断乳仔猪多系统衰竭综合征病原检测与分析

应用PCR技术,结合,临床观察、病理解剖对福建省不同地区断乳多系统衰竭综合征（PMws）发病猪群采集的病料进行多重病原检测与分离。结果表明50份病料中猪圆环病毒2型（PCV2）、猪繁殖与呼吸综合征病毒（PRRSV）、猪瘟病毒（CSFV）、猪伪狂犬病病毒（PRV）阳性率分别为40％、26％、4％、0,不同地区各病原检出率差别不大。病毒性混合感染以PCV2和PRRSV为主,感染率为22％;PCV2与CFSV混合感染率为4％;PRRSV与CFSV混合感染率为4％;PCV2、CFSV、PRRSV三种病原混合感染率为4％。研究结果表明福建大部分地区猪群PMWS的发生是PCV2、PRRSV、CSFV等多种病原混合感染的结果,其主要病原为PCV2,该病的发生没有明显地域性。     

Isolation and characterization of porcine circovirus type 2 from pigs showing signs of post-weaning multisystemic wasting syndrome in The Netherlands

Pigs with wasting syndrome were examined for macroscopic and histopathological lesions, and for porcine circovirus (PCV). Histopathological lesions were comparable to those previously documented for post-weaning multisystemic wasting syndrome (PMWS). In addition, in seven out of ten examined PMWS-affected pigs focal-to-slight mononuclear meningitis and focal cerebral mononuclear infiltrates (4 out of 10) were observed. A virus was isolated from organs and sera from pigs showing wasting syndrome. An immunoperoxidase monolayer assay and an indirect immunofluorescence assay were performed on the infected PK-15 and Dulac cell cultures, respectively, and both assays indicated the presence of PCV type 2 (PCV2). The nested-polymerase chain reaction (nPCR) technique, based on the use of PCV2 specific oligonucleotides, revealed specific amplified products of 481 bp. Nucleotide sequence analysis of the entire genome of the Dutch PCV isolate 24657 NL showed a homology with known nucleotide sequences of porcine PCV type 1 (PCV1) and PCV2 isolates of 77.1% and >96%, respectively. This is the first report of the isolation and characterization of PCV2 in PMWS-affected pigs in the Netherlands.     

Induction of porcine post-weaning multisystemic wasting syndrome (PMWS) in pigs from PMWS unaffected herds following mingling with pigs from PMWS-affected herds

In this paper we present the results from two experimental studies (I and II) investigating whether post-weaning multisystemic wasting syndrome (PMWS) can be induced in pigs from PMWS unaffected herds by mingling with pigs from PMWS-affected herds and to observe whether transportation and/or mingling of healthy pigs from unaffected herds could induce PMWS.The studies comprised pigs from 12 different herds. Eight herds had PMWS while four were unaffected. All 12 herds were found to be infected with PCV2. Pigs from PMWS-affected herds were mingled with pigs from unaffected herds in four separate compartments in both study I and study II. In addition, in study II, four groups of pigs from unaffected herds were included. Two groups with pigs transported and mingled from unaffected herds and two groups with pigs which were only transported. The PMWS diagnoses on the individual pigs were based on lymphoid depletion, histiocytic proliferation and the presence of giant cells or inclusion bodies together with the demonstration of PCV2 in lymphoid tissue.Healthy pigs, in both studies, developed PMWS 4–5 weeks after mingling with pigs clinically affected with PMWS. None of the pigs from unaffected herds which had no contact with pigs from PMWS-affected herds developed clinical signs of PMWS. Transportation and mingling of pigs from PMWS unaffected herds in combination or alone was insufficient to provoke PMWS.     

Pathological investigations of pigs with porcine multisystemic wasting syndrome (PMWS)

Extract

Pathological changes and immunohistochemical (IHC) examination for porcine circovirus-2 (PCV-2) in nine weaner pigs from a property with PMWS are reported. The pigs were in moderate to poor body condition, and several had diarrhoea and noisy respiration. The outstanding pathological changes related to microscopic lesions in lymphoid tissues, including lymph nodes, palatine tonsils, Peyer's patches, peribronchial lymphoid aggregates and spleen. While there was variability of expression of change between and within cases, the main features of lesions could be summarised as depletion of lymphoid cells, infiltration of histiocytes (often syncytial), and the presence of botryoid intra-cytoplasmic inclusion bodies in macrophages. The changes in lymphoid tissue were typical of those described in PMWS. Also characteristic of this syndrome were histiocyte-dominated cellular inflammatory infiltrates in kidney, liver and lung. There was strong correlation between IHC positivity for PCV-2 and the lesions in lymphoid, pulmonary and renal tissues. Concurrent diseases that are probably promoted by immune suppression may complicate the diagnosis of PMWS. In the present cases, salmonellosis, attaching and effacing Escherichia coli infections, and secondary bacterial bronchopneumonia were identified.     

An ORF2 protein-based ELISA for porcine circovirus type 2 antibodies in post-weaning multisystemic wasting syndrome

Porcine circovirus type 2 (PCV2) plays a crucial role in the pathogenesis of post-weaning multisystemic wasting syndrome (PMWS) in swine. As PCV2 displays significant homology with PCV1 (a non-pathogenic virus) at the nucleotide and amino-acid level, a discriminative antigen is needed for specific serological diagnosis. The ORF2-encoded capsid protein from PCV2 was used to develop an indirect enzyme-linked immunosorbent assay (ELISA). GST-fused capsid protein from PCV2 and GST alone (both expressed in recombinant baculovirus-infected cells) were used as antigens for serodiagnosis. The specificity of the ELISA for detection of PCV2 antibodies was demonstrated in sera from pigs experimentally infected with PCV1, PCV2 and other swine viruses. The semi-quantitative nature of the test was evaluated versus an immunoperoxidase monolayer assay (IPMA). The ELISA was performed on 322 sera from pigs in eight Brittany herds and compared with IPMA. The sensitivity (98.2%) and specificity (94.5%) of this test were considered suitable for individual serological detection. High PCV2 seroprevalence was found in sows and pigs at the end of the growth phase (18-19 weeks) in all eight herds. The seroprevalence in piglets (11-17 weeks) was statistically correlated with clinical symptoms of PMWS (93% in affected versus 54%, in non-affected farms). A cohort study performed in PMWS-free farms showed that 57% of piglets exhibited active seroconversion after 13 weeks, indicating that PCV2 infection occurred earlier in PMWS-affected piglets.     

Protection of pigs against post-weaning multisystemic wasting syndrome by a recombinant adenovirus expressing the capsid protein of porcine circovirus type 2

Post-weaning multisystemic wating syndrome (PMWS) associated with PCV2 was one of the most costly diseases currently faced by the swine industry. In order to develop a vaccine to control this disease, we previously constructed a recombinant adenovirus expressing the capsid of PCV2. Here, we examined the protection of swine against PMWS by the recombinant adenovirus. Eighteen 32-day-old pigs were assigned to three groups each with six. Group 1 was vaccinated subcutaneously with rAd-Cap and boosted 2 weeks later. Thirty-seven days after first vaccination, Groups 1 and 2 were oronasally challenged with virulent PCV2 isolate, 4 and 7 days later, intramuscularly exposed to keyhole limpet hemocyanin (KLH). Group 3 remained unchallenged but with KLH. The results showed that high level of PCV2-specific ELISA antibody and neutralizing antibody could be induced at 37 days after first vaccination. After challenge, pigs in vaccinated group had no clearly clinical signs, although some of them had increased rectal temperatures (>/=40 degrees C) for short time. The pyrexic phase in vaccinated group was significantly lighter than that in challenge-control group (P<0.05). The relative daily weight gain in vaccinated-challenged group was similar to that in empty control group. But it was significantly high compared to the challenge-control group (P<0.05). Mean while the pathological lesions and virema presented in vaccinated group were milder than those in control group. It indicated that the recombinant adenovirus was able to confer significant protection against clinical disease and reduce pathogenic lesions induced by PCV2 challenge, even though it could not provide complete virological protection. The recombinant adenovirus might be an attractive candidate vaccine for preventing the disease associated with PCV2 infection.     

Risk factors for porcine post-weaning multisystemic wasting syndrome (PMWS) in 149 French farrow-to-finish herds

A cross-sectional study involving 149 farms was carried out in France in 2000 and 2001 to assess the risk factors for post-weaning multisystemic wasting syndrome (PMWS). The farms were divided into three groups according to their current or past PMWS status: CASES (current and typical PMWS), CONTROLS#1 (PMWS-free farms), and CONTROLS#2 (farms which have recovered from PMWS). Two different comparisons were tested: CASES versus CONTROLS#1 and CASES versus CONTROLS#2. In the first comparison, the odds of PMWS were increased when fattening pigs tested positive for parvovirus (PPv) and porcine reproductive and respiratory syndrome (PRRS) virus (OR=4.4 and 6.5, respectively), when separate vaccines for parvovirus and Erysipela for the gilts versus associated vaccines were used (OR=2.5), and when on-farm semen collection was used versus all the semen purchased from an insemination centre (OR=4.6). Large pens in weaning facilities increased the odds of PMWS (OR=4.1); whereas long empty periods in weaning and farrowing facilities versus shorter (OR=0.2), regular treatment against external parasites (OR=0.1), and housing the sows in collective pens during pregnancy versus individual pens (OR=0.3) all decreased the odds of PMWS. The same kinds of risk factors were found with the second comparison with, in addition, a common pit for several adjacent fattening rooms versus separate pits (OR=6.7) and a high level of cross-fostering (OR=5.1). On the other hand, when farms had a self-replacement scheme for the gilts (OR=0.1), and when vaccination of the sows against E. coli was in place (OR=0.2), the odds of PMWS were decreased.     

Descriptive summary of an outbreak of porcine post-weaning multisystemic wasting syndrome (PMWS ) in New Zealand

CASE HISTORY: Investigations were conducted to determine the cause of an acute, multi-farm outbreak of porcine respiratory disease that included diarrhoea and subsequent loss of body condition in affected pigs. A definition for post-weaning multisystemic wasting syndrome (PMWS) including both clinical and pathological features, previously developed for the pig industry in New Zealand, was applied to the current outbreak. In addition to self-reporting by owners of affected farms, local veterinarians, disease and epidemiology consultants, and animal health officials from the Ministry of Agriculture and Forestry (MAF) were involved in conducting farm visits and submission of diagnostic specimens. CLINICAL FINDINGS AND DIAGNOSIS: Pathogens known to be endemic in the pig industry in New Zealand as well as likely exotic diseases were excluded as causative agents of the outbreak. Clinical signs including dyspnoea, diarrhoea, and rapid loss of body condition were consistent with the New Zealand case definition for PMWS. Interstitial pneumonia, pulmonary oedema, generalised lymph-node enlargement, and presence of porcine circovirus type 2 (PCV2) inclusion bodies were consistently identified in affected pigs. Classical swine fever virus (CSFv), Porcine reproductive and respiratory syndrome virus (PRRSv), and Influenza virus were ruled out, using molecular and traditional virological techniques. Spread of the disease between farms was hypothesised to be facilitated by locally migrating flocks of black-backed seagulls. The original source of the disease incursion was not identified. DIAGNOSIS: Based on the consistent presence of circovirus-associated lesions in lymphoid tissues in combination with generalised enlargement of lymph nodes, histiocytic interstitial pneumonia, clinical wasting, and poor response to antibiotic therapy, a diagnosis of PMWS was made. CLINICAL RELEVANCE: PMWS should be considered in the differential diagnoses of sudden onset of respiratory dyspnoea, diarrhoea, and rapid loss of body condition in young pigs in New Zealand pig herds.     

Torque teno sus virus 1 and 2 viral loads in postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS) affected pigs

Torque teno viruses (TTV) are small, non-enveloped viruses with a circular single-stranded DNA genome, which are considered non-pathogenic. However, TTVs have been eventually linked to human diseases. TTVs infecting pigs, Torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2), have been recently associated to porcine circovirus diseases (PCVD). To get more insights into such potential disease association, the aim of this study was to quantify TTSuV1 and TTSuV2 viral loads in serum of pigs affected by two PCVDs, postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). Such study was carried out by means of a newly developed real-time quantitative PCR (qPCR) method. Both TTSuVs were highly prevalent among studied pigs. TTSuV2 viral loads were significantly higher in PMWS affected animals, further supporting the previously suggested association between TTSuV2 and PMWS. On the contrary, TTSuV1 prevalence and loads were not related with the studied PCVDs.     

Descriptive summary of an outbreak of porcine post-weaning multisystemic wasting syndrome (PMWS) in New Zealand

CASE HISTORY: Investigations were conducted to determine the cause of an acute, multi-farm outbreak of porcine respiratory disease that included diarrhoea and subsequent loss of body condition in affected pigs. A definition for post-weaning multisystemic wasting syndrome (PMWS) including both clinical and pathological features, previously developed for the pig industry in New Zealand, was applied to the current outbreak. In addition to self-reporting by owners of affected farms, local veterinarians, disease and epidemiology consultants, and animal health officials from the Ministry of Agriculture and Forestry (MAF) were involved in conducting farm visits and submission of diagnostic specimens.

CLINICAL FINDINGS AND DIAGNOSIS: Pathogens known to be endemic in the pig industry in New Zealand as well as likely exotic diseases were excluded as causative agents of the outbreak. Clinical signs including dyspnoea, diarrhoea, and rapid loss of body condition were consistent with the New Zealand case definition for PMWS. Interstitial pneumonia, pulmonary oedema, generalised lymph-node enlargement, and presence of porcine circovirus type 2 (PCV2) inclusion bodies were consistently identified in affected pigs. Classical swine fever virus (CSFv), Porcine reproductive and respiratory syndrome virus (PRRSv), and Influenza virus were ruled out, using molecular and traditional virological techniques. Spread of the disease between farms was hypothesised to be facilitated by locally migrating flocks of black-backed seagulls. The original source of the disease incursion was not identified.

DIAGNOSIS: Based on the consistent presence of circovirusassociated lesions in lymphoid tissues in combination with generalised enlargement of lymph nodes, histiocytic interstitial pneumonia, clinical wasting, and poor response to antibiotic therapy, a diagnosis of PMWS was made.

CLINICAL RELEVANCE: PMWS should be considered in the differential diagnoses of sudden onset of respiratory dyspnoea, diarrhoea, and rapid loss of body condition in young pigs in New Zealand pig herds.     

Immunosuppression in postweaning multisystemic wasting syndrome affected pigs

The present review concentrates on the clinical, pathological and immunological aspects of pigs suffering from PMWS which strongly suggest that PCV2 may be, in particular conditions, a cause of secondary immunodeficiency in pigs. From a clinical point of view, the lack of antibiotic therapy response against the disease, the existence of a litter effect and the concurrence of other disease syndromes and well-known secondary pathogens, such as Pneumocystis carinii, Chlamydia spp. and Aspergillus spp., may account as features of immunosuppression in PMWS. Furthermore, pathologic, immunohistologic and flow cytometric studies also suggest that pigs with PMWS may be immunosuppressed. Lymphocyte depletion of follicular and interfollicular areas together with macrophage infiltration of lymphoid tissues is a unique lesion, which is the basic feature of PMWS affected pigs. These findings are highly correlated with the decrease of circulating B- and T-cells and the diminution of these cell types in lymphoid organs, and with the increase of macrophage/monocytes lineage cells both in peripheral blood and lymphoid tissues in both naturally and experimentally PMWS affected pigs. The altered populations of cells participating in the immune system response both in blood and tissues suggests, at least in those severely PMWS affected pigs, a transient inability of diseased pigs to mount an effective immune response. From these points of view, strong suspicions on the immunosuppressive status of PMWS affected pigs do exist; however, future studies are needed to characterise the exact role of PCV2 on the immune system of pigs affected with PMWS.     

猪断奶后多系统衰竭综合征诊断方法的系列研究

本研究对猪断奶后多系统衰竭综合征(PMWS)的诊断方法临床症状和病理变化、病毒的分离与鉴定、聚合酶链式反应(PCR)、间接免疫荧光试验(IFA)、间接酶联免疫吸附试验(IELISA)、免疫组织化学试验(IHC)等进行了系列研究,这些方法适用于猪断奶后多系统衰竭综合征的诊断.     

Coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome.

Postweaning multisystemic wasting syndrome (PMWS) is an emerging disease in swine. Recently, the disease has been reproduced with inocula containing a newly described porcine circovirus (PCV), designated PCV 2, and porcine parvovirus (PPV). In order to determine if these viruses interact in naturally acquired PMWS, affected tissues from field cases were examined by immunohistochemistry (IHC) and polymerase chain reaction (PCR) for PCV 2 and PPV, as well as by PCR for the other recognized porcine circovirus, PCV 1. Porcine circovirus 2 was detected by PCR or IHC in affected fixed or frozen tissues from 69 of 69 cases of PMWS collected over 3 years from 25 farms. Porcine parvovirus was detected in 12 of the same cases, and PCV 1 was detected in 9 of 69; however, an apparent decrease was found in the sensitivity of the PCRs used to detect the latter 2 viruses when fixed tissue from the same cases were compared with the use of frozen tissues. Porcine circovirus 2 was not detected by PCR in affected tissues from 16 age-matched pigs that had Streptococcus suis-associated disease. Electron microscopic examination of plasma pooled from 15 pigs with PMWS revealed the presence of PCV and PPV, whereas these viruses were not observed in pooled plasma from 5 age-matched clinically normal pigs. These results confirm and extend previous findings documenting a consistent association of PCV 2 with PMWS. As well, infection by PPV or PCV 1 or both may be an important cofactor in the pathogenesis of some, but apparently not all, cases of PMWS.     

Identification of porcine circovirus in tissues of pigs with porcine dermatitis and nephropathy syndrome

Thirty-three pigs affected by porcine dermatitis and nephropathy syndrome, 30 from Spain and three from the USA, were investigated in order to detect porcine circovirus (PCV) in their tissues. A standard in situ hybridisation technique using a specific DNA 317-bp probe based on a well-conserved sequence of PCV (which recognises both PCV-1 and PCV-2) was applied to formalin-fixed, paraffin-embedded tissues. Twenty-eight of the 30 Spanish pigs and all three American pigs had PCV in at least one tissue. Viral nucleic acid was detected mainly in lymphoid organs, and especially the lymph nodes. The viral genome was also found, in order of decreasing quantity, in Peyer's patches, tonsil, lung, spleen, kidney, liver, and skin. Viral nucleic acid was located mainly within the cytoplasm of monocyte/macrophage lineage cells, including follicular dendritic cells, macrophages, histiocytes and Kupffer cells. No viral nucleic acid was found in damaged glomeruli or arteriolar walls. In frozen samples available from three Spanish pigs, the virus was identified as type 2 by using the polymerase chain reaction and restriction fragment length polymorphism. Most of the pigs from which serum was available were seropositive against porcine respiratory and reproductive syndrome virus (PRRSV), and PRRSV antigen was detected in the lung of two of the Spanish pigs. These results suggested that PCV is present in tissues of almost all pigs affected by PDNS, and PCV has to be considered as a possible agent involved in the pathogenesis of the syndrome.