Genetic characterization and phylogenetic analysis of porcine circovirus type 2 strains present in Cuban swine herds

Porcine circovirus type 2 (PCV2) is the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS) considered as one of the most important swine diseases worldwide. One of the main risk factors reported for the development of PMWS is the PCV2 genotype. The presence of PCV2 in Cuban swine herds has been reported recently. However, genetic information about these viruses is not available yet. Hence, the objectives of this study were to classify the Cuban porcine circovirus type 2 sequences as well as to investigate the genetic diversity and the putative origins of the virus circulating in Cuban swine herds. PCV2 Cuban sequences appeared to be close related when an analysis of the entire viral genome sequences was performed. The main variations on amino acid sequences of the capsid protein were found within the immunoreactive areas. All the Cuban PCV2 sequences analyzed belonged to genotype 1 and were located within the same Cluster (1A). Interestingly, five of them were clustered with high confident values with those described as the PCV2 variants associated with severe porcine circovirus diseases reported in Canada from the late 2004 to 2006. Pigs imported from this source appeared to be the most probable origin of the viruses circulating in Cuban swine herds currently. The fact that one sequence was not clustered with any other group of PCV2 within genotype 1 might suggest that different introductions of the agent in the country from unknown sources have occurred.     

High prevalence of bovine viral diarrhea virus 1 in Chinese swine herds

Nested RT-PCR was used to investigate bovine viral diarrhea virus in 511 specimens collected from Chinese pigs exhibiting clinical symptoms between 2007 and 2010. Of these, 137 samples were BVDV-positive and the BVDV prevalence rate was 23.1% (9/39) in 2007, 27.7% (44/159) in 2008, 33.6% (34/101) in 2009, and 23.6% (50/212) in 2010. Twenty of 137 BVDV-positive samples were used for further genetic analysis of the 5'-UTR. Phylogenetic analysis revealed that they were BVDV-1 and subtyped into BVDV-1a, BVDV-1b, BVDV-1m, BVDV-1o and an unknown subgenotype. This study showed that BVDVs were highly prevalent in Chinese pig herds and appropriate measures should be taken to control BVDV prevalence in pig herds.     

Prevalence of porcine circovirus type 2 in aborted fetuses and stillborn piglets

A retrospective study of natural cases of abortion, recorded between October 2000 and September 2002, was made to determine the prevalence of abortions associated with porcine circovirus type 2 (PCV-2). The virus was detected by PCR, immunohistochemistry and in situ hybridisation. A total of 46 (13.1 per cent) of 350 aborted fetuses and stillborn piglets were positive for PCV-2 by PCR, and the virus was detected in fetuses at all stages of gestation. Viral antigen was detected in macrophages from the aborted fetuses and stillborn piglets by immunohistochemistry, and viral DNA was detected by in situ hybridisation.     

Spread of pseudorabies virus among breeding swine in quarantined herds.

In theory, pseudorabies virus (PRV) may be eliminated from any size of breeding herd by phased test and removal if replacement gilts are not infected with PRV, culling decisions are partially based on PRV status, and the cull rate is higher than the incidence rate of PRV. Annual cull rates are commonly at least 50%, but little information exists on the incidence of PRV within enzootically infected swine herds. The purpose of this study was to develop a method by which spread of PRV could be detected among breeding swine within enzootically infected herds and to determine the incidence of PRV infection in these herds. Data were collected from 17 herds that were quarantined for PRV and ranged in size from 120 to 1,100 sows. At each herd, within the first 5 days of introduction, a group of approximately 30 replacement gilts was identified, vaccinated with a glycoprotein X-deleted PRV vaccine, and blood sample was collected. The owner of 1 herd had a nonvaccinated breeding herd and elected to leave incoming gilts nonvaccinated. After vaccination, blood samples were collected every 1 to 2 months for an average of 13.6 months. Serum samples from vaccinated gilts were tested for antiglycoprotein X antibodies by a specific differential ELISA. Samples from nonvaccinated gilts were evaluated by serum neutralization test. Product-limit method was used to estimate the probability of not becoming infected with PRV. Spread was detected in 7 of 8 herds that had more than 400 sows and in 2 of 9 herds that had less than 400 sows.(ABSTRACT TRUNCATED AT 250 WORDS)     

猪圆环病毒和猪繁殖与呼吸综合征病毒混合感染对仔猪致病性的评估

本研究通过猪圆环病毒2型(Porcine circovirus type 2,PCV2)和猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome virus,PRRSV)强毒共感染3周龄健康仔猪来评价其致病性。试验动物随机分为3组,空白对照组(n=3头),PRRSV单独感染组(n=3头),PCV2和PRRSV共感染组(n=6头),从而比较相互之间的差异。通过临床症状、病理学变化、病原学和血清学检查,对二者混合感染仔猪的致病性进行了研究。结果表明PCV2和PRRSV共同感染能引起仔猪断奶后多系统消耗性综合征,表现为淋巴组织肿大、出血,肉芽肿性炎症,坏死性肝炎,仔猪消瘦、生长缓慢等特征性病变;混合感染能加重PRRSV对仔猪引起的间质性肺炎的严重程度。混合感染可以出现支气管肺炎和明显的肝病变,淋巴结多呈界限明显的块状出血等典型病变。     

Absence of evidence of porcine circovirus infection in piglets with congenital tremors.

Porcine circovirus types 1 (PCV1) and 2 (PCV2) have been associated with congenital tremors (CTs) in piglets in the United States. In this study, central nervous system and nonneural tissues of 40 CT piglets from Spain, the United Kingdom, Ireland, and Sweden were investigated for the presence of PCV1 and PCV2 using in situ hybridization and immunohistochemical labeling on paraffin sections. The polymerase chain reaction for PCV2 was also carried out on sera from the Spanish CT cases. No evidence of circovirus nucleic acid or antigen was found in any CT piglet. Although these results do not support the hypothesis that PCV1 or PCV2 are linked to porcine CT, they cannot disprove it.     

猪圆环病毒2型SD/2008株对仔猪的致病性

为了明确PCV2-SD/2008株对猪的致病性,将12头5周龄健康断奶仔猪随机分为PCV2组和对照组,每组6头.PCV2组经口鼻途径接种PCV2 SD/2008株第3代细胞毒(105.61 TCID50/0.1 mL),3 mL/头,对照组经相同途径接种3 mL细胞维持液.PCV2组中的1头猪从感染后14 d开始出现间断腹泻,一直到持续到试验结束.感染后第4、5周,感染组的周平均增重显著低于对照组(P＜0.05).病理组织学检查可见PCV2感染猪的淋巴结和脾脏内淋巴细胞缺失、单核/巨噬细胞和嗜酸性粒细胞浸润;间质性肺炎、坏死性肝炎和肾炎、小血管炎症等变化,并且感染后35 d比感染后14 d的病理变化更加严重.应用免疫组化方法在感染猪的肺、肝、脾、肾、淋巴结、小肠等多种组织中检测到了PCV2抗原,其中颌下淋巴结、肺、小肠中的阳性信号数与检出率最高.结果表明,PCV2 SD/2008株对仔猪具有明显的致病性.     

The survey of porcine teschoviruses, porcine circovirus and porcine transmissible gastroenteritis virus infecting piglets in clinical specimens in China

A multiplex PCR assay was developed and evaluated for its ability to simultaneously detect three viral infections of swine. Specific primers were carefully selected from articles published for each of the following three viruses: porcine circovirus type II (PCV2), porcine teschovirus (PTV) and porcine transmissible gastroenteritis virus (TGEV). Each target produced a specific amplicon with a size of 353 bp (PCV2), 168 bp (PTV) and 499 bp (TGEV). The sensitivity of the multiplex PCR using purified plasmid constructs containing the specific viral target fragments was 6.60?×?10², 8.43?×?10² and 7.30?×?10² copies for PCV2, PTV and TGEV, respectively. Among 127 samples which were collected from Heilongjiang, Jilin, Henan and Guangxi provinces, the single infection of PCV2, PTV and TGEV was 99.21, 46.88 and 65.35 %, respectively, and co-infection of the three viruses was 26.77 %. In conclusion, the multiplex PCR has the potential to be useful for routine molecular diagnosis and epidemiology.     

免疫组化Envision法检测肺组织中猪圆环病毒抗原的研究

为了对猪圆环病毒Ⅱ型(PCV2)的早期诊断、临床诊断和流行病学调查提供技术支持,本试验制备了兔抗PCV2多克隆抗体,采用免疫组织化学Envision二步法对30例疑似PCV2病猪肺组织的病毒抗原进行定位、半定量检测.结果表明,试验制备的兔抗PCV2抗体具有高纯度和良好的特异性;Envision法可原位检测病猪肺组织PCV2抗原的分布,其抗原的阳性表达主要出现在肺巨噬细胞胞浆内;依据阳性细胞百分率,进行染色结果判断,检出阳性病猪16例,阳性检出率为53.37%.实验证明Envision法具有高敏感、低背景、快速简便的特点,可在兽医病理诊断中推广应用.     

Epidemiologic assessment of porcine circovirus type 2 coinfection with other pathogens in swine

OBJECTIVE: To identify important pathogens and characterize their serologic and pathologic effects in porcine circovirus type 2 (PCV2)-infected pigs in relation to pig age and type of swine production system. DESIGN: Cross-sectional study. ANIMALS: 583 conventionally reared pigs. PROCEDURES: 3- (n = 157), 9- (149), 16- (152), and 24-week-old (125) pigs from 41 different 1-, 2-, and 3-site production systems (5 pigs/age group/farm) were euthanized and necropsied. Pigs with and without PCV2 infection were identified (via PCR assay); infection with and serologic responses to other pathogens and pathologic changes in various tissues (including lungs) were assessed. Logistic regression models were constructed for effects overall and within each age group and type of production system. RESULTS: Compared with PCV2-negative pigs, PCV2-positive pigs were more likely to have swine influenza virus (SIV) type A and Mycoplasma hyopneumoniae infections and sample-to-positive (S:P) ratios for SIV H1N1 from 0.50 to 0.99; also, PCV2-positive pigs had higher serum anti-porcine reproductive and respiratory syndrome virus (PRRSV) antibody titers and more severe lung tissue damage. Infection with SIV (but lower SIV H1N1 S:P ratio) was more likely in 3-week-old PCV2-positive pigs and evidence of systemic disease was greater in 16-week-old PCV2-positive pigs than in their PCV2-negative counterparts. By site type, associations of coinfections and disease effects between PCV2-positive and -negative pigs were greatest in 3-site production systems. CONCLUSIONS AND CLINICAL RELEVANCE: In PCV2-positive pigs, coinfections with SIV, M. hyopneumoniae, and PRRSV are important, having the greatest effect in the early to late nursery phase and in 3-site production systems.     

猪GM-CSF基因对猪圆环病毒2型DNA疫苗诱导仔猪免疫反应的调节作用

粒细胞-巨噬细胞集落刺激因子（GM-CSF）能够诱导树突状细胞的成熟和存活，并提高其抗原递呈功能，是非常有潜力的分子佐剂之一。为了探讨猪GM-CSF对猪圆环病毒2型（PCV2）DNA疫苗诱导免疫反应的调节作用，本研究将表达猪GM-CSF和PCV2衣壳蛋白的重组质粒混合物（pcDNA3．1-gm-csf和pcDNA3．1-orf2）及在同一载体上表达PCV2衣壳蛋白与猪GM-CSF的双表达重组质粒（pIRES-orf2／gm-csf），分别接种于7日龄健康仔猪，共肌肉注射3次，每次间隔2周。结果表明猪GM-CSF基因能够明显增强PCV2DNA疫苗诱导的特异性体液免疫反应，提高外周血淋巴细胞（PBLC）对ConA刺激的增殖活性，增强NK细胞的杀伤功能，影响细胞因子IL-12、IFN-γ、IL-4和IL-10的表达水平。但GM-CSF基因单独表达和与PCV2衣壳蛋白基因在同一载体上表达，对上述4个细胞因子表达的影响有一定差异。     

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.     

猪瘟病毒和猪圆环病毒混合感染病例的诊治

本文对2017年5月山东省新泰市某养殖场的一头病死猪病例进行了诊断,经临床症状检查、病理剖检、RT-PCR检测、PCR检测等,最终判定该病例为猪瘟病毒和圆环病毒混感。     

断奶前仔猪经胎盘感染猪圆环病毒2型对猪流行性腹泻病毒引起的肠炎的影响

本文报道了经胎盘感染猪圆环病毒2型对猪流行性腹泻病毒引起新生仔猪肠炎的影响。6头怀孕母猪被随机分为感染组和对照组,每组3头。感染组3头怀孕母猪在分娩前3周鼻内接种6mLPCV2组织培养毒(病毒含量为1.2×105TCID50/mL,毒株为SNUVR000470);对照组3头怀孕母猪同时接种正常细胞培养上清液。PCV2感染母猪所产的30头仔猪被随机分为A、B两组,每组15头;对照组非感染母猪所产的30头仔猪被随机分为C、D两组,每组15头。A和C组仔猪在3日龄口服2mLPEDV第3次传代病毒液(病毒含量为1×106.5TCID50/mL,毒株为SNUVR971496)。在感染后36、48和72h测量绒毛高度与隐窝深度的平均比值,A组PCV2感染母猪所产的仔猪感染PEDV与C组PCV2阴性母猪所产的仔猪感染PEDV差异显著。在感染后24h,A组PEDV感染仔猪的空肠组织中比C组检测到更多的PEDV核酸。此后,在感染后36、48、60和70h,C组PCV2阴性母猪所产的PEDV感染仔猪的空肠组织中比A组检测到更多的PEDV核酸。由此推断,经胎盘感染PCV2显著影响PEDV所致疾病的临床进程。     

Seroprevalence of porcine circovirus type 2 in swine populations in Canada and Costa Rica

Porcine circovirus (PCV) was recently divided into 2 antigenically distinct types that differ (65% amino acid identity) in the protein encoded by open reading frame 2 (ORF2). Porcine circovirus 1 is apparently non-pathogenic and, in contrast, PCV2 is associated with porcine multisystemic wasting syndrome (PMWS). Our objective was to determine the extent of exposure of normal pigs in Canada and Costa Rica to PCV2. Recombinant DNA techniques were used to produce an antigen from ORF2 of PCV2 that was suitable for the detection of antibody in swine sera. The presence of PCV2 nucleotide sequences was detected using polymerase chain reaction (PCR) techniques. Using these tests, specific antibody and nucleotide sequences were demonstrated in sera from a cohort of pigs during a PMWS outbreak. Antibody was detected in normal, healthy hogs slaughtered in Canada (82.4% of 386) and in Costa Rica (14.6% of 322). This is the first report indicating the presence of PCV2 in Latin America. More than 50% of these sera also contained PCV2 nucleotide sequence. Although these hogs were healthy when slaughtered, they were infected with PCV2 and may have previously been ill. The widespread occurrence of PCV2 in swine suggests that this virus is adapted to replication in porcine tissue.     

Experimental in utero inoculation of late-term swine fetuses with porcine circovirus type 2.

All 37 fetuses of 3 laparotomized pregnant sows at 86, 92, and 93 days of gestation were inoculated intramuscularly through the uterine wall with porcine circovirus type 2 (PCV-2). The sows were allowed to farrow, and blood and tissue samples were collected from their piglets before and after suckling colostrum. Thirteen fetuses from 2 sows at 90 and 103 days of gestation were used as controls. Of the 37 PCV-2 inoculated fetuses, 24 were grossly normal and 13 were mummified, stillborn, or weak-born at farrowing. Infection with PCV-2 was demonstrated in various tissues of grossly normal and abnormal fetuses by virus isolation, polymerase chain reaction, and immunohistochemical methods. Antibodies specific to PCV-2 were also detected from the sera or thoracic fluids of abnormal fetuses and unsuckled normal pigs. No evidence of PCV-2 infection was found in any control fetuses. The present results confirm previous findings that PCV-2 can infect late-term swine fetuses and may cause reproductive abnormalities.     

Factors associated with spread of pseudorabies virus among breeding swine in quarantined herds.

Knowledge of the factors that place susceptible gilts at highest risk of pseudorabies virus (PRV) infection in a quarantined herd is crucial to reduce spread of PRV within the herd. Cohorts of PRV seronegative gilts were monitored in 17 herds that were endemically infected with PRV to determine the location of breeding females at the time of infection with PRV and identify herd characteristics and management and housing factors that may influence spread of PRV in the breeding section of swine herds endemically infected with PRV. Blood samples were collected every 1 to 2 months for an average of 13.6 months. In addition, blood was collected from a representative sample of finishing pigs (greater than or equal to 20 weeks old) 3 times per year to determine their serologic PRV status. Incidence rates and relative risks of PRV infection were estimated for 4 areas of the breeding section: gestation barn, gilt pool, farrowing room, and breeding area. Overall, 28, 11, 8, and 2 females became infected with PRV in each of these areas, respectively. The greater number of females infected in the gestation barns, compared with the number of females infected in other locations, is probably a consequence of being at risk for a longer period rather than of a higher incidence rate. Herd size, common housing for gilts in the gilt pool and sows, and serologic pattern of PRV infection in finishing pigs were associated with the detection of spread of PRV in the breeding section of the 17 herds.(ABSTRACT TRUNCATED AT 250 WORDS)