram testadas 21 amostras de bursa que foram submetidas à Unidade de Sanidade Avícola da Universidade Federal de Viçosa com suspeita de doença infecciosa bursal e três amostras vacinais. O isolamento viral foi realizado em células VERO, sendo o vírus isolado de 14,28% (3/21) das amostras de campo e de todas as amostras vacinais. As amostras foram padronizadas pela habilidade de provocar efeito patogênico em cultivo celular e foram inoculadas em ovos embrionados de 10 dias de idade. Amostras de fígado, rins, bursa, baço e intestino de cada um dos embriões foram coletadas durante 8 dias e as alterações macroscópicas avaliadas. Metade de cada amostra coletada foi mantida à 4°C e metade foi fixada com formol 10%. As amostras fixadas foram processadas pelo método de inclusão em parafina, coradas com hematoxilina e eosina e as alterações microscópicas avaliadas. Das amostras mantidas a 4°C foi extraído o RNA total pelo método do TRIzol e testadas pela técnica de RT- PCR. Nas observações anatomopatológicas os embriões inoculados apresentavam tamanho reduzido quando comparado aos embriões controle e órgãos com lesões sugestivas de danos vasculares. Nas análises histopatológicas, os órgãos linfóides apresentaram pronunciada depleção linfóide, os hepátocitos vacuolização citoplasmática e os rins estruturas basófilas dentro de túbulos. No teste de RT-PCR foi possível detectar a presença do ácido nucléico viral em todos os tecidos, sendo que a distribuição tecidual dos isolados de campo foi maior quando comparada aos isolados vacinais. Esses resultados revelaram não haver grande diferença quando comparadas as lesões causadas pelos isolados de campo com as lesões causadas por algumas amostras vacinais. Porém há distintos padrões de distribuição em tecidos que poderia indicar uma variação na atenuação de algumas amostras vacinais. Esses resultados sugerem que as amostras vacinais podem causar lesões características da doença e que o uso de determinada cepa, mais ou menos invasiva, deve ser avaliada com cuidado pelo profissional no campo. / The infectious bursal disease virus is an important pathological agent of poultry and since 1962 it has been related to great economical losses. Until now, an efficient vaccination protocol has not been established and faults in poultry immunization are generally described. These faults motifs are being searched and various causes have been quoted, from the emergence of pathogenic strains by mutations in the gene responsible for coding VP2, to bad usage of vaccines attenuated for low passage and vaccinal reversion. The aim of this work was to examine and compare field isolates pathogenicity with vaccinal strains. Twenty-one bursa samples suspicious of infectious bursal disease submitted to Unidade de Sanidade Avícola da Universidade Federal de Viçosa and three vaccinal samples were tested. The viral isolation was accomplished in VERO cells, the virus was isolated in 14,28% (3/21) of field samples and in all vaccinal samples. The samples were standardized by its ability to provoke pathogenic effect in cellular culture and were inoculated in 10 days embryonic eggs. Liver, kidney, bursa, spleen and gut samples of each embryo were collected during 8 days and the macroscopic alterations evaluated. Half of each collected sample was stored at 4°C and the other half was fixed with 10% formol. The fixed samples were processed by paraffin xinclusion method, dyed with hematoxilin and eosin and the microscopic alterations evaluated. Total RNA extraction was performed on those samples stored at 4°C by TRIzol method and tested by RT-PCR technique. In anatomopathological observations inoculated embryos presented reduced size when compared to control embryos and organs with characteristics lesions of vascular injury. In histopathological analysis lymphoid organs presented an accentuated lymphoid depletion, hepatocytes presented citoplasmatic vacuolization and kidneys basophiles structures inside tubules. In RT-PCR test was possible to detect the presence of viral nucleic acid in each and every tissues, and the distribution of field isolates was larger when compared to vaccinal isolates. The results revealed that there is not much difference between lesions caused by field isolates with lesions caused by some of vaccinal samples. However, there are distinct distribution patterns in tissues that could indicate a variation on attenuation in some vaccinal samples. These results suggest that vaccinal samples could cause characteristics disease lesions and that the use of a determinate strain more or less invasive should be evaluated with care by the field professional.