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为改进猪圆环病毒2型的培养工艺,驯化了一株可无血清培养的全悬浮PK15细胞用于培养猪圆环病毒2型,并对病毒的敏感性、接毒时间、接毒量、收获方法进行了试验.结果表明,用该细胞培养猪圆环病毒2型,如果采用批次收获,接毒时细胞密度为0.5×106/mL,接毒量为0.1 MOI,接毒72 h后收毒,病毒滴度能达到106.4 T... 相似文献
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A miniarray system was developed for the simultaneous detection of porcine circovirus type 1 (PCV1) and type 2 (PCV2) in pigs.
The system consists of a polymerase chain reaction (PCR) step to amplify target viral DNA, followed by detection of the amplified
DNA using a membrane-anchored probe array and an avidin-alkaline phosphatase (Av-AP) indicator system. The lower limit of
detection of PCV using the miniarray was 101.9 tissue culture infectious dose 50 (TCID50)/ml and 102.08TCID50/ml for PCV1 and PCV2, respectively, and 100 viral copies/μl for both PCV1 and PCV2. We validated the miniarray system using
141 lymph node specimens from pigs with suspected postweaning multisystemic wasting syndrome or porcine dermatitis and nephropathy
syndrome. Of the 141 samples evaluated, 55 were identified as positive for PCV by the miniarray. Relative to in situ hybridization, the sensitivity and specificity of the miniarray was 100% and 98.9%, respectively. In contrast to other microarray
systems, the miniarray does not require a DNA chip reader, since the results can be determined by visual inspection of colorized
spots on a nylon membrane. This system represents an effective alternative method for the differential detection of PCV1 and
PCV2 in pigs, as well as the maintenance of PCV-free cell lines and pre-screening of commercial vaccines for possible contamination. 相似文献
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从临床疑似猪伪狂犬病发病仔猪的脑组织等病料中,经PCR扩增出大小为217 bp的伪狂犬病病毒gp50的基因片段,结果证实为猪伪狂犬病病毒(porcine pseudorabies virus,PRV)感染。随后采用BHK-21细胞进行猪伪狂犬病病毒的分离培养,该分离株经细胞传代培养5代后,能够产生典型的细胞病变,经PCR鉴定为伪狂犬病病毒,其病毒感染力达108.68 TCID50/0.1mL。最后用107.0 TCID50/mL病毒培养物接种家兔,48 h后注射部位出现典型瘙痒、皮肤破损等症状,于72 h后全部死亡。结果表明,该伪狂犬病病毒分离株对易感动物具有高致病性,为进一步开展该病毒流行病学、致病机理、疫苗免疫及诊断研究奠定了基础。 相似文献
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用间接免疫荧光检测方法评价猪瘟兔化弱毒活疫苗效力的研究 总被引：1，自引：0，他引：1
为用体外试验方法评价猪瘟兔化弱毒活疫苗效力,以猪瘟病毒抗体（兔源）为一抗、荧光素标记羊抗兔IgG 为二抗,建立了CSFV兔化弱毒株的间接免疫荧光检测方法（ IFA）。特异性试验表明,用该IFA方法检测CSFV兔化弱毒株接种的RK细胞为阳性,而检测伪狂犬病毒、猪细小病毒病、牛病毒性腹泻/粘膜病毒接种的RK细胞均为阴性。 CSFV兔化弱毒株和活疫苗的兔体感染量（ RID）用兔体测定,半数组织感染量（ TCID50）用IFA测定,拟合RID与TCID50的线性回归方程,确定1RID/mL＝（ TCID50/0．1mL＋200）/12。 相似文献
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Comparative infection efficiency of Porcine reproductive and respiratory syndrome virus field isolates on MA104 cells and porcine alveolar macrophages 
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下载免费PDF全文 Martha Fuentes de Abin Gordon Spronk Mark Wagner Mark Fitzsimmons Juan E. Abrahante Michael P. Murtaugh 《Canadian journal of veterinary research》2009,73(3):200-204
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Lynn M. Herrmann-Hoesing Howard D. Lehmkuhl Randall C. Cutlip 《Research in veterinary science》2009,87(2):329-331
The minimum intravenous infectious dose for ovine progressive pneumonia virus (OPPV) WLC1 was determined using twenty-four 6 month-old lambs. Twelve groups of two 6 month-old lambs were inoculated intravenously (i.v.) with tissue culture fluid containing ovine progressive pneumonia virus (OPPV) WLC1 titers ranging from 107.6 TCID50/lamb down to 10−3.4 TCID50/lamb and were monitored for seroconversion using the OPPV agar gel immunodiffusion assay (AGID). Fifteen of the 16 lambs given equal or greater than 100.6 TCID50 seroconverted, and virus could be isolated from peripheral blood leukocytes in 13 out of the 15 of these lambs. None of the eight lambs receiving less than 100.6 TCID50 seroconverted during the 12 months. The results of this study indicated that 100.6 or 4 TCID50/lamb given i.v. was capable of establishing infection. 相似文献
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Generation of embryonic stem‐like cells from in vivo‐derived porcine blastocysts at a low concentration of basic fibroblast growth factor 下载免费PDF全文
下载免费PDF全文 NR Han H‐Y Kim S Baek S‐H Lee J Lee E Lee C‐K Park ST Lee 《Reproduction in domestic animals》2018,53(1):176-185
Although basic fibroblast growth factor (bFGF) is an essential factor supporting the maintenance of porcine embryonic stem (ES) cell self‐renewal and pluripotency, its high cost has limited previous studies, and the development of a low‐cost culture system is required. For these systems, in vivo blastocysts were progressively cultured under various conditions consisting of different culture mediums and/or different feeder cell numbers at a low concentration of bFGF. As the results, the sequential culture of in vivo‐derived porcine blastocysts on 5.0 × 105 mouse embryonic fibroblast (MEF) feeder cells in alpha minimum essential medium‐based medium for primary culture, on 2.5 × 105 MEF feeder cells in Mixture medium for the 1st subpassage, and on 2.5 × 105 MEF feeder cells in DMEM/Ham's F10‐based medium for the post‐2nd subpassage could support the establishment and maintenance of porcine ES‐like cells at the low concentration of bFGF. The established porcine ES‐like cells showed ES cell‐specific characteristics such as self‐renewal and pluripotency. We confirmed that porcine ES‐like cells could be generated from in vivo‐derived porcine blastocysts at a low concentration of bFGF. 相似文献
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Real-time PCR to detect and analyze virulent PPV loads in artificially challenged sows and their fetuses 总被引：3，自引：0，他引：3
Lan-fei Miao Chao-fan Zhang Chang-mu Chen Shang-jin Cui 《Veterinary microbiology》2009,138(1-2):145-149
To establish a real-time polymerase chain reaction with SYBR® Green for detection and quantification of porcine parvovirus (PPV) in porcine tissues, two primers specific for the non-structural protein 1 gene were designed. The detection limit of this assay was 3–23 gene copies/reaction, equivalent to 0.001 TCID50/ml. The assay was linear over a 106 dilution range of template concentrations. Other porcine pathogens involved in reproductive disorders (porcine circovirus 2, porcine reproductive and respiratory virus, pseudorabies virus, classical swine fever virus) were negative by this assay. This assay could detect PPV titres at least 105 smaller than the hemagglutination assay. To better understand the pathogenesis of PPV, the levels of viral DNA in various tissues of artificially challenged sows and their fetuses were quantified with this method. The virus was found mainly in the heart, lung, spleen, kidney, and endometrium of sows, and mainly in the heart, spleen, lung, and testis of fetuses. This study provides a new tool for the study of PPV infection and distribution in sows and their fetuses. 相似文献
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Wilaiporn Saikruang Pattara Khamrin Natthawan Chaimongkol Boonpa Suantai Aphisek Kongkaew Sompreeya Kongkaew Hiroshi Ushijima Niwat Maneekarn 《Veterinary microbiology》2013,161(3-4):255-262
Several epidemiological studies reported the detection of rotavirus strains bearing unusual combinations of genetic background of human and porcine rotaviruses. This observation supports the hypothesis of interspecies transmission of rotaviruses in humans and pigs. The aims of this study were to investigate the genotypes and molecular characteristics of rotaviruses in piglets with diarrhea in several farms from two provinces in Thailand. A total of 207 fecal specimens collected from diarrheic piglets were screened for the presence of groups A, B, and C rotaviruses. Group A rotaviruses were detected in 41 out of 207 (19.8%) fecal specimens tested. A wide variety of G-P combination rotavirus strains were detected in this study. The G4P[6] was identified as the most prevalent genotype (39.0%), followed by G4P[23] (12.2%), G3P[23] (7.3%), G4P[19] (7.3%), G3P[6] (4.9%), G3P[13] (4.9%), G3P[19] (4.9%), G9P[13] (4.9%), G9P[19] (4.9%), G5P[6], and G5P[13] each of 2.4%. Furthermore, G5 and G9 in combinations with P-nontypeable strains were also found at each consisting of 2.4% (n = 1) of the collection. It was interesting to note that among diversified porcine rotavirus strains, novel combinations of G4P[19] and G9P[19] strains were detected for the first time in this study. Nucleotide sequences of VP4 and VP7 of these strains were closely related to human rotaviruses reported previously. The data implies that these porcine rotaviruses were probably generated in nature from the reassortment between the viruses of human and porcine origin. This study provides valuable epidemiological information and molecular characteristics of porcine rotaviruses circulating in piglets with diarrhea in northern Thailand. 相似文献
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C. C. Hwang M. Igase M. Sakurai T. Haraguchi K. Tani K. Itamoto T. Shimokawa M. Nakaichi Y. Nemoto S. Noguchi M. Coffey M. Okuda T. Mizuno 《Veterinary and comparative oncology》2018,16(2):229-238
Oncolytic virotherapy is a novel treatment involving replication‐competent virus in the elimination of cancer. We have previously reported the oncolytic effects of reovirus in various canine cancer cell lines. This study aims to establish the safety profile of reovirus in dogs with spontaneously occurring tumours and to determine a recommended dosing regimen. Nineteen dogs with various tumours, mostly of advanced stages, were treated with reovirus, ranging from 1.0 × 108 to 5.0 × 109 TCID50 given as intratumour injection (IT) or intravenous infusion (IV) daily for up to 5 consecutive days in 1 or multiple treatment cycles. Adverse events (AEs) were graded according to the Veterinary Cooperative Oncology Group‐ Common Terminology Criteria for Adverse Events (VCOG‐CTCAE) v1.1 guidelines. Viral shedding, neutralizing anti‐reovirus antibody (NARA) production and immunohistochemical (IHC) detection of reovirus protein in the tumours were also assessed. AE was not observed in most dogs and events were limited to Grade I or II fever, vomiting, diarrhoea and inflammation of the injected tumour. No infectious virus was shed and all dogs had elevated NARA levels post‐treatment. Although IHC results were only available in 6 dogs, 4 were detected positive for reovirus protein. In conclusion, reovirus is well‐tolerated and can be given safely to tumour‐bearing dogs according to the dosing regimen used in this study without significant concerns of viral shedding. Reovirus is also potentially effective in various types of canine tumours. 相似文献
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The present investigation describes detection of a mammalian-like electropherogroup A rotavirus in chicken with diarrhoea. This also records the first detection of a rotavirus in an avian species from India. During the investigation 75 diarrhoeic faecal samples collected from adult chicken were screened for the presence of group A rotavirus antigen by sandwich ELISA. All three samples positive for rotavirus antigen revealed 11 bands of RNA in polyacrylamide gel electrophoresis (PAGE). In contrast to avian group A rotavirus, segment 5 was found to migrate closer to 6 as is the case with mammalian group A rotaviruses. Segments 7, 8 and 9 were found to migrate as a tight triplet, which is characteristic of group A rotavirus. 相似文献
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Elschner M Schrader C Hotzel H Prudlo J Sachse K Eichhorn W Herbst W Otto P 《Veterinary microbiology》2005,105(2):123-129
A total of 26 rotavirus positive faecal samples of diarrhoeal foals, and 8 equine rotavirus isolates were examined. Viral RNA patterns were generated, G typing was performed by PCR, and a P[12]-specific DNA probe was developed for P typing. Furthermore, five equine rotavirus isolates were sequenced in the genomic regions coding for VP7 and part of VP4. Rotaviruses of genotype G3 P[12] were found in 22 faecal samples and G14 P[12] type could be found in 4 faecal samples. These findings confirm that in Germany G3 P[12] is the predominating type of equine rotaviruses. 相似文献
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F Samartzi I Tsakmakidis E Theodosiadou E Vainas 《Reproduction in domestic animals》2008,43(2):153-156
The effect of porcine or ovine FSH on the maturation rate of porcine oocytes and on the time course of meiotic progression was studied. Groups of 20 grade‐A cumulus oocyte complexes, aspirated from slaughterhouse cycling‐gilt ovaries, were cultured in vitro in 400 μl of Modified Parker's Medium supplemented with oestrous cow serum and porcine FSH (Folltropin®‐V, 0.50 mg/ml) or ovine FSH (OvagenTM, 0.44 iu/ml), in four‐well dishes under mineral oil, at 38.5°C, 5% CO2 in humidified air. At the end of each 3‐h interval, from 3 to 42 h of culture, the nuclear status of oocytes was assessed microscopically (1000×), after fixation (methanol/acetic acid: 3/1) and orcein (2%) staining. Oocytes were classified as (i) immature (IMM), i.e. oocytes at germinal vesicle stage, germinal vesicle break down and prophase I, (ii) metaphase I (MI) and (iii) metaphase II (MII), i.e. oocytes at anaphase I, telophase I and metaphase II. Data were analysed using regression analysis, chi‐square and t‐test. Nuclear status was assessed in 1610 oocytes (porcine FSH: 787, ovine FSH: 823). Most of the oocytes were at MI from 24 to 33 h (porcine FSH 60.27%, ovine FSH 42.80%, p < 0.001) and at MII from 36 to 42 h (porcine FSH 80.38%, ovine FSH 67.45%, p < 0.01) of culture. Significantly higher maturation rate was observed in porcine FSH than in ovine FSH treated oocytes (86.69 ± 12.97%, 71.34 ± 9.86%, mean ± SD, p < 0.05), after 42 h of culture. In conclusion, under the specific culture conditions, porcine FSH seems to support pig oocyte maturation better than ovine FSH. 相似文献
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Di Gialleonardo L Migliaccio P Teodori L Savini G 《Research in veterinary science》2011,91(2):316-320
Four groups of BTV free Frisian and cross bred calves were used to determine the length of viraemia following infection with different doses of BTV-8 Italian isolate. The first group of five animals was infected with 10 TCID50 of BTV-8, the second group of four animals with 103 TCID50 and the third group, which also included four animals, was infected with 106 TCID50. A placebo containing uninfected tissue culture medium was given to the four animals of the fourth group. The viraemia was evaluated by real time RT-PCR and virus isolation. In all infected groups, virus isolation was able to detect infectious virus up to 39 days post infection (dpi) while RT-PCR was positive up to 151–157 dpi. Infectious dose did influence neither the length nor the pattern of BTV-8 viraemia and confirmed that real time RT-PCR remains positive although no circulating virus is detectable in the peripheral circulation. 相似文献
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Contents: Immune electron microscopy, hybridization (Sandwich, Southern-blot and dot-blot hybridization) as well as two cell culture inoculation techniques to detect bovine herpesvirus 1 (BHV-1) in naturally and artificially contamined bovine semen were compared. Immune electron microscopy was not a suitable method because of its low limit of detection. Dot-blot hybridization, as the most sensitive hybridization technique, could detect 150 pg BHV-1 DNA/semen straw (～ 106TCID50/ 500 μl semen). Best results were obtained by using cell culture techniques. With the first technique embryonic bovine lung cells (EBLC) on microtiterplates were inoculated with an ultracentrifuged pellet of seminal plasma (pelleting method). The second procedure comprised a dilution of 250 μl (1/2 content of a straw) in 6,25 ml cell culture medium which were overlayed on a confluent layer of EBLC in a 25 cm2 flask. After an incubation of 4 hours at 37°C the medium was decanted and replaced by 6,5 ml fresh medium (dilution method). With both tissue culture methods as little as 5 TCID50 of BHV-1 in 500 μl of artificially contaminated semen were detectable. However the dilution method was better suited than the pelleting method because toxic effects to cells were less pronounced. It was even possible to detect BHV-1 in naturally contaminated semen samples where all other methods failed. The DNA of virus isolates from semen showed three different restriction patterns. Inhalt: Vergleich dreier Methoden zum Nachweis von bovinem Herpesvirus Typ 1 (BHV-1) in natürlich und experimentell kontaminiertem Rindersamen Die Immunelektronenmikroskopie, die Hybridisation (Sandwich-, Southern-Blot- und die Dot-Blot Hybridisation) sowie zwei Zellkultur-Inokulationstechniken wurden zum Nachweis von bovinem Herpesvirus Typ-1 (BHV-1) in natürlich und künstlich kontaminiertem Stierensamen verglichen. Die Immunelektronenmikroskopie erwies sich dabei als zu wenig empfindliche Methode. Die Dot-Blot Hybridisation war die beste der Hybridbationsmethoden. Es lieβen sich damit bis zu 150 pg BHV-1 DNA/Paillette (～ 106 TCID 50 /500 μl Samen) nachweisen. Am besten eigneten sich Zellkulturmethoden. Eine erste Methode bestand darin, embyonale bovine Lungenzellen in Microtiterplatten mit einem Ultrazentrifugensediment des Samenplasmas zu inokulieren (pelleting method). In einem zweiten Verfahren wurde 1/2 Inhalt einer Pailette (250 μl) in 6,25 ml Zellkulturmedium verdünnt und damit ein Zellrasen von embyonalen bovinen Lungenzellen überschichtet. Nach 4 h bei 37°C wurde das Medium abgegossen und durch 6,5 ml neues Medium ersetzt (Verdünnungsmethode). Mit beiden Methoden lieβen sich 5 TCID50 BHV-1 in 500 μl Rünstlich kontaminiertem Samen nachweisen. Die Verdünnungsmethode war aber besser geeignet, weil der toxische Effekt auf die Zellen geringgradiger war. Mit der Verdünnungsmethode lieβ sich aus Ejakulaten BHV-1 isolieren, auch in Fällen, in denen alle andern Methoden versagten. Die DNA's von Virusisolaten aus Rindersamen wiesen drei verschiedene Restriktionsmuster auf. 相似文献