scholarly journals Possibility for use of RT-PCR technique in establishing presence of bovine viral diarrhea virus in sperm of breeding bulls

2005 ◽  
Vol 59 (3-4) ◽  
pp. 371-381
Author(s):  
Tamas Petrovic ◽  
Sava Lazic ◽  
Milovan Jovicin ◽  
Bosiljka Djuricic
Keyword(s):  
Cell Culture ◽  
Microtiter Plate ◽  
Bovine Viral Diarrhea ◽  
Rt Pcr ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Culture Cells ◽  
Three Samples ◽  
Pcr Method ◽  
Set Up

The bovine viral diarrhea (BVD) virus is a significant health-economic pathogen in cattle which can be excreted and spread also through sperm of persistently or acutely infected bulls. Native sperm of 6 bulls, found to be negative to the BVD virus by isolating the virus and using the RT-PCR method, was experimentally infected with a tenfold dilution of the non-cytopathogen 22146 strain of the BVD virus with a titer of 105,5. This way, dilutions of the BVD virus from 10-1 to 10-6 (5 x 104 TCID/50 do 0,5 TCID/50 in 0.1 ml native sperm were obtained. From sperm infected in this way, the virus was reisolated on FTB cell culture in a microtiter plate with 96 pools in which each sample of the infected sperm was set up in three samples, and each of them was titrated to a dilution of 1:2 to 1:256. The presence of the BVD virus was proven using the technique of fluorescent antibodies in a second blind passage on FTB culture cells. For cell culture an extremely toxic effect of native sperm to a dilution of 1:64 was established. The BVD virus was reisolated from sperm in all three sperm samples with 5 x 104, 5 x 103 i 5 x 102 TCID/50, and it was not reisolated from sperm with 50, with 5, and with 0.5 TCID/50 BVD virus in 0.1 ml native sperm. At the same time, the presence of the BVD viral genome was proved using the RT-PCR method in the same samples of artificially infected native sperm of bulls. A positive re suit was established in native sperm with 5 x 104, 5 x 103, 5 x 102 and 50 TCID/50 BVD virus n 0.1 ml native sperm. The experiment proved that the RT-PCR method has advantages over the isolation of the BVD virus from samples of native sperm of bulls. These are: shortterm investigations (1 to 2 days) and greater sensitivity (10 times bigger than the isolation of the virus). The isolation of the virus takes at least 10 days, and its greater sensitivity is primarily a result of the cyrotoxic effect of native sperm of bulls on cell culture.

scholarly journals Circulation of bovine viral diarrhea virus – 1 (BVDV-1) in dairy cattle and buffalo farms in Ismailia Province, Egypt

2015 ◽  
Vol 9 (12) ◽  
pp. 1331-1337 ◽  
Author(s):  
Mohamed Ahmed Soltan ◽  
Rebecca P Wilkes ◽  
Mohamed Nagy Elsheery ◽  
Mahmoud Mohy Elhaig ◽  
Matthhew C Riley ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Dairy Cattle ◽  
Real Time ◽  
Bovine Viral Diarrhea ◽  
Rt Pcr ◽  
Cattle Farm ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Three Samples ◽  
Pcr Products

Introduction: Bovine viral diarrhea (BVD) is one of the most economically significant diseases in the bovine industry causing losses due to diarrhea, reproductive disorders, immunosuppression and mortalities. The aim of our investigation was to detect and subtype BVDV from calves on two dairy cattle and two buffalo farms in Ismailia province, Egypt as an indicator of BVDV infection status in the province. Methodology: A total of 298 blood samples were collected and tested using an optimized one-step, real-time multiplex Taqman-based RT-PCR. All the positive samples by the multiplex real-time RT-PCR were tested using conventional RT-PCR to amplify multiple areas of the genome for further phylogenetic analysis and subtyping. Results: Thirty one (10.4%) of the tested samples were positive for BVDV-1. Only three samples, all from a single dairy cattle farm, had enough viral RNA to be amplified by RT-PCR. The PCR products were sequenced and phylogenetic analysis revealed detection of BVDV-1b. The detected strain is closely related to worldwide BVDV-1b strains, making it difficult to trace its origin. Nucleotide and amino acid alignments of the E2 glycoprotein region of the detected strain with other BVDV-1b strains showed high divergence, with identity ranging from 81.3% to 93.6% and 85.3% to 93.6%, respectively. Conclusion: To our knowledge, this is the first report describing the circulation of BVDV-1b in Egyptian dairy cattle populations.

scholarly journals Laboratory tests of the VetMAX BVDV Screening RT-PCR test system for detecting the genome of the bovine viral diarrhea virus by ThermoFisher

2020 ◽  
pp. 28-34
Author(s):  
A. S. Preobrazhenskaya ◽  
Z. S. Devrishova ◽  
T. P. Lobova ◽  
V. V. Mikhailova ◽  
A. A. Varentsova ◽  
...  
Keyword(s):  
Cell Culture ◽  
Coefficient Of Variation ◽  
Reference Strain ◽  
Test System ◽  
Bovine Viral Diarrhea ◽  
Rt Pcr ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus ◽  
Pcr Test

Relevance. Currently, BVD is widespread in almost all countries of the world with intensive livestock farming. The special relevance of the problem lies in the large economic damage that consists of a decrease in milk yield during the disease, the death of young animals from serous pneumonia, the loss of live weight gain in young animals, the loss of productivity and reproduction of animals and, as a result, abortions and stillbirths, the birth of non-viable calves, as well as preventive, quarantine and liquidation measures. An important link in preventing the spread of viral diarrhea in cattle remains the rapid conduct of laboratory research. One of the most technologically advanced diagnostic methods is real-time polymerase chain reaction.Methods. The diagnostic significance of the VetMAX BVDV Screening (Thermo Fisher) RT-PCR test system was assessed by sensitivity, specificity, and precision in conditions of repeatability and reproducibility. To determine repeatability, 5 samples of PT-80 cell culture infected with the reference strain "Oregon 24" and 7 samples of PT-80 cell culture infected with Ressa isolate were studied in tenfold dilutions from 10-1 to 10-7, by one operator in three parallel studies on the same equipment. To determine reproducibility and sensitivity, 5 samples of PT-80 cell culture infected with the reference strain "Oregon 24" and 7 samples of PT-80 cell culture infected with «Ressa» isolate were studied in ten-fold dilutions from 10-1 to 10-7 , by three operators on different days on the same equipment.To determine the specificity, studies were conducted on 3 samples that did not contain the virus of viral diarrhea — bovine adenovirus type 1 strain Bovina — 10, rhinotracheitis virus of cattle strain "Orenburg" and parainfluenza virus 3 strain ZKSM.Results. After mathematical processing of the results of PCR formulation for the assessment of reproducibility, the following results were obtained: the coefficient of variation (CV) for the VetMAX BVDV Screening (Thermo Fisher) test system was from 1.0–4.0 %; the Coefficient of variation (CV) for the assessment of repeatability was 1–3 %. The specificity of the test systems VetMAX BVDV Screening (Thermo Fisher) was 100%. The VetMAX BVDV Screening (Thermo Fisher) test system is sensitive to detecting the genome of the bovine viral diarrhea virus. So when setting up PCR samples of the Ressa isolate in the 10-7 dilution, the CT values were determined at 38.18–39.24 cycles, and the reference VD virus "Oregon 24" in the 10–5 dilution at 37.85–39.45 amplification cycles.

scholarly journals Is contamination of bovine-sourced material with bovine viral diarrhea virus still a problem in countries with ongoing eradication campaigns?

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Aleksandra Antos ◽  
Jerzy Rola ◽  
Michał Bednarski ◽  
Michał Konrad Krzysiak ◽  
Julia Kęsik-Maliszewska ◽  
...  
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Genetic Material ◽  
Elisa Test ◽  
Bovine Viral Diarrhea ◽  
Rt Pcr ◽  
Serum Samples ◽  
Viral Pathogens ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

AbstractIn this report, we describe the detection of bovine viral diarrhea virus (BVDV) contamination in commercial animal-derived sera and vaccines against animal viral pathogens on the market in Poland. Antibodies against BVDV were detected in 4/45 sera samples (8.9%) using an ELISA test. The presence of BVDV antigen using ELISA was found using ELISA in 3/45 serum samples (6.6%) and 18/172 vaccine samples (10.5%). An RT-PCR was conducted using primers targeting two genome regions, the five prime untranslated region (5’UTR) and N-terminal protease (Npro). BVDV RNA was detected in 33/45 (73.3%) of sera, and 11/172 samples (6.4%) of collected vaccines, of which one vaccine did not declare BVDV strain in its composition. A single serum showed the presence of an infectious virus and only one was contaminated with all 3 species of BVDV. The most frequent species in sera was BVDV-3 (75.5%), whereas in vaccines only BVDV-1 was identified. Sequence analysis showed that the tested commercial sera and one vaccine were contaminated by six genotypes of BVDV: -1a, -1b, -1c, -1d, -2a, and -3. Identification of BVDV and its genetic material in animal-derived products is important due to the possibility of pestivirus transmission as well as the chance of falsifying the results of a diagnostic test. It also demonstrates the necessity of rigorous monitoring of the bioproducts used in the laboratory and industry level.

270 EFFICIENCY OF SPERM SEPARATORY TECHNIQUES FOR BOVINE VIRAL DIARRHEA VIRUS REMOVAL FROM FROZEN BOVINE SEMEN SAMPLES

2010 ◽  
Vol 22 (1) ◽  
pp. 292
Author(s):  
A. G. Galuppo ◽  
L. L. Almeida ◽  
B. Meyrer ◽  
N. S. Arruda ◽  
O. Sicco ◽  
...  
Keyword(s):  
Cell Culture ◽  
Statistical Analysis ◽  
Virus Isolation ◽  
Sperm Concentration ◽  
Bovine Viral Diarrhea ◽  
Percoll Gradient ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus ◽  
Sperm Separation

It has been recognized that systemic and local infections of the reproductive tract, as well as the inadvertent introduction of microorganisms during processing, may potentially contribute to the contamination of semen (Bielanski 2007 Theriogenology 68, 1-22). A number of viral pathogens have been identified in association with semen, such as bovine viral diarrhea virus (BVDV). The aim of the study was evaluate the capacity of sperm separation procedures Percoll gradient and swim-up, as proposed by Bielanski et al. (1992 Reprod. Domest. Anim. 27, 303-306) and also a combination of swim-up and Percoll, to reduce or eliminate BVDV from experimentally infected semen samples. We used frozen semen straws (0.5 mL) and the virus sample was cytopathic type BVDV (106.68 TCID50mL-1). The experimental infection was performed immediately after semen thawing in a 1 : 1 volume proportion semen/viral suspension. The experimental groups were Percoll control (Pc); swim-up control (Sc); Swim up followed by Percoll control (SPc); Percoll and virus (Pv); swim-up and virus (Sv); and swim-up followed by a Percoll and virus (SPv). The Percoll gradient was prepared with 0.5 mL 90% under 0.5 mL 45% Percoll solution, centrifuged at 200 × g for 10 min; the pellet was collected and washed with Sperm-TALP. The swim-up was prepared with 0.3 mL of semen under 1 mL of Sperm-TALP. After 60 min of incubation at 38.5°C, 0.8 mL of the supernatant was collected and centrifuged at 200 × g for 10 min. The combination of techniques was prepared first with swim-up, and then the swim-up pellet was submitted to a Percoll, as previous described. The final pellet of each separation procedure was evaluated for sperm motility, sperm concentration, and virus isolation in cell culture. We used Student’s t-test for statistical analysis (P < 0.05). Preliminary results showed no significant statistical difference between groups of the same sperm separation techniques when analyzing sperm motility (Pc = 90%; Sc = 90%; SPc = 90%; Pv = 80%; Sv = 90%, SPv = 90%) and sperm concentration (million mL-1; Pc = 28; Sc = 11; SPc = 3; Pv = 18, Sv = 11, SPv = 4). The virus isolation in cell culture presented the following results (3 replicates): Pv = 103.92a, 103.8b, 102.46c TCID50 mL-1; Sv = 103.62, 103.13, 102 TCID50 mL-1; and SPv = undetectedd, undetectede, 102.92f TCID50mL-1 (different letters for means indicate statistical significance). The control groups of all techniques presented a cytotoxic effect probably because of the contact with sperm, Percoll, and sperm-TALP residues in the analyzed sample. Because of those cytotoxc effects in the cell culture, molecular biology techniques such as RT-PCR will be used as a complementary test to confirm the presence of BVDV in the samples. A large number of repetitions will be performed for a better statistical analysis. However, these preliminary results showed that the combination of swim-up followed by Percoll promoted a significant reduction in the number of viral particles in the semen samples compared with Percoll alone, considering that in 2 of the 3 repetitions it was not possible to detect the virus in cell culture. CNPq, CAPES.

scholarly journals Multiplex real-time RT-PCR assay for bovine viral diarrhea virus type 1, type 2 and HoBi-like pestivirus

2016 ◽  
Vol 229 ◽  
pp. 1-7 ◽  
Author(s):  
Viviana Mari ◽  
Michele Losurdo ◽  
Maria Stella Lucente ◽  
Eleonora Lorusso ◽  
Gabriella Elia ◽  
...  
Keyword(s):  
Real Time ◽  
Bovine Viral Diarrhea Virus ◽  
Bovine Viral Diarrhea ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

scholarly journals Padronização da técnica de imunoperoxidase para detecção do vírus da diarréia bovina a vírus em cultura de células: Standardization of immunoperoxidase test to detection bovine viral diarrhea virus in cell culture

2002 ◽  
Vol 54 (6) ◽  
pp. 568-574 ◽  
Author(s):  
G.I. Andrade ◽  
Z.I.P. Lobato ◽  
R.C. Leite ◽  
E.F. Barbosa-Stancioli
Keyword(s):  
Cell Culture ◽  
Bovine Viral Diarrhea Virus ◽  
Bovine Viral Diarrhea ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

Este estudo teve como objetivo a padronização do ensaio de imunoperoxidase em monocamada de células (IPM) para o diagnóstico etiológico da diarréia bovina a vírus (DBV). O teste foi padronizado em monocamada de cultivo primário de pulmão fetal bovino (PFB) inoculada com as amostras clássicas, citopatogênica (CP) e não citopatogênica (NCP), do vírus da DBV e testado em amostras biológicas suspeitas processadas no teste clássico de isolamento viral (IV). O método de IPM identificou o vírus da DBV, apresentando melhores resultados com a utilização do calor como agente fixador, a soroalbumina bovina a 4% em PBS como bloqueador e a revelação com o cromógeno 3-amino-9-etil-carbazol (AEC). Como anticorpos primários, tanto o anticorpo policlonal como o monoclonal forneceram bons resultados.

scholarly journals First Evidence of Bovine Viral Diarrhea Virus Infection in Wild Boars

2018 ◽  
Vol 44 (1) ◽  
pp. 5 ◽  
Author(s):  
Matheus Nunes Weber ◽  
Eloisa Helena Moreira Pino ◽  
Carine Kunzler Souza ◽  
Ana Cristina Sbaraini Mósena ◽  
José Paulo Hiroji Sato ◽  
...  
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Disease Transmission ◽  
Animal Species ◽  
Bovine Viral Diarrhea ◽  
Southern Brazil ◽  
Rt Pcr ◽  
Wild Boars ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

Background: The farming of wild boars has growing due to the interest of the human consumption of this exotic meat. Such a development may pose an increased risk of disease transmission between boars and domestic animals. The wild boar population has increased in South America in the last years due the absence of predator causing economic losses due to direct damage to crops and risk of disease transmission. The genus Pestivirus within the family Flaviviridae are composed by four recognized species by the International Committee on the Taxonomy of Viruses (ICTV): classical swine fever virus (CSFV), border disease virus (BDV), bovine viral diarrhea virus type 1 (BVDV-1) and 2 (BVDV-2). Other putative species denoted as atypical pesitiviruses have been reported as ‘HoBi’-like virus, giraffe pestivirus, Bungowannah pestivirus, Pronghorn antelope virus, atypical porcine pestivirus (APPV), Norwegian rat pestivirus (NrPV) and Rhinolophus affinis bat pestivirus (RaPestV-1). CSFV is commonly detected in wild boars, but despite positive serology, bovine viral diarrhea virus (BVDV) was never detected in this animal species. Thereby, the present communication describes the first detection of BVDV in the lungs of captive boars using RT-PCR and DNA sequencing.Materials, Methods & Results: Forty lung samples from farmed wild boars were collected after slaughter in a commercial abattoir. The organs were crushed separately, centrifuged, and the supernatant was stored for further analysis. The total RNA was isolated using a phenol-based protocol and RT-PCR protocol that amplified 118 bp of 5’ untranslated region (5’UTR) was carried out. One out 40 samples resulted positive. The positive sample had partial fragments of 5’UTR and N terminal autoprotease (Npro) sequenced and analyzed. The strain LV Java/2012 presented 99% of identity in 5’UTR and 98% in Npro region with a BVDV-2 previously reported in bovines in Southern Brazil. In both 5’UTR and Npro phylogenetic analysis, the strain LV Java/2015 clustered with BVDV-2 strains and was most closely related to subtype 2b identified in bovines in Southern Brazil grouping in the same terminal node.Discussion: Wild boars are commonly associated to pathogen transmission to domestic animals. This animal species is considered a reservoir of the pestivirus CSFV and important keys in CSFV control and eradication programs in Europe. Despite indirect presence of BVDV was reported in wild boars by serology tests, the direct detection of the viral agent was never reported. The present study showed the presence of BVDV-2 genomic segments obtained by RT-PCR followed by DNA sequencing in captive wild boars. The reported data suggests a possible importance of this animal species in the epidemiology of ruminant pestiviruses which could interfere in control and eradication programs of these important pathogens for cattle worldwide. The strain LV Java/2012 was closely related to BVDV-2b and presented highest identity with a strain detected in cattle from Southern Brazil. This data suggests that wild boars and bovines could be sharing this pathogen due the similarity of the strains and that both were reported in the same region. It can lead to need of inclusion of wild swines in BVDV control programs since boars can circulate between different regions and carry this pathogen to different cattle herds. The present study reported the first molecular evidence of BVDV in wild boars in the literature. The data generated herein suggests a possible importance of boars in the epidemiology of ruminant pestiviruses.

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