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1.
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2.
本研究从疑似牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)感染牛的分泌物与排泄物中分离鉴定1株牛病毒性腹泻病毒,并进行E2基因序列分析。结果表明,分离株病毒命名为JN株;Reed-Muench法测定分离株病毒TCID50为10-7.5/0.1 mL;病毒中和试验结果表明,BVDV JN分离株可被BVDV阳性血清特异性中和,而不能被BVDV阴性血清中和;分离株病毒E2基因序列测序结果表明,该分离毒株属于BVDVⅠa亚型。  相似文献   

3.
The genetic characteristics, of 38 field isolates of bovine viral diarrhoea virus (BVDV) collected in 1999 from sick or healthy and persistently infected cattle of dairy farms situated in northern Italy, were investigated. A partial 5-untranslated region (5-UTR) sequence of each isolate was determined and a phylogenetic analysis was performed. All the isolates were classified as belonging to the BVDV-1 genotype and could be assigned to different BVDV-1 groups, namely BVDV-1b (n = 20), BVDV-1d (n = 6) and BVDV-1e (n = 10). Two remaining isolates could be classified as BVDV-1f and BVDV-1h, respectively. These results provided evidence for genetic heterogeneity of BVDV in Italy, and contribute to a better knowledge of the circulation of BVDV strains, and to their classification.  相似文献   

4.
Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus within the family Flaviviridae. NS3 is one of the immunodominance regions of the BVDV viral proteins. To identify the potential B-cell linear antigenic epitopes within BVDV NS3 region, serial overlapping truncations covering the whole region were expressed and purified, and screened by multistep of Western-blot. We found (1VCKKITEHERCHVNI15), (20AFFGVMPRGTTPRAPVR36), (46RRGLETGWAYTHQGGI61), (281EGDMATGITYASYGYFC297), (426YSGEDPANLRVVTSQSPYVVVATNAIESGV455) and (481FIVTGLKRMAVTVGEQA497) can be recognized by the BVDV infected bovine serum. These proteins have been confirmed by indirect enzyme-linked immunosorbent assay (I-ELISA). The results of this study might open new perspectives on the structure and antibody–antigen reaction of the non-structural proteins and may aid in the clinical application as well.  相似文献   

5.
Pestiviruses isolated from sheep and goats in India thus far have been bovine viral diarrhoea virus 1 (BVDV-1) or BVDV-2. During routine genetic typing of pestiviruses in the years 2009-10, border disease virus (BDV) was detected in eight Indian sheep of a flock showing clinical signs of BD by real time RT-PCR. All the samples yielded positive virus isolates in cell culture but were found negative by a BVDV antigen ELISA. A representative BDV isolate was characterized at genetic and antigenic level. Phylogenetic analysis carried out in 5′-UTR, Npro and E2 regions of genome typed the Indian BDV isolate as BDV-3. A more detailed analysis in Npro and entire region coding structural proteins showed that the Npro (168), C (100 aa), Erns (227 aa), E1 (195 aa) and E2 (373 aa) proteins were of size characteristic for BDV reference strain X818. Antigenic differences were evident between the BDV-3 isolate and previously reported BDV-1, BDV-5 and BDV-7 strains. Although origin of BDV-3 in India is not clear, the results reflect probable introduction through trade in sheep between India and other countries or BDV-3 may be more widely distributed. Additionally, this study suggests that for diagnosis of BDV infection, the commercial BVDV Ag-ELISA should be used with caution. This is the first identification of BDV in sheep in India which highlights the need for continued pestivirus surveillance and assessing its impact on sheep and goat production.  相似文献   

6.
Previous studies have shown that bovine viral diarrhoea virus type 1 (BVDV-1) subtype b is predominantly circulating in Indian cattle. During testing for exotic pestiviruses between 2007 and 2010, BVDV-2 was identified by real time RT-PCR in two of 1446 cattle blood samples originating from thirteen states of India. The genetic analysis of the isolated virus in 5′ UTR, Npro, entire structural genes (C, Erns, E1 and E2), nonstructural genes NS2-3 besides 3′ UTR demonstrated that the nucleotide and amino acid sequences showed highest similarity with BVDV-2. The entire 5′ and 3′ UTR consisted of 387 and 204 nucleotides, respectively, and an eight nucleotide repeat motif was found twice within the variable part of 3′ UTR that may be considered as a characteristic of BVDV-2. The phylogenetic analysis revealed that the cattle isolate and earlier reported goat BVDV-2 isolate fall into separate clades within BVDV-2a subtype. Antigenic typing with monoclonal antibodies verified the cattle isolate also as BVDV-2. In addition, cross-neutralization tests using antisera raised against Indian BVDV strains circulating in ruminants (cattle, sheep, goat and yak) displayed significant antigenic differences only between BVDV-1 and BVDV-2 strains. This is the first identification of BVDV-2 in Indian cattle that may have important implications for immunization strategies and molecular epidemiology of BVD.  相似文献   

7.
Llamas and alpacas are domesticated South American camelids (SACs) important to ancestral population in the Altiplano region, and to different communities worldwide where they have been introduced. These ungulates have shown to be susceptible to several livestock viral pathogens such as members of the Pestivirus genus, in particular Bovine Viral Diarrhea (BVDV), but there is little data available on Pestivirus infections in SACs. In this study we aimed to detect and identify Pestivirus genotypes and subgroups infecting SACs in both wild and confined environments. Samples were collected from 136 llamas and 30 alpacas from different areas in the Chilean Altiplano (wild animals), and from 22 llamas and 26 alpacas diagnosed as Pestivirus positive from the Metropolitana region in Chile (confined animals). Seroneutralization tests showed titers lower than 2 in all 166 samples from Chilean Altiplano. These samples were also negative to BVDV isolation, indicating that these animals have not been exposed to Pestivirus. After reactivation of positive samples from the Metropolitana region, the 5′ non-codifying region (5′NCR) and E2 glycoprotein were amplified by RT-PCR from the Pestivirus genome. Viral sequences were pairwise compared and phylogenetic trees were constructed. The 5′NCR analysis showed that all 12 sequenced isolates belonged to BVDV-1. Of particular interest, isolates from eight llama and two alpaca were BVDV-1j and two alpacas were BVDV-1b. In agreement with these results, E2 phylogenetic analysis rendered a similar grouping indicating that all 16 isolates belong to BVDV-1. However, the lower availability of E2 sequences determines the creation of a smaller number of sub-groups than the 5′NCR sequences. Based on the E2 sequences, the 5′NCR BVDV 1j group consisting of all the llamas and 3 alpacas are completely included in the E2 BVDV 1e group. Due to the universal availability of the 5′NCR segment, we propose the classification of these Chilean llamas and alpacas Pestivirus isolates as BVDV 1j and BVDV 1b respectively. Thus, this is the first time BVDV-1j is obtained in SACs. In addition, these results indicate Pestivirus infection in llamas and alpacas is associated with bovine population as genotypes and sub-groups are the same as those affecting Chilean livestock.  相似文献   

8.
The aim of this study was to determine the kinetics of noncytopathic bovine viral diarrhoea virus (BVDV) multiplication and synthesis of BVDV specific RNA and proteins in ovine cells (SFT-R) during a one-step growth curve. The virus titre and RNA level were determined by focus-forming assay and real time RT-PCR. The RNA synthesis was detected by Northern blot while synthesis of E2 and NS3 proteins was assayed by immunohistochemistry and Western blot. The results showed that synthesis of viral RNA is initiated at 4 h, NS3 and E2 proteins are detectable at 6-7 h and the replication cycle is complete at 10-12 h. Additionally, we provide evidence that NS2-3 protein was cleaved in ovine cells early during infection and in proliferated leukocytes of acutely infected sheep. This study showed that synthesis of BVDV RNA and proteins in ovine cells occurs at similar times as found in bovine cells.  相似文献   

9.
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10.
Antibodies against non-structural protein 3 (NS3, p80) of bovine viral diarrhoea virus (BVDV) were determined in milk from cows vaccinated with an inactivated BVDV vaccine and compared to serum antibody levels. Animals in one herd were vaccinated with an inactivated BVDV vaccine according to the standard protocol and animals from a second herd with an intensive schedule. Serum and milk samples were tested for BVDV NS3 antibodies using five commercial ELISAs. With a few exceptions, vaccination according to the standard schedule did not induce BVDV NS3-specific antibodies in serum or milk. However, after vaccination according to the intensive schedule, anti-NS3 antibodies were detected for a short time in serum and, to a lesser extent, in milk. Bulk milk was a suitable substrate for BVDV monitoring of herds vaccinated with the inactivated BVD vaccine.  相似文献   

11.
The BVDV envelope glycoprotein E(rns)/gp48 and the C terminal 79 amino acids of the capsid protein coding region were expressed in a baculovirus system and antigenically characterized. Western blot assay was used to detect recombinant E(rns) (r-E(rns)) in infected insect cells using specific monoclonal antibodies. The r-E(rns) was then used in an indirect ELISA to detect BVDV specific antibodies in a panel of 540 well-characterized sera. Results of the r-E(rns) ELISA were compared to those obtained with a commercially available competitive ELISA targeting anti-NS2/3 antibodies. A good correlation was observed between the 2 ELISA (kappa = 0.916, 95% C.I.: 0.876, 0.956). Using the commercial NS2/3 ELISA as the reference test, the relative sensitivity of r-E(rns) ELISA was 97.5% (95% C.I.: 94.3%, 99.1%) and the relative specificity was 93.9% (95% C.I.: 89.4%, 96.9%), while relative specificity was 100% (95% C.I.: 97%, 100%) using true negative sera (derived from a negative herd). All but 1 antigen positive animals (n = 36) tested negative in the r-E(rns) ELISA; among them all 22 confirmed PI animals were negative by r-E(rns) ELISA. The ability of r-E(rns) ELISA to identify cattle immunized with inactivated vaccine was also demonstrated in a small group of cattle, compared to an NS2/3 antibody ELISA. Results suggest that r-E(rns) ELISA represents an alternative test for antibody generated by natural infection or BVDV vaccination.  相似文献   

12.
Bovine viral diarrhoea virus: its effects on ovarian function in the cow   总被引：1，自引：0，他引：1  
Bovine viral diarrhoea virus (BVDV) is a major cattle pathogen responsible for a spectrum of symptoms, including reproductive failure. In this paper we investigate how BVDV interacts with the ovary. The viruses' tropism for the pre-ovulatory oocyte was studied by indirect immunohistochemistry. Two monoclonal antibodies, raised against the non-structural protein NS3 and the envelope glycoprotein E2 were used to probe cryo-sections cut from the ovaries of three persistently infected heifers. NS3 and E2 antigens were widely distributed within the ovarian stroma and follicular cells. NS3 was also localised within the proportion of oocytes. Overall 18.7% of the oocyte population had detectable levels of NS3. What is more, the proportion of antigen positive oocytes remained constant (P>0. 05) throughout the different stages of oocyte maturation.In a subsequent study seven cows were challenged with non-cytopathogenic BVDV (strain Pe515: 5x10(6) TCID(50)) to determine the oestradiol and progesterone responses to an acute infection. The sensitivity of the endogenous luteolytic mechanism was also established by analysing plasma prostaglandin F2alpha metabolite (PGFM) levels following an exogenous oxytocin (50 IU) challenge. The inoculation was given 2 days before a synchronised oestrus and was timed to ensure that viraemia occurred during the initial stage of corpora luteal development. Seven cows inoculated with non-infectious culture medium served as control animals and remained BVDV naive throughout the study. The BVDV challenge was followed by leucopenia, viraemia and sero-conversion. The virus also significantly (P<0.01) reduced plasma oestradiol levels between day 6 and day 11 post-inoculation (i.e. between day 4 and day 9 post-oestrus). However, the infection did not alter (P>0.05) progesterone secretion throughout the oestrous cycle or the plasma concentration of PGFM. These data indicate that bovine follicular cells and oocytes are permissive to BVDV at all stages of follicular development. They also show that a transient fall in oestradiol secretion may accompany an acute infection. In conclusion, this work has identified two potential routes through which BVDV can reduce fertility in the cow, namely impairment of oocyte quality and disruption of gonadal steroidogenesis.  相似文献   

13.
The immunodominant NS3 (p80) protein of the pestivirus bovine viral diarrhea virus (BVDV) functions as a serine protease and a RNA helicase. To identify antigenic domains of the BVDV NS3, a panel of monoclonal antibodies (mAbs) was tested against fragments of the protein expressed in E. coli. Two large overlapping NS3 fragments, A (amino acids [aa] 1-434) and B (aa 368-683) which together contain all NS3 sequences, were used to screen mAbs for reactivity. Two mAbs, 21.5.8 and 1.11.3, were reactive to fragment A (in ELISA only) and one mAb, 20.10.6, was reactive to fragment B (in ELISA and Western blotting). Further mapping demonstrated that the smallest fragment mAbs 21.5.8 and 1.11.3 bound to was comprised of aa 205-369 (domain A). In Western blotting, the smallest fragment reactive with mAb 20.10.6 was comprised of aa 368-549 (domain B). However, in indirect ELISA, mAb 20.10.6 also demonstrated high reactivity to a smaller fragment comprising aa 368-512 (domain B'). This indicated that the epitope of mAb 20.10.6 was conformational and not linear. Blocking ELISAs using these mAbs and type 1 and type 2 BVDV antisera demonstrated that an immunodominant region of the NS3 protein in cattle is defined by aa 205-549.  相似文献   

14.
Phylogenetic analyses of bovine viral diarrhea virus (BVDV) were performed based on the nucleotide sequences of the 5' untranslated region (5'-UTR) and E2-coding gene. Thirty-six BVDV detected from naturally infected cattle in the northern region of Japan were divided into three genotypes, BVDV1a, BVDV1b and BVDV2, in a 5'-UTR phylogenetic tree. In a phylogenetic tree constructed from the E2-coding gene, BVDV1c was identified and the viruses classified in BVDV1c were included in BVDV1a in the 5'-UTR phylogenetic tree. Moreover, BVDV1a and BVDV1b in the E2-phylogenetic tree clustered closer together than in the 5'-UTR tree. These results suggested that phylogenetic analysis of the E2 gene was more useful for identification of subgenotypes within BVDV1.  相似文献   

15.
To develop a vaccine to prevent diseases caused by Mycobacterium tuberculosis and bovine viral diarrhea virus (BVDV) simultaneously, recombinant Bacillus Calmette–Guerin (rBCG) vaccines expressing different regions of the BVDV E2 gene were constructed. Using DNASTAR 6.0 software, potential antigenic epitopes were predicted, and six regions were chosen to generate recombinant plasmids with the pMV361 vector (pMV361-E2-1, pMV361-E2-2, pMV361-E2-3, pMV361-E2-4, pMV361-E2-5 and pMV361-E2-6, respectively). The recombinant plasmids were transformed into BCG, and protein expression was thermally induced at 45 °C. Mice were immunized with 5 × 106 CFU/200 µL of each rBCG strain. Compared with other groups, BVDV E2 specific antibody titers were higher in mice immunized with rBCG-E2-6. Ratios and numbers of CD4+, CD8+ and IL-12 expressing spleen lymphocytes of the rBCG-E2-6 group also were higher than those of other groups. Thus, the rBCG-E2-6 vaccine showed the highest immunogenicity of all groups based on the humoral and cellular responses to vaccination.  相似文献   

16.
A study to evaluate BVDV-prevalence, recent -contact and -vaccine use in dairy herds in the "Entre Douro e Minho" (EDM) region in North Portugal was carried out in 124 dairy herds in 2003. Herds were visited to ascertain BVDV-vaccine use and to collect a bulk tank milk (BTM) sample and serum from 1268 cattle to analyse BVDV-antibodies using an NS2-3 ELISA. Fifty-three percent of farmers used inactivated BVDV-vaccines whilst the remaining farmers were not presently using BVDV-vaccines. BMT-antibody results included 35% positives, 25% negative and 39% inconclusive, and were similar in vaccinated and non-vaccinated herds (p>0.05) and allowed estimating a 10% BVDV herd-prevalence from prior knowledge of the relationship between BMT-antibody results and probability of PI cattle in the herd. Overall individual seroprevalence was 27% and was 23% in non-vaccinated and 36% in vaccinated cattle (p<0.05). Contact of the herd with BVDV was assessed according to seroprevalence in young and adult cattle in the herd and it was estimated that 35% of herds were infected or had recent contact with BVDV, 40% were not infected and did not have recent contact with BVDV and the BVDV-infection and -contact status of remaining herds was undetermined. The results from this study indicate BVDV is endemic and BVDV-vaccines are widespread in the dairy-cattle population in EDM region in Portugal.  相似文献   

17.
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18.
Immunosuppressive effects of an intranasal challenge with non-cytopathic bovine viral diarrhea virus (BVDV) 2a (strain 1373) were assessed through acquired and innate immune system responses to ovalbumin (OVA). Concurrent BVDV infection was hypothesized to delay and reduce the humoral response to ovalbumin (administered on days 3 and 15 post-inoculation). Infected animals followed the expected clinical course. BVDV titers, and anti-BVDV antibodies confirmed the course of infection and were not affected by the administration of OVA. Both the T-helper (CD4+) and B-cell (CD20+) compartments were significantly (P < 0.05) reduced in infected animals, while the gamma-delta T-cell population (Workshop cluster 1+, WC1+) decreased slightly in numbers. Infection with BVDV delayed the increase in OVA IgG by approximately 3 d from day 12 through day 21 post-inoculation. Between days 25 and 37 post-inoculation following BVDV infection the IgM concentration in the BVDV− group decreased while the OVA IgM titer still was rising in the BVDV+ animals. Thus, active BVDV infection delays IgM and IgG responses to a novel, non-infectious antigen.  相似文献   

19.
1. Two experiments were conducted to quantify heat stress (HS) acclimation effects on heat production (H), evaporative heat loss (E), sensible heat loss (S) and change in heat content (HC) of 24 food‐deprived and precision fed broilers.

2. In experiment 1, heat stressed fasted HS acclimated birds (group 1) exhibited lower H (22–6 v. 25–5 kJ/kgW0.66 per h), E (7.5 v. 8.8 kJ/kgW0.66 per h), core body temperature (41.8 v. 42.4

C) and respiration rate (129 v. 160 breaths/min) than nonacclimated controls (group 2).

3. In contrast to the first experiment, precision fed HS acclimated birds (group 1) exhibited a higher H (29.3 v. 28.0 kJ/kgW0.66 per h) and E (10.5 v. 96 kJ/kgW0.66 per h) during HS and elevated H during thermoneutral periods than their non‐acclimated counterparts (group 2). The elevated H became more pronounced with each successive HS exposure.

4. These results indicate that H increases when broilers are fed, that broilers preferentially dissipate heat as S when environmental conditions permit, and that food and/or energy consumption level markedly influences the bird's capacity to exhibit a HS acclimation response, and in fact has the ability to mask it.  相似文献   


20.
Semen samples, collected from bulls pesistently infected with bovine viral diarrhea virus (BVDV) and containing BVDV (titer 105 - 106TCID50/ml), were subjected to sperm separation procedures (washing, swim up, Percoll gradient, glass wool filtration, glass beads filtration) that are commonly used prior to in vitro fertilization (IVF) to determine if these procedures would yield spermatozoa free from BVDV. The final sperm pellets from frozen and fresh ejaculates were tested for the presence of BVDV by the immunoperoxidase technique; all tests were positive for BVDV in the range of 103 - 104TCID50/ml). The study shows that when semen containing BVDV at the level of 105 - 106TCID50/ml) is used for IVF, the virus is not completely removed by any of the simple physical methods commonly used to prepare sperm for IVF. Inhalt: Feldversuch das bovine Diarrhoe Virus (BVDV) aus Bullensperma durch Swim up oder andere Trennverfahren in Verbindung mit der In vitro Fertilisation zu entfernen Spermaproben, die von Bullen gewonnen wurden, welche dauerhaft mit dem Virus der bovinen Virus-Diarrhoe (BVDV) infiziert waren und einen Titer zwischen 105 - 106TCID50/ml enthielten, wurden verschiedenen Trennverfahren unterzogen (Waschen, swim up, Percoll gradient, Glaswollenfiltration, Glaskugelfiltration). Diese Verfahren werden üblicherweise für die Vorbereitung zur In vitro Befruchtung verwendet, und es sollte geprüft werden, ob diese Verfahren auch das Sperma von dem BVD-Virus befreien können. Das endgültige Spermapellet von gefrorenen/aufgetauten und frischen Ejakulaten wurde in Gegenwart des BVD-Virus und mit Hilfe der immunoperoxidase Technik getestet. Alle Testergebnisse waren positiv für BVD-Virus in einem Bereich von 103 - 104 TCID50/ml. Die Studie zeigt, daß Sperma, wenn es 105-106TCID50/ml des Virus enhält, nicht vollständig mit den für die IVF üblichen, einfachen physikalischen Methoden von dem Virus befeit werden kann.  相似文献   

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