Single-Tube Single-Enzyme Reverse Transcriptase PCR Assay for Detection of Bovine Viral Diarrhea Virus in Pooled Bovine Serum

Weinstock, Daniel; Bhudevi, Bodreddigari; Castro, Anthony E.

doi:10.1128/jcm.39.1.343-346.2001

Cited by 76 publications

(60 citation statements)

References 14 publications

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“…Expression was confirmed by immunostaining with anti-Pk-Tag mAb (MCA1360P, Serotec) and CTL recognition of virus-infected iSF. All vaccine preparations were screened for the presence of bovine viral diarrhea virus (27) and mycoplasma (Mycoplasma Detection kit version 2.0; American Type Culture Collection).…”

Section: Ifn-␥ Elispot and 51mentioning

confidence: 99%

Theileria parvacandidate vaccine antigens recognized by immune bovine cytotoxic T lymphocytes

Graham

Pellé

Honda

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

115

131

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East Coast fever, caused by the tick-borne intracellular apicomplexan parasite Theileria parva, is a highly fatal lymphoproliferative disease of cattle. The pathogenic schizont-induced lymphocyte transformation is a unique cancer-like condition that is reversible with parasite removal. Schizont-infected cell-directed CD8 ؉ cytotoxic T lymphocytes (CTL) constitute the dominant protective bovine immune response after a single exposure to infection. However, the schizont antigens targeted by T. parva-specific CTL are undefined. Here we show the identification of five candidate vaccine antigens that are the targets of MHC class I-restricted CD8 ؉ CTL from immune cattle. CD8 ؉ T cell responses to these antigens were boosted in T. parva-immune cattle resolving a challenge infection and, when used to immunize naïve cattle, induced CTL responses that significantly correlated with survival from a lethal parasite challenge. These data provide a basis for developing a CTL-targeted anti-East Coast fever subunit vaccine. In addition, orthologs of these antigens may be vaccine targets for other apicomplexan parasites.cattle ͉ East Coast fever ͉ immunoscreening ͉ protozoan parasite ͉ vaccination A single inoculation with a potentially lethal dose of Theileria parva sporozoites and simultaneous treatment with a longacting oxytetracycline induces solid immunity to homologous and, in certain instances, heterologous parasite challenge (1, 2). This methodology has been adopted as a live vaccine for the control of East Coast fever (ECF) (3). The long-lasting immunity to ECF contrasts with the partial immunity to malaria that develops after only several years of exposure to T. parva-related Plasmodium spp. (4). Manufacture and delivery of the live ECF vaccine is difficult to sustain, but it has enabled elucidation of the dominant protective immune response against the disease. Kinetic and adoptive cell transfer studies (5, 6) have demonstrated that protection of cattle is mediated by MHC class I-restricted CD8 ϩ cytotoxic T lymphocytes (CTL) that destroy schizontinfected lymphocytes, the pathogenic life-cycle stage of T. parva. In addition, there is a strong correlation between the specificity of the CTL response and cross-immunity profiles of distinct parasite strains (2). The identification of schizont antigens targeted by CTL from T. parva-immune cattle has been elusive but should pave the way for the development of a subunit vaccine against ECF and provide a long-term solution to a socioeconomically important constraint to livestock agriculture in Africa (7). We adopted two approaches to antigen identification, both dependent on screening of transiently transfected antigenpresenting cells with fully characterized CTL (8, 9) from live vaccine-immunized cattle of diverse bovine leukocyte antigen (BoLA) MHC class I genotypes. First, in a targeted gene approach, we immunoscreened genes that were predicted by using preliminary sequence data from one of the four T. parva chromosomes (10) to contain a secretion signal. The approach was ...

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Section: Ifn-␥ Elispot and 51mentioning

confidence: 99%

Theileria parvacandidate vaccine antigens recognized by immune bovine cytotoxic T lymphocytes

Graham

Pellé

Honda

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

115

131

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“…To optimization of technique, the following conditions were evaluated: i) two pairs of primers, 103 / 372 (WEINSTOCK et al, 2001) and 324 / 326 (VILCEK et al, 1994), ii) four methods of nucleic acid extraction (phenol/chloroform/isoamyl alcohol; silica/guanidine isothiocyanate; a combination of the two previous methods; and TRIzol ™ ) and iii) different concentrations and compositions of reagents and time/temperature of the reactions. Between the alternatives tested that resulted in the amplification of the 290 bp product that was easily visualized in ethidium bromide stained 2% agarose gel was that presented the following conditions: i) primers 103 and 372; ii) initial volume and clinical sample: 50 mL of blood serum; iii) extraction of nucleic acid: silica/guanidine isothiocyanate method; iv) reverse transcription: 9 mL extracted nucleic acid, 1xPCR buffer (20 mM TrisHCl pH 8.4 and 50 mM KCl), 1.5 mM MgCl 2 ; 60 units of reverse transcriptase enzyme M-MLV, RNA denaturation at 97°C / 4 min, and reverse transcription at 42°C / 30 min; v) PCR: primers 103 / 372 with anneling temperature at 59°C.…”

Section: Introductionmentioning

confidence: 99%

Comparação de diferentes protocolos para a detecção do vírus da diarréia viral bovina por RT-PCR em grupos de sangue total e de soro sangüíneo, artificialmente contaminados

Pilz

Alfieri

2005

Sem. Ci. Agr.

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ResumoA técnica da RT-PCR foi otimizada e avaliada para a detecção da região 5' terminal não-traduzida do genoma do vírus da diarréia viral bovina (BVDV) em amostras clínicas de bovinos, constituídas por soro sangüíneo e sangue total, artificialmente contaminadas com a estirpe NADL do BVDV. Para a otimização da técnica foram avaliados: i) dois pares de primers, 103 / 372 (WEINSTOCK et al., 2001) e 324 / 326 (VILCEK et al., 1994) ii) quatro métodos de extração do ácido nucléico (fenol / clorofórmio / álcool isoamílico; sílica / isotiocianato de guanidina; uma combinação dos dois métodos anteriores e TRIzol ™ ) e iii) diferentes concentrações e composições de reagentes, tampões e tempo/temperatura das reações. Entre todas as alternativas testadas a que resultou na amplificação de um produto com 290 pb, que foi facilmente visualizado em gel de agarose 2% com brometo de etídeo, foi a que empregou as seguintes condições: i) primers 103 e 372; ii) volume inicial e amostra clínica: 50 mL de soro sangüíneo; iii) extração do ácido nucléico: método da sílica / isotiocianato de guanidina, iv) transcrição reversa: 9 mL do ácido nucléico extraído, 1xPCR buffer (20 mM Tris-HCl pH 8,4 e 50 mM KCl), 1,5 mM MgCl 2 , 60 unidades da enzima transcriptase reversa M-MLV, desnaturação do RNA a 97°C / 4 min e transcrição reversa a 42°C / 30 min e v) PCR: primers 103 / 372 com temperatura de anelamento de 59°C. A utilização da RT-PCR nas condições otimizadas nesse estudo possibilitou a amplificação da estirpe NADL do BVDV (10 3,56

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“…During this period, samples of blood and nasal swab were collected and analyzed using virus neutralization (VN) and RT-PCR, respectively (Weinstock et al, 2001), proving that the animals were seronegative and free of viremia.…”

Section: Experimental Designmentioning

confidence: 99%

“…A conventional PCR for virus identification was performed using sense primers 103 (5'-TAG CCA TGC CCT TAG TAG GAC -3') and 392 antisense (5'-ACT CCA TGT GCC ATG TAC AGC -3') which amplifies a product of 290 bp (Weinstock et al, 2001).…”

Section: Rt-pcrmentioning

confidence: 99%

Experimental Infection and Evaluation of Airborne Transmission And Nose-To-Nose Contact of Bovine Viral Diarrhea Virus Among Weaned Piglets

2017

Aust. J. Basic & Appl. Sci.

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Single-Tube Single-Enzyme Reverse Transcriptase PCR Assay for Detection of Bovine Viral Diarrhea Virus in Pooled Bovine Serum

Cited by 76 publications

References 14 publications

Theileria parvacandidate vaccine antigens recognized by immune bovine cytotoxic T lymphocytes

Theileria parvacandidate vaccine antigens recognized by immune bovine cytotoxic T lymphocytes

Comparação de diferentes protocolos para a detecção do vírus da diarréia viral bovina por RT-PCR em grupos de sangue total e de soro sangüíneo, artificialmente contaminados

Experimental Infection and Evaluation of Airborne Transmission And Nose-To-Nose Contact of Bovine Viral Diarrhea Virus Among Weaned Piglets

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