الملخص
@#Objective To investigate the effect of amplification culture of micro-carrier Vero cells from 30 L bioreactor to 300 L bioreactor after extra-tank trypsinization on the virus-producing ability of rabies virus(RABV)CTN-1Ⅴ strain.Methods The 140-passage of Vero cells were cultured at 37 ℃ for 72-120 h,then amplified by passaging at a cell density ratio of 1∶4 into the 10 × cell factory. After incubation at 37 ℃ for 72-120 h,the monolayer cells were detached and inoculated into the 30 L bioreactor with micro-carriers 7-10 g/L,culture temperature 37 ℃,pH 7. 0-7. 4,dissolved oxygen 30%-80%,stirring speed 10-50 r/min,and continuous perfusion culture 72-120 h. Total three batches of microcarrier Vero cells were cultured,which were amplified to the 300 L bioreactor after extra-tank trypsinization,with microcarrier5-8 g/L,culture temperature 37 ℃,pH 7. 0-7. 4,dissolved oxygen 30%-80%,stirring speed 30-80 r/min,and perfusion culture 72-120 h. RABV CTN-1Ⅴ was inoculated at the MOI of 0. 05,and the virus solution was harvested every 24 h and detected for the virus titer and antigen content. Results The cell density was about 1 × 10~7 cells/mL after culture for 96 h in the 30 L bioreactor,and was about 7. 4 × 10~6 cells/mL after culture for 96 h in the 300 L bioreactor. At 96 h after virus inoculation,the virus harvest solution reached the peak potency,with the average virus titer of 6. 8 lgLD_(50)/mL and the average antigen content of 2. 58 IU/mL. Conclusion The scale-up culture process of micro-carrier Vero cells after extra-tank trypsinization from 30 L bioreactor to 300 L bioreactor is stable and feasible,with no significant effect on the virus-producing ability of RABV CTN-1Ⅴ strain,which provides a reference for the large-scale production of inactivated RABV vaccine
الملخص
@#Objective To develop a large-scale culture process for rabies virus(RABV)in 150 L bioreactor,and lay a foundation for the further development of a larger-scale and high-density microcarrier reactor process.Methods Vero cells and RABV strain CTN-1V were cultured in 30 L(model:C30-2)and 150 L(model:VESSEL FERMENTER 300L)bioreactors by perfused culture with 20 g/L Cytodex-1 microcarrier and DO 20%-60%,at culture temperature 36-38 ℃ and pH 7.0-7.4.During the culture process,the cell density and virus titer were measured.The virus culture media was harvested for consecutive 13 d and detected for the sterility,mycoplasma,and the residues of antigen,host cell protein(HCP),bovine serum albumin(BSA)and DNA.Results The density of cultured cells in 30 L and 150 L bioreactors all reached above 1.2 ×10~7cell/mL.There was no significant difference in cell density at different time points during the culture(t = 0.225-2.173,P = 0.096-0.833).The highest virus titer(8.5 lgLD_(50)/mL)was found in the both bioreactors 6 d after infection with no significant difference(t = 1.000,P = 0.374).The residues of antigen,HCP,BSA and DNA in the virus suspension from the two bioreactors were basically the same.Conclusion 150 L bioreactor can be used for the large-scale culture of RABV,and the harvested virus conformed to the relevant standards in Chinese Pharmacopoeia(Volume Ⅲ,2020 edition).
الملخص
ABSTRACT: This research reports the use of different diagnostic tests in cattle, naturally infected by Rabies lyssavirus (RABV), and correlates the positivity of the tests with the clinical moment of euthanasia, the intensity of the inflammatory lesion and viral load. It also highlights the possibility of euthanasia in early stages of the disease as a way to improve animal welfare. For that, samples of 34 bovine brains were collected for analysis, preserved in 10% buffered formaline and refrigerated with subsequent freezing. The samples were subjected to direct immunofluorescence antibody technique (DFAT) tests, viral isolation in cell culture (VICC), histopathology with hematoxylin and eosin staining (HE), immunohistochemistry (IHC), Shorr stainied neural tissue smears (DSS), Reverse transcription polymerase chain reaction (RT-PCR) and polymerase chain reaction by quantitative reverse transcriptase (qRT-PCR). The areas used for analysis were the cerebellum, parietal telencephalon and thalamus. Samples with Negri bodies (NBs) or immunostaining in at least one of the analyzed areas were considered positive. For the study of the intensity of histological lesions, the lesions were classified into grades 0, 1, 2 and 3 and the positivity of the test in the presence or absence of NBs in one of the three areas analyzed. To verify the influence of the disease clinical evolution, 4-four groups of analysis were created according to the animals clinical status at moment of the euthanasia, being: M1 = animal euthanized while standing, M2 = euthanized when in sternal recumbence, M3 = euthanized when in lateral recumbence, M4 = animal with natural death. Of the 34 brains evaluated, IHC was positive in 100% of cases, DFAT was positive in 97.05% of them, and in this negative sample the presence of RABV was confirmed by VICC. NBs ere seen in 88.23% of the cases, and the DSS test was positive in 82.35% of them. All diagnostic techniques showed positive cases in all groups analyzed. Each case was positive in at least two diagnostic methods. All cases that contained NBs were positive for rabies in the other tests. In this study, it was observed that the variables analyzed (intensity of injury and clinical evolution at the moment of euthanasia) had an influence only on HE and DSS techniques, which are based on NB research to form the diagnosis, but did not interfere with the effectiveness of the diagnosis performed by detecting the viral antigen performed by DFAT and IHC. All isolated RABV samples included in the present study have a genetic lineage characteristic of hematophagous bats Desmodus rotundus. The evaluation of qRT-PCR showed that the amount of virus did not interfere in the positivity of the tests. This work shows that IHC and DFAT are safe diagnostic techniques. They are capable of detecting RABV even in euthanized animals in the early stages of clinical evolution with mild intensities of histological lesions.
RESUMO: Esta pesquisa relata a utilização de diferentes testes de diagnóstico em bovinos, naturalmente infectados pelo Rabies lyssavirus (RABV), e correlaciona a positividade dos testes com o momento clínico da eutanásia, a intensidade da lesão inflamatória, e a carga viral. Salienta também a possibilidade da eutanásia em estágios precoces da doença como forma de melhorar o bem-estar animal. Para isso amostras de 34 encéfalos bovinos foram coletados para análise, conservadas em formol tamponado 10% e sob refrigeração com posterior congelamento. As amostras foram submetidas aos testes de imunofluorescência direta (IFD), isolamento viral em cultivo de células (IVCC), histopatologia com coloração de hematoxilina e eosina (HE), imuno-histoquímica (IHQ), esfregaço direto com coloração de Shorr (EDS), reação da polimerase em cadeia por transcriptase reversa (RT-PCR) e reação da polimerase em cadeia por transcriptase reversa quantitativo (qRT-PCR). As áreas utilizadas para análise foram o cerebelo, telencéfalo parietal e tálamo. Foram consideradas positivas as amostras que apresentaram Corpúsculo de Negri (CNs) ou imuno-marcação em ao menos uma das áreas analisadas. Para o estudo da intensidade das lesões histológicas, as lesões foram classificadas em graus 0, 1, 2 e 3 e a positividade do teste na presença ou ausência de CN em uma das três áreas analisadas. Para verificar a influência da evolução clínica da doença foram criados 4 grupos de análise conforme o estado clínico do animal no momento da eutanásia, sendo: M1 = animal eutanasiado em estação, M2 = eutanasiado em decúbito esternal, M3 = eutanasiado em decúbito lateral, M4 = animal com morte natural. Dos 34 encéfalos avaliados a IHQ foi positiva em 100% dos casos, a IFD foi positiva em 97,05%, sendo que na amostra negativa a presença de RABV foi confirmada por IVCC. A histologia com HE, através da visualização das CNs, foi positiva em 88,23 % dos casos, e o teste de EDS, foi positivo em 82,35%. Todas as técnicas de diagnóstico apresentaram casos positivos em todos os grupos analisados. Cada caso foi positivo em, pelo menos, dois métodos de diagnóstico. Todos os casos que continham CN foram positivos para raiva nos demais testes. Nesse estudo observou-se que as variáveis analisadas intensidade de lesão e evolução clínica no momento da eutanásia tiveram influência somente nas técnicas de HE e EDS, que se baseiam na pesquisa do CN para formação do diagnóstico, mas não interferiram na eficácia do diagnóstico realizado através da detecção do antígeno viral realizado por IFD e IHQ. Todas as amostras RABV isoladas incluídas no presente estudo apresentam linhagem genética característica de morcegos hematófagos Desmodus rotundus. A avaliação de qRT-PCR demostrou que a quantidade de vírus não interferiu na positividade dos testes. Esse trabalho mostra que a IHQ e a IFD são técnicas seguras de diagnóstico e que mesmo em animais eutanasiados em estágios iniciais de evolução clínica com intensidades leve de lesões histológicas, são capazes de detectar o RABV.
الملخص
This research reports the use of different diagnostic tests in cattle, naturally infected by Rabies lyssavirus (RABV), and correlates the positivity of the tests with the clinical moment of euthanasia, the intensity of the inflammatory lesion and viral load. It also highlights the possibility of euthanasia in early stages of the disease as a way to improve animal welfare. For that, samples of 34 bovine brains were collected for analysis, preserved in 10% buffered formaline and refrigerated with subsequent freezing. The samples were subjected to direct immunofluorescence antibody technique (DFAT) tests, viral isolation in cell culture (VICC), histopathology with hematoxylin and eosin staining (HE), immunohistochemistry (IHC), Shorr stainied neural tissue smears (DSS), Reverse transcription polymerase chain reaction (RT-PCR) and polymerase chain reaction by quantitative reverse transcriptase (qRT-PCR). The areas used for analysis were the cerebellum, parietal telencephalon and thalamus. Samples with Negri bodies (NBs) or immunostaining in at least one of the analyzed areas were considered positive. For the study of the intensity of histological lesions, the lesions were classified into grades 0, 1, 2 and 3 and the positivity of the test in the presence or absence of NBs in one of the three areas analyzed. To verify the influence of the disease clinical evolution, 4-four groups of analysis were created according to the animal's clinical status at moment of the euthanasia, being: M1 = animal euthanized while standing, M2 = euthanized when in sternal recumbence, M3 = euthanized when in lateral recumbence, M4 = animal with natural death. Of the 34 brains evaluated, IHC was positive in 100% of cases, DFAT was positive in 97.05% of them, and in this negative sample the presence of RABV was confirmed by VICC. NBs ere seen in 88.23% of the cases, and the DSS test was positive in 82.35% of them. All diagnostic techniques showed positive cases in all groups analyzed. Each case was positive in at least two diagnostic methods. All cases that contained NBs were positive for rabies in the other tests. In this study, it was observed that the variables analyzed (intensity of injury and clinical evolution at the moment of euthanasia) had an influence only on HE and DSS techniques, which are based on NB research to form the diagnosis, but did not interfere with the effectiveness of the diagnosis performed by detecting the viral antigen performed by DFAT and IHC. All isolated RABV samples included in the present study have a genetic lineage characteristic of hematophagous bats Desmodus rotundus. The evaluation of qRT-PCR showed that the amount of virus did not interfere in the positivity of the tests. This work shows that IHC and DFAT are safe diagnostic techniques. They are capable of detecting RABV even in euthanized animals in the early stages of clinical evolution with mild intensities of histological lesions.(AU)
Esta pesquisa relata a utilização de diferentes testes de diagnóstico em bovinos, naturalmente infectados pelo Rabies lyssavirus (RABV), e correlaciona a positividade dos testes com o momento clínico da eutanásia, a intensidade da lesão inflamatória, e a carga viral. Salienta também a possibilidade da eutanásia em estágios precoces da doença como forma de melhorar o bem-estar animal. Para isso amostras de 34 encéfalos bovinos foram coletados para análise, conservadas em formol tamponado 10% e sob refrigeração com posterior congelamento. As amostras foram submetidas aos testes de imunofluorescência direta (IFD), isolamento viral em cultivo de células (IVCC), histopatologia com coloração de hematoxilina e eosina (HE), imuno-histoquímica (IHQ), esfregaço direto com coloração de Shorr (EDS), reação da polimerase em cadeia por transcriptase reversa (RT-PCR) e reação da polimerase em cadeia por transcriptase reversa quantitativo (qRT-PCR). As áreas utilizadas para análise foram o cerebelo, telencéfalo parietal e tálamo. Foram consideradas positivas as amostras que apresentaram Corpúsculo de Negri (CNs) ou imuno-marcação em ao menos uma das áreas analisadas. Para o estudo da intensidade das lesões histológicas, as lesões foram classificadas em graus 0, 1, 2 e 3 e a positividade do teste na presença ou ausência de CN em uma das três áreas analisadas. Para verificar a influência da evolução clínica da doença foram criados 4 grupos de análise conforme o estado clínico do animal no momento da eutanásia, sendo: M1 = animal eutanasiado em estação, M2 = eutanasiado em decúbito esternal, M3 = eutanasiado em decúbito lateral, M4 = animal com morte natural. Dos 34 encéfalos avaliados a IHQ foi positiva em 100% dos casos, a IFD foi positiva em 97,05%, sendo que na amostra negativa a presença de RABV foi confirmada por IVCC. A histologia com HE, através da visualização das CNs, foi positiva em 88,23 % dos casos, e o teste de EDS, foi positivo em 82,35%. Todas as técnicas de diagnóstico apresentaram casos positivos em todos os grupos analisados. Cada caso foi positivo em, pelo menos, dois métodos de diagnóstico. Todos os casos que continham CN foram positivos para raiva nos demais testes. Nesse estudo observou-se que as variáveis analisadas intensidade de lesão e evolução clínica no momento da eutanásia tiveram influência somente nas técnicas de HE e EDS, que se baseiam na pesquisa do CN para formação do diagnóstico, mas não interferiram na eficácia do diagnóstico realizado através da detecção do antígeno viral realizado por IFD e IHQ. Todas as amostras RABV isoladas incluídas no presente estudo apresentam linhagem genética característica de morcegos hematófagos Desmodus rotundus. A avaliação de qRT-PCR demostrou que a quantidade de vírus não interferiu na positividade dos testes. Esse trabalho mostra que a IHQ e a IFD são técnicas seguras de diagnóstico e que mesmo em animais eutanasiados em estágios iniciais de evolução clínica com intensidades leve de lesões histológicas, são capazes de detectar o RABV.(AU)
الموضوعات
Animals , Cattle , Cattle/injuries , Euthanasia , Viral Load/veterinary , Rabies virus , Wounds and Injuries/diagnosis , Encephalitisالملخص
ABSTRACT: The present study investigated the frequency and magnitude of neutralizing antibodies to rabies virus (RABV) in dogs with and without historic of vaccination in Santa Maria/RS. Group A included serum samples from 440 dogs with recent historic of vaccination against rabies, obtained during the 2015 rabies vaccination campaign. Group B included 300 serum samples from dogs submitted to the Veterinary Hospital of the Universidade Federal de Santa Maria in 2015, whose historic of rabies vaccination was unknown. Serum samples were submitted to the rapid fluorescent focus inhibition test (RFFIT) to detect neutralizing antibodies against RABV. In group A, 70.6% (310/440) of the samples had neutralizing antibody titers ≥0.5 international units per milliliter (IU mL-1), considered an indicative of protection against rabies by the World Health Organization. However, approximately 30% of the dogs did not contain antibodies in adequate levels. In group B, 42.3% (127/300) of the samples contained neutralizing antibody titers ≥0.5IU mL-1 and 57.7% (173/300) were negative or contained titers below of the value considered immunized. These results demonstrate that an important proportion of vaccinated dogs (~30%) did not develop adequate antibody levels, mainly those receiving a single vaccine dose. Serologic testing of animals with unknown historic of vaccination revealed relatively low vaccine coverage in the general dog population. Thus, reformulation of immunization strategies - especially the recommendation of a boost vaccination 30 days after the primary dose - and extension of vaccination campaigns are necessary to reach adequate levels and coverage of immunity against RABV in the canine population.
RESUMO: O presente estudo investigou a frequência e a magnitude dos anticorpos neutralizantes do vírus da raiva (RABV) em cães com e sem histórico de vacinação em Santa Maria/RS. O Grupo A incluiu amostras de soro de 440 cães com histórico recente de vacinação contra a raiva, obtidos durante a campanha de vacinação contra a raiva de 2015. O Grupo B incluiu 300 amostras de cães submetidos ao Hospital Veterinário da Universidade Federal de Santa Maria em 2015, cujo histórico de vacinação antirrábica era desconhecido. As amostras de soro foram submetidas ao teste rápido de inibição de focos fluorescentes para detecção de anticorpos neutralizantes do RABV. No grupo A, 70,6% (310/440) das amostras possuíam títulos de anticorpos neutralizantes ≥0,5 unidades internacionais por mililitro (UI mL-1), considerado um indicador de proteção contra a raiva pela Organização Mundial da Saúde. No entanto, aproximadamente 30% dos animais não continham anticorpos em níveis adequados. No grupo B, 42,3% (127/300) das amostras continham títulos de anticorpos neutralizantes ≥0,5UI mL-1 e 57,7% (173/300) eram negativas ou continham títulos abaixo do valor considerado imunizado. Estes resultados demonstram que uma proporção importante de cães vacinados (~30%) não desenvolveu níveis adequados de anticorpos, principalmente aqueles que receberam uma única dose de vacina. O teste sorológico de animais com histórico de vacinação desconhecido revelou uma cobertura vacinal relativamente baixa na população geral de cães. Assim, a reformulação das estratégias de imunização - especialmente a recomendação de uma vacinação de reforço 30 dias após a primeira dose - e a extensão das campanhas de vacinação são necessárias para atingir níveis e cobertura adequados de imunidade contra o RABV na população canina.
الملخص
Oral rabies vaccination (ORV) program for the wild animals in rabies risk regions of Korea has been conducted since 2000. Evaluation of ORV program under field condition and information concerning the incidence of exposure to canine distemper and canine parvovirus (CPV) are needed in wild raccoon dogs (Nyctereutes procyonoides koreensis). Ninety four sera of wild raccoon dogs were screened for antibodies against rabies, canine distemper virus (CDV) and CPV in Korea. The overall prevalence of antibodies against rabies virus (RABV), CDV and CPV in wild raccoon dogs was 35.1%, 89.4% and 24.5%, respectively. Comparisons of sero-prevalences of RABV, CDV and CPV were assayed in two regions (Gyeonggi-do and Gangwon-do). The Gyeonggi-do (36.4%) showed higher sero-positive rate against CPV than Gangwon-do (20.8%). In contrast, Gangwon-do (41.7% and 97.2%) showed higher sero-positive rates against RABV and CDV than Gyeonggi-do (13.6% and 63.6%). These results indicate that there was severe circulation of CDV and CPV among wild raccoon dogs in the two regions of Korea. Furthermore, raccoon dogs showing a protective antibody titer (0.5 IU/ml) were 15.9%, suggesting that new rabies control program such as trap-vaccination-release (TVR) should be launched urgently in rabies risk regions.