Experimental infection of 3-week-old conventional colostrum-fed pigs with porcine circovirus type 2 and porcine parvovirus

This report describes an experimental infection with porcine circovirus type 2 (PCV2) in combination with porcine parvovirus (PPV) in 3-week-old conventional colostrum-fed pigs with maternal antibodies to both viruses. Two groups of four pigs each were inoculated with PCV2 and PPV. One of the groups received also a commercial inactivated vaccine against porcine pleuropneumonia to evaluate possible effects of the stimulation of the immune system of pigs on the infection. Another group of four pigs was kept as uninfected control. Clinical signs, rectal temperatures and body weights were recorded. Serum antibody titers to PCV2 and PPV were determined at weekly intervals. Pigs were killed 42 days after inoculation and tissue samples were examined for the presence of gross and microscopic lesions. Tissues were also analyzed for the presence of PCV2 and PPV DNA by PCR, and for the presence of PCV2 antigen by immunohistochemistry (IHC). All the pigs had serum antibodies to PCV2 and PPV at the beginning of the trial. None of them developed clinical symptoms or pathological lesions typical of post-weaning multisystemic wasting syndrome (PMWS), a disease associated to PCV2 infection. However, IHC and/or PCR analyses showed that clinically silent PCV2 infection developed in five of the eight inoculated pigs, regardless of the administration of the vaccine. In particular, PCV2 DNA and/or antigen were detected in most of the tissues examined in the two pigs with the lowest titer of maternal PCV2 antibodies at the beginning of the trial. PPV DNA was not detected in any of the samples examined. The five pigs with PCR and/or IHC evidence of PCV2 infection had a mean weight gain during the experiment lower than that of the inoculated PCR-negative pigs considered together and that of the control pigs. In conclusion, it would appear that passive immunity against PCV2 can play a role in preventing the development of PMWS, but is not able to prevent the establishing of clinically silent PCV2 infections. The dissemination and persistence of the virus in the tissues may depend on the level of PCV2 antibodies at the time of inoculation.     

Effect of porcine parvovirus vaccination on the development of PMWS in segregated early weaned pigs coinfected with type 2 porcine circovirus and porcine parvovirus

The objectives of this study were to determine if coinfection of segregated early weaned (SEW) pigs with porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) induces an increase in the incidence of post-weaning multisystemic wasting syndrome (PMWS) compared to singular PCV2 infection, and to determine if vaccination against PPV protects pigs against PMWS associated with PCV2/PPV coinfection in SEW pigs. Seventy, 3-week-old, SEW pigs were randomly assigned to one of the five groups. Pigs in group 1 (n = 14) served as the negative controls, group 2 pigs (n = 14) were inoculated with PCV2, group 3 pigs (n = 12) were inoculated with PPV, groups 4 (n = 16) and 5 (n = 14) pigs were inoculated with both PCV2 and PPV. Pigs in groups 1-3 and 5 were vaccinated with two doses of a killed parvovirus-leptospira-erysipelothrix (PLE) vaccine prior to inoculation. The PCV2/PPV-coinfected pigs (groups 4 and 5) had significantly (P < 0.05) higher and more persistent fevers than the singular PCV2-infected pigs. One pig in each of the coinfected groups developed clinical disease (fever, respiratory disease, jaundice, weight loss) consistent with PMWS. Lymphoid depletion was significantly (P < 0.05) more severe in the dually-infected pigs at 42 days post-inoculation (DPI). Vaccinated, coinfected pigs (group 5) remained viremic significantly (P < 0.05) longer and had higher copy numbers of genomic PCV2 DNA in sera at 28, 35, and 42 DPI compared to the unvaccinated coinfected pigs (group 4). PPV-viremia was detected only in the unvaccinated group 4 pigs. PLE-vaccination prevented PPV-viremia but did not prevent clinical PMWS or reduce the severity of lymphoid depletion in PCV2/PPV-coinfected pigs. Evidence of increased incidence of clinical PMWS due to vaccination was not observed in this model.     

Hepatitis and staging of hepatic damage in pigs naturally infected with porcine circovirus type 2

A total of 100 liver samples from pigs with postweaning multisystemic wasting syndrome (PMWS) were studied. All livers were evaluated microscopically and were staged based on the severity and localization of lesions. Presence of porcine circovirus type 2 (PCV-2) was evaluated using an in situ hybridization technique. Eighty-eight of 100 livers (88%) had a variable degree of lymphohistiocytic hepatitis, with apoptotic bodies, disorganization of hepatic plates, and/or perilobular fibrosis. Twelve pigs did not have microscopic liver lesions. Four stages of hepatic damage were established based on intensity and distribution of the lesions. Viral nucleic acid was detected in 70 of 100 livers (70%). Target cells for PCV-2 infection included Kupffer cells, hepatocytes, and inflammatory infiltrates. According to distribution of PCV-2 nucleic acid, four basic labeling patterns were identified. This study shows that liver damage is a frequent microscopic finding in cases of PMWS and hepatocytes are a target cell for PCV-2 infection and replication. Therefore, PCV-2 should be considered a new hepatitis-inducing viral agent in pigs.     

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.     

Fetal infections and antibody profiles in pigs naturally infected with porcine circovirus type 2 (PCV2)

The aim of this study was to describe early infections with porcine circovirus type 2 (PCV2) in naturally infected piglets and the piglets' serologic profiles. A total of 20 sows (15 PCV2-vaccinated and 5 unvaccinated) and 100 newborn piglets were studied. Colostrum and serum of the sows and serum of the presuckling piglets were obtained on the day of parturition. Milk samples were collected on day 20 postpartum. Blood samples were taken and the piglets weighed on days 1, 20, 42, 63, and 84 postpartum. Colostrum and milk were evaluated for infectious PCV2 and for PCV2 total antibody (TA), neutralizing antibody (NA), and IgA. Serum samples were evaluated for PCV2 TA, NA, IgA, IgM, and DNA. The sows had high levels of TA and NA in serum and colostrum; however, 11 and 5, respectively, of the 20 colostrum and milk samples contained infectious PCV2. In the serum, PCV2 DNA and IgM were detected in 17 and 5, respectively, of the 20 sows. Nine piglets were born with PCV2 antibodies, which indicates in utero transmission of PCV2 after the period of immunocompetence (> 70 d of gestation). On day 1 postpartum, PCV2 DNA was detected in 29 of the 100 serum samples from the piglets. There was no difference between the weights of viremic and nonviremic piglets throughout the study. In conclusion, even on farms with sows that have high PCV2 antibody titers, vertical transmission of PCV2 may occur, resulting in piglet infection.     

实时定量PCR对猪圆环病毒2型感染猪体内病毒分布规律的研究

根据发表的猪圆环病毒2型（PCV2）ORF2基因序列设计了一对特异引物,并以克隆重组质粒作为阳性标准品,采用SYBR-GreenⅠ嵌合荧光染料建立了一种用于检测PCV2的实时定量PCR方法。该方法在10^7～10^1范围内具有良好的线性关系,相关系数为R2=0．997,扩增效率为E=0．920;对质粒标准品检测下限值为8．2拷贝／μL,检测变异系数低于2．0％。该方法与常规PCR及过氧化物酶单层细胞试验（IPMA）比较,其敏感性提高10^3倍以上。采用该法对PCV2人工感染猪的心、肝、脾、肺、肾、腹股沟淋巴结等组织中病毒核酸载量检测结果表明,在多种脏器中均可检9n,4到病毒,其中腹股沟淋巴结、扁桃体和脾脏中病毒含量较高（为3．4&#215;10^9拷贝儋～1．7&#215;10^10拷贝／g）,这表明病毒主要在免疫器官增殖,导致淋巴细胞耗损。对来自国内不同地区的28份临床发病猪病料进行病毒DNA定量检测,有半数以上病料的病毒载量达到10^9-10拷贝／g。实验表明,实时定量PCR可用于PCV2核酸定量检测,为该病毒体内外定量检测提供了一种技术手段。     

Proliferative and necrotising pneumonia and severe vascular lesions in pigs naturally infected with porcine circovirus type 2

Severe disease induced by porcine circovirus type 2 (PCV2) was observed in three pigs originating from a large herd affected by respiratory and digestive signs as well as wasting. Proliferative and necrotising pneumonia (PNP) was diagnosed in two animals, while severe acute interstitial pneumonia characterised by the presence of abundant hyaline membrane in the alveoli and fibrin in the bronchioles was found in one pig. In all cases, large amounts of PCV2 antigen were found in each tissue sample collected from the lungs and mediastinal lymph nodes. Neither porcine reproductive and respiratory syndrome virus (PRRSV) nor swine influenza virus (SIV) was detected, and no bacteria could be cultured in any of the cases. Vascular lesions, e.g. degeneration of endothelial cells, perivascular and intramural oedema, fibrinoid necrosis, vasculitis, perivasculitis, and vascular thrombi were observed in all cases, associated with the presence of PCV2 antigen. The viral antigen was present in the intravascular mononuclear cells, endothelial cells, myocytes and infiltrating inflammatory cells in lymph and blood vessels. In one case, obliterating thrombi in the lymph and blood vessels were directly connected to areas of tissue necrosis and were associated with abundant PCV2 antigen. The results further suggest the causative role of PCV2 infection in PNP, and the importance of the vascular system in the pathogenesis of PCV2-associated diseases of swine.     

Efficacy of bivalent vaccines of porcine circovirus type 2 and Mycoplasma hyopneumoniae in specific pathogen-free pigs challenged with porcine circovirus type 2d

BackgroundPorcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (MHP) are economically significant pathogens in the pig industry. The use of combined vaccines against PCV2 and MHP is one of the most effective ways of protecting pigs from both diseases, and it has become a part of general management.ObjectivesThis study evaluated the efficacy of two new bivalent vaccines of PCV2 and MHP (Myco-X and Myco-XD) in SPF pigs. Myco-X and Myco-XD are a combined vaccine of MHP with PCV2b and PCV2d, respectively.MethodsSixteen pigs were divided into four groups: Myco-X-vaccinated challenged, Myco-XD-vaccinated challenged, unvaccinated challenged, and unvaccinated unchallenged. Two milliliters of Myco-X were administered intramuscularly, and 0.5 mL of Myco-XD was injected intradermally at 3 wk of age. The pigs were challenged with virulent PCV2d via the intramuscular and intranasal route 4 wk post-vaccination.ResultsAll vaccinated pigs showed effective reduction of the clinical signs, the PCV2d load in the blood and nasal swab samples, as well as lung and lymphoid tissue lesions in the challenge test. Compared to unvaccinated challenged animals, the vaccinated challenged animals showed significantly higher (p < 0.05) levels of anti-PCV2 IgG, PCV2d-specific interferon-γ (IFN-γ), and anti-MHP IgG.ConclusionsBased on clinical, microbiological, serological, and pathological assessments, this study confirmed that both combined vaccines could protect pigs against PCV2 infection caused by PCV2d. On the other hand, further research on the efficacy evaluation of these new vaccines against the MHP challenge and PCV2d/MHP co-challenge is needed.     

猪圆环病毒2型检测与免疫技术研究进展

对年近来国内外对猪圆环病毒2型(PCV2)的检测技术进展和疫苗研究进展作了简要回顾。PCV2实验室检验技术有间接免疫荧光(IFA)、间接酶联免疫吸附试验(ELISA)、免疫过氧化物酶单层细胞试验(IPMA)、单克隆抗体以及多种聚合酶链式反应(PCR)技术等。在疫苗研究方面,国外已研发多种PCV2灭活疫苗,国内亦有人正在研发PCV2灭活苗(SH株)。在PCV2基因工程疫苗研究方面主要有重组活载体疫苗和核酸疫苗两类。     

Enteritis associated with porcine circovirus 2 in pigs

This report describes the diagnosis of porcine circovirus 2 (PCV2)-associated enteritis in 6 weaned pigs without postweaning multisystemic wasting syndrome by histopathology, virus isolation, and in situ hybridization. The most unique lesions were granulomatous inflammation affecting Peyer's patches, characterized by infiltrates of epithelioid macrophages and giant multinucleated cells. Large, multiple, basophilic or amphophilic, grape-like intracytoplasmic inclusion bodies were often seen in the cytoplasm of histiocytic cells and giant multinucleated cells. No microscopic lesions were observed in the lymphoid tissue, such as lymph node, spleen, and tonsil. A strong hybridization signal for PCV2 was detected in the cytoplasm of histiocytes and giant multinucleated cells in Peyer's patches. Porcine circovirus 2 was isolated from homogenates of the small and large intestines in 2 weaned pigs. The presence of diarrhea and granulomatous enteritis, and abundant PCV2 DNA associated with the microscopic lesions is suggestive of PCV2-associated enteritis. Thus, PCV2-associated enteritis could be a distinct clinical manifestation of PCV2.     

Experimental reproduction of severe disease in CD/CD pigs concurrently infected with type 2 porcine circovirus and porcine reproductive and respiratory syndrome virus

Three-week-old cesarean-derived colostrum-deprived (CD/CD) pigs were inoculated with porcine circovirus type 2 (PCV2, n = 19), porcine reproductive and respiratory syndrome virus (PRRSV, n = 13), concurrent PCV2 and PRRSV (PCV2/PRRSV, n = 17), or a sham inoculum (n = 12) to compare the independent and combined effects of these agents. Necropsies were performed at 7, 10, 14, 21, 35, and 49 days postinoculation (dpi) or when pigs became moribund. By 10 dpi, PCV2/PRRSV-inoculated pigs had severe dyspnea, lethargy, and occasional icterus; after 10 dpi, mortality in this group was 10/11 (91%), and all PCV2/ PRRSV-inoculated pigs were dead by 20 dpi. PCV2-inoculated pigs developed lethargy and sporadic icterus, and 8/19 (42%) developed exudative epidermitis; mortality was 5/19 (26%). PRRSV-inoculated pigs developed dyspnea and mild lethargy that resolved by 28 dpi. Microscopic lesions consistent with postweaning multisystemic wasting syndrome (PMWS) were present in both PCV2- and PCV2/PRRSV-inoculated pigs and included lymphoid depletion, necrotizing hepatitis, mild necrotizing bronchiolitis, and infiltrates of macrophages that occasionally contained basophilic intracytoplasmic inclusion bodies in lymphoid and other tissues. PCV2/ PRRSV-inoculated pigs also had severe proliferative interstitial pneumonia and more consistent hepatic lesions. The most severe lesions contained the greatest number of PCV2 antigen-containing cells. PRRSV-inoculated pigs had moderate proliferative interstitial pneumonia but did not develop bronchiolar or hepatic lesions or lymphoid depletion. All groups remained seronegative to porcine parvovirus. The results indicate that 1) PCV2 coinfection increases the severity of PRRSV-induced interstitial pneumonia in CD/CD pigs and 2) PCV2 but not PRRSV induces the lymphoid depletion, granulomatous inflammation, and necrotizing hepatitis characteristic of PMWS.     

猪圆环病毒2型感染对猪肺泡巨噬细胞生物学活性的影响

将猪圆环病毒2型（PCV2）BF株经口、鼻接种40日龄健康仔猪，在接种后不同时间宰杀，收集猪肺泡巨噬细胞（PAM），同时设立对照。用FITC—Annexin V／PI双染色流式细胞术和琼脂糖凝胶电泳检测PAM凋亡现象，通过EA花环试验测定Fc受体数目，通过吞噬鸡红细胞试验测定吞噬功能，分析PCV2感染对PAM生物学活性的影响。结果显示，在整个试验期内2种方法均没有检测到PAM凋亡，表明PCV2感染不会诱发PAM凋亡。PAM的Fc受体数和吞噬鸡红细胞数的变化规律一致，与对照组相比，两者数量在接种后第3d明显下降，第7d有所回升，之后基本恢复，表明PCV2感染后PAM吞噬和清除病原的功能出现短暂下降。     

Coinfection by Cryptosporidium parvum and porcine circovirus type 2 in weaned pigs

Routine histopathological diagnosis of one representative 3-month-old pig from a group suffering from diarrhoea revealed a massive degree of parasitation by Cryptosporidium parvum, with a concomitant infection by porcine circovirus type 2 (PCV2), that was confirmed by immunohistochemical procedures. The areas of intestine where parasites were more numerous presented abundant PCV2 infected cells in mucosa and submucosa. The concurrence of C. parvum, a rare primary intestinal pathogen in post-weaning and growing pigs, and PCV2 infections suggest an increased susceptibility as a result of an immunosuppression state.     

Commercially produced spray-dried porcine plasma contains increased concentrations of porcine circovirus type 2 DNA but does not transmit porcine circovirus type 2 when fed to naive pigs

The porcine circovirus type 2 (PCV2) antibody and DNA status of porcine plasma products collected during the commercial spray-drying process were evaluated. Samples evaluated included 52 pooled liquid plasma (fresh) samples collected at 14 regional abattoirs before transport to 1 of 2 spray-drying facilities, 32 pooled liquid plasma (concentrated) samples collected after arrival at the spray-drying facilities at different stages before the spray-drying process, and 32 samples in powdered form (spray-dried) collected after spray drying. All 116 samples were positive for PCV2 antibody, with PCV2 ELISA sample-to-positive ratios ranging from 9.2 to 13.6 on a DM basis. Porcine circovirus type 2 DNA (4.5 to 7.9 log(10) PCV2 copies/mL, DM basis) was present in 82.7% (43/52) of the fresh plasma samples, 71.9% (23/32) of the concentrated plasma samples and 78.1% (25/32) of the spray-dried plasma samples, with a greater prevalence of PCV2b than PCV2a. To determine the infectivity of PCV2 DNA-positive commercial spray-dried plasma, nine 10-wk-old 68-kg PCV2-na?ve pigs were randomly assigned to 1 of 3 treatment groups and rooms: 1) a negative control (no plasma in the feed, not inoculated with PCV2); 2) a positive control (no plasma in the feed, inoculated with PCV2); and 3) plasma-fed pigs (4% porcine plasma in the feed for 42 d, not inoculated with PCV2). All positive control pigs became viremic by 7 d postinoculation and seroconverted by 42 d postinoculation, whereas pigs in the negative control group and in the spray-dried plasma group were PCV2 PCR negative and did not seroconvert to PCV2 for the duration of the study. The results indicate that PCV2 DNA and antibodies are commonly found in commercial spray-dried plasma. However, no evidence of infectivity of the PCV2 DNA was found in na?ve pigs when commercial spray-dried plasma was included in the diet under the conditions of this study.     

Transient correlation between viremia levels and IL-10 expression in pigs subclinically infected with porcine circovirus type 2 (PCV2)

Immunological impairment by porcine circovirus type 2 (PCV2) infection is well documented in pigs suffering from postweaning multisystemic wasting syndrome. Nonetheless, little is known about immune status of pigs that remain PCV2 subclinically infected. Thus, seven pigs successfully infected in an experimental inoculation and without developing disease and nine control non-inoculated pigs were examined. Serological, virological and immunological determinations were done throughout ten weeks post-infection (PI). At week 3 PI, inoculated animals presented the peak of viremia and produced higher levels of IL-10 than the controls; correlation between viral load and IL-10 amounts was observed (p<0.05). Also, the ratio IgM/IgG suffered a shift skewing IgM production towards an IgG response. By 10 weeks PI, levels of IL-10 disappeared and the viremia decreased. In summary, subclinically PCV2-infected pigs developed a transient PCV2-specific IL-10 response during the viremic phase of infection which coincided with the inversion of the IgM/IgG ratio.     

Genetic diversity of porcine circovirus type 2 from pigs in Republic of Korea

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS). In this study, 38 PCV2 cases obtained from different pig farms with different health conditions in Republic of Korea were sequenced and analyzed. Phylogenetic analysis showed that our cases had a greater variation and the existence of two PCV2 groups with at least four subgroups (1A, 1C, 2D, and 2E). Most cases obtained from PMWS-affected herds were in group 1, whereas cases with no clinical signs compatible with PMWS (wasted non-PMWS) were included within group 2. Moreover, four cases from the wasted non-PMWS belonged to PCV2-group 1. Therefore, our results suggest that PCV2-group 1 is more related to PMWS than group 2.     

Effect of porcine circovirus type 2 (PCV2) vaccination on PCV2-viremic piglets after experimental PCV2 challenge

The objective of this study was to evaluate the effect of porcine circovirus type 2 (PCV2) vaccines on PCV2-viremic and -seropositive piglets born from naturally PCV2-infected sows against postnatal PCV2 challenge. The experimental design was aimed at mimicking commercial swine rearing conditions to evaluate the response of the PCV2 vaccine on PCV2-viremic and -seropositive piglets after experimental PCV2 challenge. PCV2a (or 2b)-viremic piglets received a PCV2 vaccine at 21 days of age followed by a PCV2b (or 2a) challenge at 49 days of age (28 days post vaccination). The PCV2 vaccines elicited a high level of humoral (as measured by immunoperoxidase monolayer assay and neutralizing antibody titers) and cellular (as measured by the frequency of PCV2-specific interferon-γ-secreting cells) immune response in the PCV2-viremic piglets after vaccination even in the presence of maternally derived antibodies (MDA). The initial infection of PCV2 in the pigs was not affected by PCV2 vaccination, however the challenging PCV2 was reduced by PCV2 vaccination on PCV2-viremic pigs. The results from this study demonstrate that the PCV2 vaccine used in this study is effective at reducing PCV2 viremia and lymphoid PCV2 DNA, even for PCV2-viremic pigs with passively acquired MDA at the time of vaccination.