我国牛病毒性腹泻流行现状与防控策略

我国牛病毒性腹泻病(Bovine viral diarrhea,BVD)的流行比较复杂,其病原BVDV (BVDV-1和BVDV-2)不仅仅局限于已知易感动物牛群感染,其他动物种群中感染BVDV-1和BVDV-2的现象也值得注意,如猪群中BVDV感染很大程度上混淆了猪瘟等病原的监测,从而加剧病程发展。牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)可致持续感染(Persistent infection,PI),这一特性导致该病的净化面临巨大困难,对整个养殖场的健康发展形成了严峻威胁。BVDV抗原变异速率非常快,目前BVDV-1已有22个亚型,BVDV-2有4个亚型,鉴于病原在自然界的适应和演进特性,对该病的防控措施迟后其病原的变异速度。因此,定期摸清BVDV-1和BVDV-2在我国的流行现状是实施疫病净化的第一步和关键步骤,进一步借鉴国外BVD净化成功经验,综合考虑我国国情,采取适宜的防控策略,逐步净化该病原感染,有助于促进国内养殖业的健康发展。     

四川省部分牛病毒性腹泻/粘膜病血清中和抗体检测

应用细胞中和试验对四川黄牛、水牛、奶牛、牦牛共243份血清进行病毒性腹泻/粘膜病(BVD/MD)的中和抗体检测,结果显示,水牛阳性率较高,达50.0%(21/42),其次为牦牛,达37.5%(15/40),奶牛达26.6%(38/143),黄牛最低,为22.2%(4/18).表明该病有蔓延和扩大的趋势,值得高度重视.     

Entry of bovine viral diarrhea virus into ovine cells occurs through clathrin-dependent endocytosis and low pH-dependent fusion

Although mechanisms of bovine viral diarrhea virus (BVDV) entry into bovine cells have been elucidated, little is known concerning pestivirus entry and receptor usage in ovine cells. In this study, we determined the entry mechanisms of BVDV-1 and BVDV-2 in sheep fetal thymus cells. Both BVDV-1 and BVDV-2 infections were inhibited completely by chlorpromazine, β-methyl cyclodextrin, sucrose, bafilomycin A1, chloroquine, and ammonium chloride. Simultaneous presence of reducing agent and low pH resulted in marked loss of BVDV infectivity. Moreover, BVDV was unable to fuse with ovine cell membrane by the presence of reducing agent or low pH alone, while combination of both led to fusion at low efficiency. Furthermore, sheep fetal thymus cells acutely infected with BVDV-1 or BVDV-2 were found protected from heterologous BVDV infection. Taken together, our results showed for the first time that entry of both BVDV-1 and BVDV-2 into ovine cells occurred through clathrin-dependent endocytosis, endosomal acidification, and low pH-dependent fusion following an activation step, besides suggesting the involvement of a common ovine cellular receptor during attachment and entry.     

Giraffe strain of pestivirus: its taxonomic status based on the 5'-untranslated region

The 5'-untranslated region (5'-UTR) of the 'Giraffe' strain of pestivirus was sequenced for comparison with those of other pestiviruses from cattle, sheep, goats, and swine. A phylogenetic tree constructed with these strains suggested that the 'Giraffe' strain was allocated to a new taxon. This observation was also confirmed by a newly proposed method based on palindromic nucleotide substitutions (PNS) at the three variable regions in the 5'-UTR. Other reported pestivirus strains isolated from deer were assigned as bovine viral disease virus (BVDV)-1 according to the PNS as well as phylogenetic analysis, suggesting that BVDV-1 strains can cross-infect deer as well as cattle, sheep, goats, and swine, and that wild deer may serve as a reservoir of BVDV-1. We also identified the genovar of a deer isolate, SH9/11, as BVDV-1c by the PNS method.     

Detection of copy number variations and their effects in Chinese bulls

Background

Copy number variations (CNVs) are a main source of genomic structural variations underlying animal evolution and production traits. Here, with one pure-blooded Angus bull as reference, we describe a genome-wide analysis of CNVs based on comparative genomic hybridization arrays in 29 Chinese domesticated bulls and examined their effects on gene expression and cattle growth traits.

Results

We identified 486 copy number variable regions (CNVRs), covering 2.45% of the bovine genome, in 24 taurine (Bos taurus), together with 161 ones in 2 yaks (Bos grunniens) and 163 ones in 3 buffaloes (Bubalus bubalis). Totally, we discovered 605 integrated CNVRs, with more “loss” events than both “gain” and “both” ones, and clearly clustered them into three cattle groups. Interestingly, we confirmed their uneven distributions across chromosomes, and the differences of mitochondrion DNA copy number (gain: taurine, loss: yak & buffalo). Furthermore, we confirmed approximately 41.8% (253/605) and 70.6% (427/605) CNVRs span cattle genes and quantitative trait loci (QTLs), respectively. Finally, we confirmed 6 CNVRs in 9 chosen ones by using quantitative PCR, and further demonstrated that CNVR22 had significantly negative effects on expression of PLA2G2D gene, and both CNVR22 and CNVR310 were associated with body measurements in Chinese cattle, suggesting their key effects on gene expression and cattle traits.

Conclusions

The results advanced our understanding of CNV as an important genomic structural variation in taurine, yak and buffalo. This study provides a highly valuable resource for Chinese cattle’s evolution and breeding researches.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-480) contains supplementary material, which is available to authorized users.     

Function of bovine CD46 as a cellular receptor for bovine viral diarrhea virus is determined by complement control protein 1

The pestivirus bovine viral diarrhea virus (BVDV) was shown to bind to the bovine CD46 molecule, which subsequently promotes entry of the virus. To assess the receptor usage of BVDV type 1 (BVDV-1) and BVDV-2, 30 BVDV isolates including clinical samples were assayed for their sensitivity to anti-CD46 antibodies. With a single exception the infectivity of all tested strains of BVDV-1 and BVDV-2 was inhibited by anti-CD46 antibodies, which indicates the general usage of CD46 as a BVDV receptor. Molecular analysis of the interaction between CD46 and the BVD virion was performed by mapping the virus binding site on the CD46 molecule. Single complement control protein modules (CCPs) within the bovine CD46 were either deleted or replaced by analogous CCPs of porcine CD46, which does not bind BVDV. While the epitopes recognized by anti-CD46 monoclonal antibodies which block BVDV infection were attributed to CCP1 and CCP2, in functional assays only CCP1 turned out to be essential for BVDV binding and infection. Within CCP1 two short peptides on antiparallel beta strands were identified as crucial for the binding of BVDV. Exchanges of these two peptide sequences were sufficient for a loss of function in bovine CD46 as well as a gain of function in porcine CD46. Determination of the size constraints of CD46 revealed that a minimum length of four CCPs is essential for receptor function. An increase of the distance between the virus binding domain and the plasma membrane by insertion of one to six CCPs of bovine C4 binding protein exhibited only a minor influence on susceptibility to BVDV.     

Molecular Cloning of Bovine FABGL Gene and Its Effects on Bovine Bioeconomic Traits

The complete CDS sequence of the bovine FABGL gene was determined by homology cloning approach combined with RT-PCR and 3′- and 5′-RACE. The results of sequence analysis and bioinformatics study showed that this cDNA contained 994 nucleotides, with a 780 bp open reading frame (ORF) flanked by a 16 bp 5′-UTR (incompletely) and a 198 bp 3′-UTR. The deduced amino acid sequence (260 AA) shows 88% identity with the corresponding sequence in humans. Two single nucleotide substitutions, one located in intron 5 (I5) at position 1 065 bp (Y = C/T) (GenBank: DQ409814) and the other in intron 8 (I8) at position 1 792 bp (R = A/G), were detected using the PCR-SSCP method. Analysis of the allele frequencies of the two polymorphic sites in three different cattle breeds (Angus, Hereford, and Simmental) with different genotypes showed large differences: in locus I8, cattle with the GG genotype showed higher beef performance index (BPI) (4.283 ± 0.475 kg/cm) in comparison with cattle with the AA genotype (4.008 ± 0.465 kg/cm) (P = 0.01). Regarding the ribeye area, cattle with the GG genotype showed significantly higher ribeye area (73.380 ± 13.005 cm²) compared with cattle with the AA genotype (67.744 ± 12.777 cm²) (p = 0.05). In locus I5, some associations for the average daily gain (ADG) were found at the significance level of 0.01 between three different genotypes (CC, CT, TT): cattle with the TT genotype showed the highest ADG (0.652 ± 0.330 kg/d), whereas cattle with the CC genotype showed the lowest ADG value (0.421 ± 0.178 kg/d).     

Bovine and buffalo in vitro embryo production using oocytes derived from abattoir ovaries or collected by transvaginal follicle aspiration

This study was undertaken in order to evaluate the effect of oocyte source (live animals and abattoir ovaries) on subsequent embryo development in buffalo (Bubalus bubalis). Cow ovaries were also collected as oocyte donors for in vitro embryo production (IVEP).Three hundred thirty-eight oocytes were recovered by ovum pick up (OPU, Group A) from 8 pluriparous buffalo cows, while 1127 and 1457 oocytes were aspirated, respectively, from buffalo (Group B) and bovine (Group C) slaughterhouse ovaries. Cumulus enclosed oocytes (COCs) suitable for IVEP were in vitro matured (IVM), fertilized (IVF) and cultured (IVC) to the tight morula (Tm) and blastocyst (Bl) stage.Within buffalo species Group A had a higher Bl yield (29.7 % versus 19.9%; P<0.05) and a lower proportion of embryos arrested at Tm stage (11.1% versus 22.3%; P<0.05) than Group B.Within slaughterhouse groups cattle oocytes had a higher cleavage rate (83.9% versus 64.8%; P<0.05) and yielded 49.2% more blastocysts than buffalo. However, when data are related to the total number of cleaved oocytes, only 13.7% more blastocysts were produced in cattle than in buffalo.In conclusion, in buffalo species the source of oocytes significantly affected post-fertilization embryo development, as demonstrated by the higher Bl yields derived from OPU-derived oocytes.A higher overall IVEP efficiency, mainly related to the higher cleavage rate, was recorded in cattle compared with buffalo when ovaries from an abattoir were used as oocyte donors.     

The amino-terminal domain of bovine viral diarrhea virus Npro protein is necessary for alpha/beta interferon antagonism

The alpha/beta interferon (IFN-alpha/beta) system is the first line of defense against viral infection and a critical link between the innate and adaptive immune responses. IFN-alpha/beta secretion is the hallmark of cellular responses to acute RNA virus infections. As part of their survival strategy, many viruses have evolved mechanisms to counteract the host IFN-alpha/beta response. Bovine viral diarrhea virus (BVDV) (genus Pestivirus) was reported to trigger interferon production in infected cultured cells under certain circumstances or to suppress it under others. Our studies with various cultured fibroblasts and epithelial bovine cells indicated that cytopathic (cp) BVDV induces IFN-alpha/beta very inefficiently. Using a set of engineered cp BVDVs expressing mutant Npro and appropriate controls, we found that the IFN-alpha/beta response to infection was dependent on Npro expression and independent of viral replication efficiency. In order to investigate whether the protease activity of Npro is required for IFN-alpha/beta antagonism, we engineered Npro mutants lacking protease activity by replacement of amino acid E22, H49, or C69. We found that E22 and H49 substitutions abolished the ability of Npro to suppress IFN, whereas C69 had no effect, suggesting that the structural integrity of the N terminus of Npro was more important than its catalytic activity for IFN-alpha/beta suppression. A catalytically active mutant with a change at a conserved Npro region near the N terminus (L8P) in both BVDV biotypes did not antagonize IFN-alpha/beta production, confirming its involvement in this process. Taken together, these results not only provide direct evidence for the role of Npro in blocking IFN-alpha/beta induction, but also implicate the amino-terminal domain of the protein in this function.     

An Experimental Study of a Concurrent Primary Infection with Bovine Respiratory Syncytial Virus (BRSV) and Bovine Viral Diarrhoea Virus (BVDV) in Calves

Experimental infections with bovine respiratory syncytial virus (BRSV) and bovine viral diarrhoea virus (BVDV) were performed to study the effect of concurrent BRSV and BVDV infections. Twelve seronegative calves, in 3 groups, were inoculated on a single occasion with pure BRSV (group A), BRSV and noncytopathogenic BVDV (group B) or mock infected (group C). Mild respiratory symptoms were recorded 4 to 5 days post inoculation (dpi) in group A and group B calves. One calf in group A was severely affected and required medical treatment. In group B, fever (40.7-41.4 °C) was prominent 7 to 8 dpi. Only calves in group B were BVDV positive in purified lymphocytes at 2 to 14 dpi and showed increased serum interferon levels, with a peak at 4 dpi, indicating BVDV to be responsible for inducing the rise. BRSV was detected in lung lavage fluids up to 7 dpi for group A calves, compared to 11 dpi for group B and calves in this group also seroconverted later displaying lower BRSV titers. The time lag before an antibody response and the titers recorded in group B, indicated that the duration of BVDV infection in lymphocytes negatively influenced the capacity to mount a BRSV antibody response. kw|Keywords|k]cattle; k]respiratory disease; k]interferon; k]PCR     

Epidemiology of brucellosis in cattle and dairy farmers of rural Ludhiana,Punjab

Brucellosis is a zoonotic disease imposing significant impacts on livestock production and public health worldwide. India is the world’s leading milk producer and Punjab is the state which produces the most cattle and buffalo milk per capita. The aim of this study was to investigate the epidemiology of bovine brucellosis to provide evidence for control of the disease in Punjab State, India. A cross-sectional study of dairy farms was conducted in humans and livestock in rural Ludhiana district using a multi-stage sampling strategy. The study suggests that brucellosis is endemic at high levels in cattle and buffalo in the study area with 15.1% of large ruminants testing seropositive and approximately a third of dairy farms having at least one animal test seropositive. In total, 9.7% of those in direct contact with livestock tested seropositive for Brucella spp. Persons that assisted with calving and/or abortion within the last year on a farm with seronegative livestock and people which did not assist with calving/abortion had 0.35 (95% CI: 0.17 to 7.1) and 0.21 (0.09 to 0.46) times the odds of testing seropositive compared to persons assisting with calving/abortion in a seropositive farm, respectively. The study demonstrated that persons in direct contact with cattle and buffalo in the study area have high risk of exposure to Brucella spp. Control of the disease in livestock is likely to result in benefits to both animal and public health sectors.     

Investigation of transferability of BovineSNP50 BeadChip from cattle to water buffalo for genome wide association study

Cattle and water buffalo belong to the same subfamily Bovinae and share chromosome banding and gene order homology. In this study, we used genome-wide Illumina BovineSNP50 BeadChip to analyze 91 DNA samples from three breeds of water buffalo (Nili-Ravi, Murrah and their crossbred with local GuangXi buffalos in China), to demonstrate the genetic divergence between cattle and water buffalo through a large single nucleotide polymorphism (SNP) transferability study at the whole genome level, and performed association analysis of functional traits in water buffalo as well. A total of 40,766 (75.5 %) bovine SNPs were found in the water buffalo genome, but 49,936 (92.5 %) were with only one allele, and finally 935 were identified to be polymorphic and useful for association analysis in water buffalo. Therefore, the genome sequences of water buffalo and cattle shared a high level of homology but the polymorphic status of the bovine SNPs varied between these two species. The different patterns of mutations between species may associate with their phenotypic divergence due to genome evolution. Among 935 bovine SNPs, we identified a total of 9 and 7 SNPs significantly associated to fertility and milk production traits in water buffalo, respectively. However, more works in larger sample size are needed in future to verify these candidate SNPs for water buffalo.     

Viral and Bacterial Pathogens in Bovine Respiratory Disease in Finland

Pathogens causing bovine respiratory tract disease in Finland were investigated. Eighteen cattle herds with bovine respiratory disease were included. Five diseased calves from each farm were chosen for closer examination and tracheobronchial lavage. Blood samples were taken from the calves at the time of the investigation and from 86 calves 3–4 weeks later. In addition, 6–10 blood samples from animals of different ages were collected from each herd, resulting in 169 samples. Serum samples were tested for antibodies to bovine parainfluenza virus-3 (PIV-3), bovine respiratory syncytial virus (BRSV), bovine coronavirus (BCV), bovine adenovirus-3 (BAV-3) and bovine adenovirus-7 (BAV-7). About one third of the samples were also tested for antibodies to bovine virus diarrhoea virus (BVDV) with negative results. Bacteria were cultured from lavage fluid and in vitro susceptibility to selected antimicrobials was tested. According to serological findings, PIV-3, BAV-7, BAV-3, BCV and BRSV are common pathogens in Finnish cattle with respiratory problems. A titre rise especially for BAV-7 and BAV-3, the dual growth of Mycoplasma dispar and Pasteurella multocida, were typical findings in diseased calves. Pasteurella sp. strains showed no resistance to tested antimicrobials. Mycoplasma bovis and Mannheimia haemolytica were not found.     

Environmental Factors Influencing White-Tailed Deer (Odocoileus virginianus) Exposure to Livestock Pathogens in Wisconsin

White-tailed deer (Odocoileus virginianus) are commonly exposed to disease agents that affect livestock but environmental factors that predispose deer to exposure are unknown for many pathogens. We trapped deer during winter months on two study areas (Northern Forest and Eastern Farmland) in Wisconsin from 2010 to 2013. Deer were tested for exposure to six serovars of Leptospira interrogans (grippotyphosa, icterohaemorrhagiae, canicola, bratislava, pomona, and hardjo), bovine viral diarrhea virus (BVDV-1 and BVDV-2), infectious bovine rhinotracheitis virus (IBR), and parainfluenza 3 virus (PI3). We used logistic regression to model potential intrinsic (e.g., age, sex) and extrinsic (e.g., land type, study site, year, exposure to multiple pathogens) variables we considered biologically meaningful to exposure of deer to livestock pathogens. Deer sampled in 2010–2011 did not demonstrate exposure to BVDV, so we did not test for BVDV in subsequent years. Deer had evidence of exposure to PI3 (24.7%), IBR (7.9%), Leptospira interrogans serovar pomona (11.7%), L. i. bratislava (1.0%), L. i. grippotyphosa (2.5%) and L. i. hardjo (0.3%). Deer did not demonstrate exposure to L. interrogans serovars canicola and icterohaemorrhagiae. For PI3, we found that capture site and year influenced exposure. Fawns (n = 119) were not exposed to L. i. pomona, but land type was an important predictor of exposure to L. i. pomona for older deer. Our results serve as baseline exposure levels of Wisconsin white-tailed deer to livestock pathogens, and helped to identify important factors that explain deer exposure to livestock pathogens.