猪萨佩罗病毒原位杂交检测方法的建立

为了直观、原位且特异性的检测猪萨佩罗病毒(porcine sapelovirus,PSV)在感染仔猪后器官和组织中病毒核酸的分布,本研究根据GenBank中PSV的5′端非编码区基因的保守序列设计并合成1对引物,利用PCR地高辛探针合成的方法制备成原位杂交检测探针,建立了PSV原位杂交组织切片检测的方法。应用该方法检测PSV感染仔猪小肠组织,结果表明阳性信号主要存在与在于肠绒毛上皮细胞和固有层淋巴细胞中,阴性对照无显色。该方法可以用于组织切片中的PSV核酸的定位,为猪萨佩罗病毒在感染仔猪后器官和组织中病毒核酸分布及致病机理研究奠定基础。     

猪圆环病毒2型原位杂交检测方法的建立

参照GenBank发表的PCV2ORFl基因序列设计了1对引物,利用PCR地高辛探针合成的方法制备了长度为494bp的特异性探针,经检验具有良好的特异性和敏感性,可检测最低质粒DNA质量浓度为0．9728ug／L。用该探针建立了原位杂交组织切片检测方法,并用来检测PCV2感染猪的扁桃体和淋巴结组织,结果表明阳性信号主要存在于巨噬细胞胞浆中,信号强、背景良好,阴性对照无显色,说明该方法可作为PCV2实验室诊断和机理研究的一种有效检测方法。     

应用原位杂交技术研究母源抗体对PRRS病毒在体内增殖的影响

挑选母源抗体阳性及母源抗体阴性仔猪各2头,人工感染PRRSV,感染后不同时间采集扁桃体、胸腺、脑、肺脏、肩前淋巴结、脾脏、肝脏等组织做成多聚甲醛固定、石蜡包埋的组织切片。应用针对PRRS病毒（PRRSV）基因ORF6片段的特异性探针进行原位杂交检测,结果表明,2头母源抗体阴性猪的组织中均可检出病毒阳性信号,而2头母源抗体阳性猪仅可在少数组织中检出病毒阳性信号。以上结果表明,母源抗体会影响同种病毒在体内的增殖。     

猪场母猪与仔猪猪圆环病毒2型感染情况调查

为了解目前母猪和仔猪猪圆环病毒2型(PCV2)感染情况,本试验对来自于15个猪场的85份母猪血清和29个猪场的55份仔猪可疑病料(脾脏、淋巴结、肾脏等),分别采用间接ELISA和PCR法进行PCV2抗体和病毒核酸检测。结果显示,在15个猪场中,1个猪场母猪抗体呈阴性,其他14个猪场母猪抗体均呈阳性,猪场PCV2阳性检出场为93.3%(14/15),85份母猪血清抗体总阳性率为75.3%(64/85)。29个猪场的仔猪,PCV2核酸检测阳性猪场为19个,猪场阳性率为65.5%(19/29),55份仔猪可疑病料病毒核酸检测总阳性率61.8%(34/55)。可见,母猪和仔猪感染PCV2相当普遍。对母猪群PCV2抗体阳性率及其仔猪群病毒核酸阳性率进行比较发现,母猪群PCV2抗体阳性率越高其仔猪群病毒核酸阳性率却越低。     

猪圆环病毒3型荧光定量PCR检测方法的建立及其在猪体中的组织分布研究

为了解猪圆环病毒3型(PCV3)在猪体中的组织分布情况,针对PCV3 Cap蛋白基因设计筛选出一对特异性引物和探针,通过对荧光PCR引物、探针浓度进行优化,建立了基于TaqMan探针实时荧光定量PCR技术的PCV3检测方法,并应用该方法对人工感染PCV3猪的多种组织器官进行病毒含量检测。结果显示,建立的PCV3实时荧光定量PCR检测方法特异性较好且灵敏度高,可检测低至1 copy/μL的PCV3病毒核酸量,而对其他几种常见猪病毒病原的检测结果均为阴性。PCV3主要存在于肺脏、淋巴结,在扁桃体和脾脏中有少量存在。试验表明,所建立的荧光定量PCR检测方法可用于PCV3的快速定量检测,对PCV3在猪体中的组织分布情况的研究为进一步揭示PCV3的组织嗜性和致病机理提供理论依据。     

PCR和核酸探针检测猪源沙门氏菌四环素耐药基因tetC的研究

对沙门氏菌的四环素耐药性进行了检测，结果表明，沙门氏菌对四环素类抗生素产生了广泛的耐药性，耐药率达100％，对其四环素耐药基因tetC进行了扩增，结果获得以质粒为模板的特异性产物，与药敏试验结果阳性符合率75％，具有较高的检出率。用光生物素对PcR产物进行标记，制备核酸探针，采用菌落原位杂交的方法对沙门氏菌进行检测，结果表明13株阳性、3株阴性，杂交结果与PCR结果阳性符合率为93．75％，具有较高的特异性。通过条件的优化，建立的四环素耐药基因tetC的PCR和核酸探针检测技术，为四环素耐药性的分子流行病学监测提供了有效的途径。     

地高辛标记cDNA探针检测猪繁殖与呼吸综合征病毒

本研究以猪繁殖与呼吸综合征病毒(PRRSV)S1株ORF7基因为模板,通过RT-PCR技术扩增出394 bp的cDNA。用PCR纯化试剂盒纯化PCR产物,并以此为模板制备出地高辛标记的cDNA探针。特异性试验表明,该探针仅与PRRSV核酸反应,与PPV、PRV、FMDV、HCV及PCV2的核酸反应为阴性。敏感性检测表明,采用CSPD化学发光检测时,硝酸纤维素膜上最低检出量为62.5 pg;尼龙膜上最低检出量为0.5 pg。应用该探针,在尼龙膜上对10份PRRSV阳性病料的组织RNA样品进行检测,结果均为阳性,与RT-PCR检测结果一致,证明该方法可用于PRRSV临床样品检测。     

用地高辛标记的核酸探针检测猪伪狂犬病毒野毒感染的研究

利用PCR技术从带有伪狂犬病毒（PRV）gE基因的重组质粒pMD18-T-gE中扩增回收约304bp大小的片段,并制备出地高辛标记的gE基因核酸探针。特异性检测结果表明,该探针能与重组质粒DNA发生特异性杂交,而与对照的PRVBartha-k61株疫苗毒DNA、猪细小病毒（PPV）DNA、猪圆环病毒（PCV）DNA、猪繁殖与呼吸综合征病毒（PRRsV）cDNA、猪瘟病毒（CSFV）cDNA的杂交反应均为阴性;敏感性检测结果表明,该探针对PRV野毒的最低检出量为4pg。应用该探针对11份繁殖障碍病料进行了杂交检测,共检出4份阳性病料,该结果与PCR检测结果一致,表明该核酸探针可用于猪伪狂犬病野毒感染的临床诊断。     

病死猪圆环病毒2型检测及全基因序列分析

为准确检测猪圆环病毒2型（PCV2）并分析其基因序列情况，在序列比对分析的基础上，设计一对简并引物和一条探针，经体系优化建立了PCV2的荧光PCR方法。该方法可检出10拷贝的PCV2质粒DNA，不与猪瘟病毒等多种病毒发生交叉反应。对送检的2批次45份病死猪脏器进行检测，检出25份阳性样品，与套式PCR方法的复合率为95.6%；对3份强阳性样品使用一对全基因组扩增引物，扩增了PCV2型全基因片段；经克隆测序后分析，发现3个病毒基因分别属于2种基因亚型（PCV2b和PCV2d）。本研究对于PCV2的准确检测及其变异特征的了解具有参考意义。     

鹅细小病毒VP1-VP3非重叠序列地高辛探针的制备和应用

根据GPV H1株核苷酸序列，设计了扩增VP1-VP3基因非重叠序列的1对引物，对其结构蛋白VP1与VP3非重叠核苷酸序列进行PCR扩增，将PCR产物纯化、回收后制备出GPV VP1-VP3基因DIG标记核酸探针，其标记效率达到0．1pg／μl。特异性检测结果表明，该探针能与GPV不同毒株核酸发生特异性杂交，而与对照的DPV、GPMV等病毒的核酸杂交反应均为阴性；敏感性检测结果表明该探针对GPV的最低检出量为0．032ng。上述试验结果表明该探针可以用于GPV感染临床病料的检测。     

Experimental inoculation of porcine circoviruses type 1 (PCV1) and type 2 (PCV2) in rabbits and mice

The objective of this work was to investigate the susceptibility of rabbits and mice experimentally inoculated with porcine circoviruses type 1 (PCV1) and type 2 (PCV2) to infection and development of disease and/or lesions. Forty six New Zealand rabbits and 50 ICR-CDI mice were both divided into two groups comprising PCVI and PCV2 inoculated animals, and a third group inoculated with non-infected cell culture medium. Rabbits were inoculated intranasally while mice were inoculated intraperitoneally. Clinical signs and body weights were recorded at the start of the experiment and at necropsy. Animals were bled, euthanised and necropsied at days 0, 3, 7, 10, 14 and 20 post-inoculation and samples were collected for histopathological, serological, in situ hybridisation and PCR analysis. No clinical signs or gross and microscopic lesions compatible with PCV2 infections such as those seen in pigs were observed. No presence of PCV2 nucleic acid was detected in rabbits and mice by in situ hybridisation. Only one mouse inoculated with PCV1 seroconverted on day 20 P1. PCV1 and PCV2 genome was detected in serum by PCR in mice inoculated with each porcine circovirus, while rabbits were negative for both viral types. These studies indicated that porcine circoviruses did not cause any disease or microscopic lesions in inoculated rabbits and mice during the experimental period. However, intraperitoneally inoculated mice might have harboured PCV2 in circulation without evidence of viral replication.     

Lack of an effect of a commercial vaccine adjuvant on the development of postweaning multisystemic wasting syndrome (PMWS) in porcine circovirus type 2 (PCV2) experimentally infected conventional pigs

The objective of this study was to evaluate the effect of a commercial vaccine adjuvant on the clinical and pathological outcome of PCV2 experimentally infected 8 to 9-week-old conventional pigs. Forty-four pigs were divided into four groups: non-infected control pigs, pigs that received a vaccine adjuvant, pigs inoculated with PCV2, and pigs inoculated with PCV2 together with the vaccine adjuvant. Infection was monitored until 69 days post-inoculation (PI). Some PCV2 inoculated pigs had hyperthermia, but no other clinical signs were recorded. No characteristic PMWS gross or microscopic lesions were observed in any of the pigs. PCV2 DNA was detected in lymphoid tissues by in situ hybridisation in 6 PCV2 inoculated pigs on day 69 PI. All PCV2 inoculated pigs seroconverted between days 21 and 49 PI, shortly after viremia detection. Moreover, viremia was detected between days 7 and 69 PI using PCR. A peak of the virus load was detected by real-time quantitative PCR between days 14 and 21 PI. There were no significant differences in the proportion of PCV2 positive serum and in the viral load between PCV2 and PCV2 + adjuvant inoculated pigs. Although PMWS was not reproduced in neither PCV2 nor PCV2 + adjuvant inoculated pigs, viremia detection and seroconversion indicated that all PCV2 inoculated pigs developed a chronic long-term asymptomatic infection. An increase of PCV2 replication was not observed in pigs inoculated with the adjuvant. These results indicate that the principle of immunostimulation may not be applicable under the experimental conditions used, suggesting that not all adjuvants used in commercial vaccines are capable of triggering mechanisms for PMWS development.     

A sequential study of experimental infection of pigs with porcine circovirus and porcine parvovirus: immunostaining of cryostat sections and virus isolation

The sequential tissue distribution of virus was investigated using virus isolation and immunofluorescence tests in 1-day-old piglets inoculated with porcine circovirus 2 (PCV2) and/or porcine parvovirus (PPV). Enlarged mesenteric lymph nodes were seen in the pig inoculated with PCV2 alone and killed at 26 days post-inoculation (PI). One of the pigs inoculated with PCV2 and PPV and killed at 21 days PI had an enlarged liver. The pig killed at 26 days PI in this group had enlarged liver, kidneys and heart. Histopathological changes were seen in lymphoid tissues of the pigs inoculated with PCV2 alone and killed at 14 and 26 days PI. Similar, but more severe, lesions were observed in the pigs infected with PCV2 and PPV and killed from 10 days PI onwards. Histological lesions of nephritis, pneumonia and hepatitis were also apparent in these animals. Mild nephritis was also seen in the pigs infected with PPV alone and killed at 14 and 26 days PI. Moderate amounts of PPV antigen were detected in tissues from the pigs inoculated with PPV alone and killed at 14 days PI. Low levels of PCV antigen were detected, mainly in lymphoid tissues, in the pigs inoculated with PCV alone and killed at 14 days PI. Low to moderate amounts of PCV antigen were detected in a wider range of tissues in the pig in this group killed at 26 days PI. In the pigs inoculated with both viruses, PPV antigen was detected in tissues of pigs killed from 3 to 26 days PI with maximal amounts detected between 6 and 14 days PI. PCV2 antigen was detected in low to moderate amounts in the tissues of pigs killed at 14 days PI. Large amounts of PCV2 antigen were detected in most of the tissues from pigs in this group killed between 17 and 26 days PI. Virus isolation results for PCV2 generally correlated well with the results for immunofluorescent staining. PPV was isolated from almost all tissues from pigs inoculated with PCV2 and PPV, a much higher incidence of positive tissues than observed for immunofluorescent staining.     

Porcine circovirus type 2 in muscle and bone marrow is infectious and transmissible to naïve pigs by oral consumption

Pork products are a possible source of introduction of PCV2 isolates into a pig population. However, limited work has been done on the transmission through meat of porcine circovirus type 2 (PCV2), a virus associated with several disease syndromes in pigs. The objectives of this study were to determine if pork products from PCV2-infected pigs contain PCV2 DNA/antigen and to determine if the PCV2 present in the tissues is infectious by performing in vitro and in vivo studies. Skeletal muscle, bone marrow, and lymphoid tissues from pigs experimentally inoculated with PCV2 were collected 14 days post-inoculation (DPI). The tissues were tested for presence of PCV2 DNA by quantitative real-time PCR, for PCV2 antigen by immunohistochemistry (IHC), and for presence of infectious PCV2 by virus isolation and inoculation of PCV2 naïve pigs. Lymphoid tissues contained the highest amount of PCV2 (positive by PCR, IHC, and virus isolation), bone marrow contained a lower amount of PCV2 (positive by PCR and IHC but negative by virus isolation), and skeletal muscle contained the lowest amount of PCV2 (positive by PCR but negative by IHC and virus isolation). Naïve pigs fed for three consecutive days with either skeletal muscle, bone marrow, or lymphoid tissues all became PCV2 viremic as determined by quantitative real-time PCR on serum starting at 7 DPI. The pigs also seroconverted to PCV2 as determined by PCV2 IgM and IgG ELISA. In addition, PCV2 antigen was detected by IHC stains in lymphoid tissues and intestines collected from the majority of these pigs. Results from this study indicate that uncooked PCV2 DNA positive lymphoid tissues, bone marrow, and skeletal muscle from PCV2 viremic pigs contain sufficient amount of infectious PCV2 to infect naïve pigs by the oral route.     

The effects of transplacental porcine circovirus type 2 infection on porcine epidemic diarrhoea virus-induced enteritis in preweaning piglets

The effects of transplacental porcine circovirus type 2 (PCV2) infection on porcine epidemic diarrhoea virus (PEDV)-induced enteritis were examined in neonatal piglets. Six pregnant sows were randomly allocated to an infected (n=3) or control group (n=3). Three pregnant sows were inoculated intranasally with 6 mL of tissue culture fluid containing 1.2 x 10(5) tissue culture infective doses 50% (TCID(50))/mL of PCV2 strain SNUVR000470 three weeks before the expected farrowing date. Three control pregnant sows were similarly exposed to uninfected cell culture supernatants. Thirty piglets from PCV2-infected sows were randomly assigned to two groups (A and B) of 15 piglets each. Another 30 piglets from noninfected sows were randomly assigned to two groups (C and D) of 15 piglets each. The piglets in groups A and C were dosed orally at three days of age with 2mL of virus stock (1 x 10(6.5) TCID(50)/mL) of the PEDV strain, SNUVR971496, at the third passage. The mean villous height and crypt depth (VH:CD) ratio in PEDV-infected piglets from PCV2-infected sows (group A) were significantly different from those of the PEDV-infected piglets from PCV2 negative sows (group C) at 36, 48, and 72 h post-inoculation (hpi) (P<0.05). In PEDV-infected piglets from PCV2-infected sows (group A), significantly more PEDV nucleic acid was detected in the jejunal tissues (P<0.05) at 24 hpi than in the same tissues of the PEDV-infected piglets from PCV2 negative sows (group C). Thereafter, at 36, 48, 60, and 70 hpi significantly more PEDV nucleic acid (P<0.05) was detected in the jejunal tissues of the PEDV-infected piglets from PCV2 negative sows (group C) than those of the PEDV-infected piglets from the PCV2-infected sows (group A). It is concluded that the clinical course of PEDV disease was markedly affected by transplacental infection of PCV2.     

Experimental reproduction of postweaning multisystemic wasting syndrome in cesarean-derived, colostrum-deprived piglets inoculated with porcine circovirus type 2 (PCV2): investigation of quantitative PCV2 distribution and antibody responses.

Sixteen cesarean-derived, colostrum-deprived piglets were inoculated intranasally with porcine circovirus type 2 (PCV2), originally isolated from a pig affected with postweaning multisystemic wasting syndrome (PMWS). At 1 day postinoculation (PI), 3 of the 5 piglets in the uninoculated control group were moved to the room of inoculated piglets for contact exposure. Porcine circovirus type 2 was detected by polymerase chain reaction (PCR) in swabs from inoculated piglets from 1 day PI and from contact piglets from 2 days after cohabitation. Porcine circovirus type 2 was also detected in all serum samples but not in control piglets 7 days PI. Until the end of study, PCV2 was detected in swabs and serum samples by PCR but not in the control piglets. One inoculated piglet died suddenly without clinical signs 19 days PI. Beginning at 14 days PI, 5 piglets, including 1 contact piglet, had clinical signs of depression, anorexia, and icterus, and 1 inoculated piglet died 21 days PI. Most of the piglets exhibiting the above clinical signs became moribund and were necropsied 21 and 28 days PI. In the piglets that showed clinical signs, gross lesions, including icterus of liver and hemorrhage in stomach, and typical histopathological lesions of PMWS, such as lymphoid depletion and basophilic intracytoplasmic inclusion bodies in lymph nodes and other tissues, were observed. Porcine circovirus type 2 was detected by PCR in all tissue samples except in those of the control piglets. Porcine circovirus type 2 was recovered from several tissue samples of the piglets necropsied until 35 days PI. In particular, PCV2 was recovered in high titer from most of the tissue samples of the piglets exhibiting clinical signs. Serum antibody against PCV2 was mostly detected in inoculated piglets and in contact piglets 14 and 21 days PI by an indirect fluorescence antibody test but was not detected in the piglets exhibiting clinical signs until 28 days PI. These results indicate that PCV2 was able to induce clinical PMWS in the absence of other swine pathogens and that there were significant differences in both the quantitative PCV2 distribution in tissues and the antibody response between the piglets that were infected and developed PMWS and those that were infected but remained healthy.     

Viremia and effect of fetal infection with porcine viruses with special reference to porcine circovirus 2 infection

This publication reviews some pathogenetic features of the transplacental infection with porcine viruses in sows. Viremia either with virus freely circulating or associated to peripheral blood mononuclear cells (PBMC) is an essential part of such pathogenesis. Virus replication occurs either in fetal tissues only or both in fetal and maternal tissues and the outcome may be different.Since porcine circovirus 2 (PCV2) has been associated with reproductive failure in sows, the question was asked what type of viremia PCV2 causes and what the effect of PCV2 is on the pregnant uterus. Seronegative gilts were oronasally inoculated and plasma and PBMC were monitored for infectious virus and for quantity of viral DNA copies. Infectious virus was found in plasma only at 21 days post-inoculation (DPI). Virus associated to PBMC was detected between 14 and 49 DPI. Viral DNA was found in plasma between 14 and 49 DPI and associated to PBMC between 7 and 63 DPI (end of experiment). Direct intra-fetal inoculation at 57, 75 and 92 days of gestation and collection of fetuses 21 days later showed that the virus replicates highly in fetal tissues, particularly in the heart. Fetal death occurred in the 57 days sows while virus and antibodies were observed in the 75- and 92-day inoculated sows. Inoculation at 57 and 75 days of gestation and collection of the piglets at the end of pregnancy showed that intrauterine spread had occurred to fetuses adjacent to the inoculated ones and that fetal death occurred also in the presence of antibodies. The pregnancy was not interrupted.This study shows that PCV2 causes viremia which is largely cell-associated and that virus replication in fetuses causes fetal death with mummification. Whether such transplacental infection occurs in the immune sow population is questionable.     

Retrospective study on porcine circovirus type 2 infection in pigs from 1985 to 1997 in Spain

A retrospective survey was performed to detect lesions of Postweaning multisystemic wasting syndrome (PMWS) and nucleic acid of porcine circovirus type 2 (PCV2) in archived formalin-fixed, paraffin-embedded tissues from 189 pigs, and antibodies to this virus in sera of 388 pigs from the Spanish livestock between the years 1985 and 1997. PCV2 nucleic acid was detected by in situ hybridization (ISH) in tissues from 78 of 189 (41.3%) examined pigs. Variable amount of viral genome was detected in association with slight to severe microscopic lymphoid lesions consisting of lymphocyte depletion and histiocytic infiltration. The first positive case of PMWS with typical lesions and ISH positive corresponded to a pig necropsied in 1986. Two hundred and eighty-two of 388 (72.7%) sera were positive by immunoperoxidase monolayer assay. Serological and pathological data of the present study indicate that PCV2 was a enzootic infection in Spain since 1985, suggesting that the introduction of this virus in the livestock occurred previously.     

Identification of porcine circovirus type 2 in retrospective cases of pigs naturally infected with porcine epidemic diarrhoea virus

The identification of porcine circovirus type 2 (PCV2) was studied in fresh intestinal tissues by polymerase chain reaction (PCR) and in formalin-fixed, paraffin-wax-embedded intestinal tissues by in situ hybridisation. The tissues came from pigs naturally infected with porcine epidemic diarrhoea virus (PEDV). A total of 35 (32.7%) of 107 small intestinal samples from pigs naturally infected with PEDV were found to be positive using PCR. Positive signals for PCV2 were detected in 32 (29.9%) of 107 small intestinal samples from pigs naturally infected with PEDV by in situ hybridisation. The distribution of positive cells in the jejunum and ileum was multifocal or patchy. Distinct positive labelling was found throughout the lamina propria in the small intestines. The results of this study indicate that PCV2 is highly prevalent in pigs naturally infected with PEDV.