Porcine circovirus 2 replication in colostrum-deprived piglets following experimental infection and immune stimulation using a modified live vaccine against porcine respiratory and reproductive syndrome virus

Porcine circovirus type 2 (PCV2) infection is now recognized as the major factor in the development of post-weaning multisystemic wasting syndrome (PMWS). Although Koch's postulates have been fulfilled for PCV2 and PMWS, the severe clinical expression of the disease observed in field cases has been difficult to reproduce experimentally. Some studies have demonstrated that immune stimulation associated with the use of some commercially available swine vaccines may trigger progression of PCV2 infection to disease and lesions characteristic of PMWS. Here we describe the effects on PCV2 infection in an experimental model following the use of a commercially available modified live vaccine to porcine respiratory and reproductive syndrome virus (PRRSV). Although none of the piglets infected with PCV2 developed clinical PMWS, the severity of microscopical lesions and the PCV2 antigen load associated with these lesions were higher in the PRRSV-vaccinated piglets compared with those detected in the PCV2 only infected animals.     

猪圆环病毒研究进展

近年来猪圆环病毒感染给养猪业的健康发展带来巨大威胁。猪感染圆环病毒后可引起的疾病有猪断奶后多系统衰弱综合征(PMWS)、猪皮炎和肾病综合征(PDNS)、猪呼吸系统衰弱综合征(PRDC)等。近年来,基于PMWS而对PCV所做的研究工作不断深入,对病毒的相关特性有了较为清晰的认识。论文就近年来猪圆环病毒的致病性、流行病学、诊断及防控等方面的研究进展做一综述。     

A review of porcine circovirus 2-associated syndromes and diseases

Clinical expression of porcine circovirus 2 (PCV2) infection in swine may result in several distinct syndromes and diseases including post-weaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, porcine respiratory disease complex, granulomatous enteritis, necrotizing lymphadenitis, and possibly exudative epidermitis. Association of PCV2 with congenital tremor in piglets is still controversial. The extent of the involvement of PCV2 in swine disease other than PMWS is currently poorly understood. This review concentrates on PCV-2-associated syndromes and diseases other than PMWS.     

猪圆环病毒感染及动物模型研究进展

1974年德国学者Tischer从多株连续传代的猪肾细胞(PK-15)中发现了猪圆环病毒(PCV),其致病性及其疾病直到1991年才由Clark报道,称为断奶仔猪多系统衰竭综合征(PMWS).随后,许多学者对PCV-2感染以及PMWS的动物模型建立进行了广泛研究,证实了免疫激发及PCV-2与其他病原混合感染对PMWS的...     

Postweaning multisystemic wasting syndrome (PMWS) in pigs. A review

Postweaning multisystemic wasting syndrome, caused by porcine circovirus type 2 (PCV2), is a recently described clinical condition which affects nursery and growing pigs. PMWS was initially recognized in Canada in 1991 and nowadays is considered to be worldwide distributed. Clinically, PMWS most representative symptoms include wasting, unthriftness, paleness of the skin, respiratory distress, diarrhea and sometimes icterus. PCV2 infection occurs in both PMWS affected and non-affected farms, and viral seroconversion shows a typical pattern, with declining of colostral antibodies during the lactating and nursery periods, with the lowest levels at the end of the nursery period, and active seroconversion of almost all pigs during the grower period. Although antibodies to PCV2 have been detected as early as 1969, no explanation for the emergence of this disease in the 90s has been established. Macroscopic lesions associated with PMWS are quite unspecific, but histopathological lesions in lymphoid tissues (lymphocyte depletion with histiocytic infiltration) are almost unique for this disease. These lesions together with other clinical and laboratorial findings suggest that severely affected pigs may be immunosuppressed. The criteria used for the diagnosis of PMWS include the existence of compatible clinical signs, presence of characteristic microscopic lesions and detection of PCV2 within these lesions. Because of the lack of appropriate treatment or vaccination against PCV2, zootechnical changes have been proposed in affected farms to reduce the so-called "infection pressure" due to PCV2 as well as to any other pathogen.     

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.     

Reproduction of postweaning multisystemic wasting syndrome in pigs by prenatal porcine circovirus 2 infection and postnatal porcine parvovirus infection or immunostimulation

Postweaning multisystemic wasting syndrome (PMWS) was reproduced in prenatally porcine circovirus 2 (PCV2)-infected pigs by either postnatal infection with porcine parvovirus (PPV) or by immunostimulation. Twenty-four randomly selected piglets from 3 sows, which had been experimentally infected during gestation with PCV2, were randomly divided into 3 groups; group 1 (prenatal PCV2 infection, with postnatal PPV infection), group 2 (prenatal PCV2 infection, with postnatal keyhole limpet hemocyanin, emulsified in incomplete Freund's adjuvant [KLH/ICFA] injection), and group 3 (prenatal PCV2 infection only). Twenty-four randomly selected piglets from 3 uninfected sows were randomly divided into 3 groups; group 4 (no prenatal infection, with postnatal PCV2 and PPV infection), group 5 (no prenatal infection, with postnatal PCV2 infection), and group 6 (negative control pigs). Body weight in negative control pigs (group 6) was increased significantly compared with pigs in groups 1, 2, and 4 at 49, 52, 56, 59, and 63 days of age. The granulomatous inflammatory reaction and lymphoid depletion that are typical lesions in pigs with PMWS were observed in the lymph node of piglets in groups 1, 2, and 4 at 63 days of age. Pigs in group 3 had significantly fewer PCV2-positive cells than those from groups 1, 2, 4, or 5. When the prenatally PCV2-infected pigs were infected with PPV or injected with immunostimulant in the postnatal period, they developed PMWS. Thus, factors that potentiate the progression of prenatal PCV2 infection to PMWS are postnatal infection with PPV or immune stimulation.     

猪圆环病毒2型疫苗研究进展

猪圆环病毒2型(PCV2)感染后会导致断奶仔猪多系统衰竭综合征(PMWS)等"猪圆环病毒相关疾病"(PCVADs),给全球养猪业造成了巨大的经济损失。疫苗免疫是防控该病的有效手段,近年来市场上推出了PCV2灭活疫苗、嵌合疫苗和亚单位疫苗。PCV2变异较快,主要临床流行毒株从PCV2b逐渐演变为PCV2d,现有商业化疫苗免疫效果需进一步研究证实。论文就近年来国内外PCV2传统疫苗、新型基因工程疫苗、PCV2疫苗与其他疫苗联合免疫的研究进展进行综述,旨在为猪圆环病毒相关疾病的防控及疫苗的开发与利用提供参考。     

Postweaning multisystemic wasting syndrome: a review of aetiology, diagnosis and pathology

This review paper concentrates on the aetiology, diagnosis, and pathological aspects of postweaning multisystemic wasting syndrome (PMWS). PMWS was first recognized in Canada in 1996 as a new emerging disease which caused wasting in postweaned pigs. Since then, PMWS has been recognized in pigs in many countries. The syndrome is caused by a DNA virus referred to as porcine circovirus 2 (PCV2), which is classified in the family Circoviridae. PMWS primarily occurs in pigs between 25 and 120 days of age with the highest number of cases occurring between 60 and 80 days of age. The diagnosis of PMWS must meet three criteria: (i) the presence of compatible clinical signs, (ii) the presence of characteristic microscopic lesions, and (iii) the presence of PCV2 within these lesions. In order to establish the diagnosis, techniques are required that link virus and tissue lesions, such as immunohistochemistry and in situ hybridization, but not polymerase chain reaction or virus isolation. The three criteria considered separately are not diagnostic of PMWS. For example, the detection of PCV2 alone does not indicate PMWS but merely PCV2 infection. A hallmark of microscopic lesions of PMWS is granulomatous inflammation in the lymph nodes, liver, spleen, tonsil, thymus, and Peyer's patches. Large, multiple, basophilic or amphophilic grape-like intracytoplasmic inclusion bodies are often seen in the cytoplasm of macrophages and multinucleated giant cells.     

A comparison of virus isolation, polymerase chain reaction, immunohistochemistry, and in situ hybridization for the detection of porcine circovirus 2 and porcine parvovirus in experimentally and naturally coinfected pigs.

Virus isolation, polymerase chain reaction (PCR), immunohistochemistry, and in situ hybridization were compared for the detection of porcine circovirus 2 (PCV2) and porcine parvovirus (PPV) from experimentally and naturally coinfected pigs. All coinfected pigs developed postweaning multisystemic wasting syndrome (PMWS), characterized by sudden onset of depression and anorexia. Microscopically, granulomatous inflammation with intracytoplasmic inclusion bodies was present in lymph node from all coinfected pigs at 32 days postinoculation. Of the 200 tissues from 20 experimentally coinfected pigs evaluated, 99 and 58 tissues were positive for PCV2 and PPV, respectively, by 4 techniques. Virus isolation, PCR, immunohistochemistry, and in situ hybridization identified PCV2 infection in 137, 148, 103, and 129 tissues and PPV infection in 107, 132, 59, and 94 tissues. Of the 200 tissues from 20 naturally coinfected pigs evaluated, 109 and 45 tissues were positive for PCV2 and PPV, respectively, by 4 techniques. Virus isolation, PCR, immunohistochemistry, and in situ hybridization identified PCV2 infection in 144, 155, 113, and 139 tissues and PPV infection in 93, 109, 45, and 82 tissues. Because the characteristic microscopic lesions are important criteria for the diagnosis of clinical PMWS, immunohistochemistry and in situ hybridization for the detection of PCV2 and PPV in formalin-fixed, paraffin-embedded tissues provide confirmation of a histopathological diagnosis of PMWS.     

Reproduction of postweaning multisystemic wasting syndrome in pigs experimentally inoculated with a Swedish porcine circovirus 2 isolate.

In recent years, porcine circovirus type 2 (PCV2)-associated postweaning multisystemic wasting syndrome (PMWS) has been reported worldwide. However, to date, PMWS has not been reported in Sweden despite the demonstration of serum antibodies to a PCV2-like virus in Swedish pigs. This communication reports the experimental reproduction of clinical PMWS after inoculation of colostrum-deprived (CD) pigs, derived from a Northern Ireland herd, with an isolate of PCV2 virus recovered from a clinically normal Swedish pig that was necropsied in 1993. The clinical disease and histological lesions observed in CD pigs inoculated with this virus were indistinguishable from those observed in previous studies on CD pigs inoculated with a PCV2 virus isolate recovered from pigs with PMWS. These results highlight the disease potential of PCV2 isolated from regions apparently free of PMWS and suggest that the status of the host and its environment is an important factor in the development of clinical PMWS.     

Temporal distribution of porcine circovirus 2 genogroups recovered from postweaning multisystemic wasting syndrome affected and nonaffected farms in Ireland and Northern Ireland.

Porcine circovirus type 2 (PCV2) is now recognized as the essential infectious component of porcine postweaning multisystemic wasting syndrome (PMWS). PMWS was first recognized in high-status, specific pathogen-free pigs in Canada in 1991 and is now an economically important disease that affects the swine industry around the world. Recently, reports of genomic studies on PCV2 viruses indicated that 2 distinctive genogroups of PCV2 exist.4,10 This report involves the results of a study on the distribution of predominant PCV2 genogroups recovered from samples taken from PMWS-affected and PMWS-nonaffected farms on the island of Ireland over a 9-year period and the results of a study on PCV2 genogroup recovery from fecal samples taken from a farm in Northern Ireland from 2003 to 2005 that was first diagnosed as PMWS positive in August 2005. The results indicate that, although at least 2 distinct genogroups of PCV2 have been circulating on pig farms on the island of Ireland, there does not appear to be a direct relationship between infection with these different genogroups of PCV2 and the development of PMWS.     

Experimental reproduction of postweaning multisystemic wasting syndrome (PMWS) in pigs in Sweden and Denmark with a Swedish isolate of porcine circovirus type 2

An experimental model using 3-day-old snatch-farrowed colostrum-deprived piglets co-infected with porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) is at present one of the best methods to study factors affecting development of postweaning multisystemic wasting syndrome (PMWS). A Swedish isolate of PCV2 (S-PCV2) retrieved in 1993 from a healthy pig has been used in this model to reproduce PMWS in pigs from Northern Ireland. This virus has been present in the Swedish pig population for at least a decade without causing any known PMWS disease problems, despite its potential pathogenicity. The reasons for this are unknown, but could be related to genetics, absence of triggers for PCV2 upregulation (infectious agent and/or management forms) within Swedish pig husbandry. In order to confirm the pathogenicity of S-PCV2, Swedish and Danish pigs were experimentally infected with this isolate according to the established model. Swedish pigs were also infected with a reference isolate of PCV2 (PCV2-1010) to compare the severity of disease caused by the two isolates in Swedish pigs. Both Danish and Swedish pigs developed PMWS after the experimental infection with S-PCV2. Antibodies to PCV2 developed later and reached lower levels in serum from pigs infected with S-PCV2 than in pigs inoculated with PCV2-1010. In general, pigs infected with S-PCV2 showed more severe clinical signs of disease than pigs infected with PCV2-1010, but pigs from all PCV2-inoculated groups displayed gross and histological lesions consistent with PMWS. All pigs inoculated with PPV, alone or in combination with PCV2, displayed interleukin-10 responses in serum while only pigs infected with PPV in combination with PCV2 showed interferon-alpha in serum on repeated occasions. Thus, the pathogenicity of S-PCV2 was confirmed and a role for cytokines in the etiology of PMWS was indicated.     

The presence of co-infections in pigs with clinical signs of PMWS in The Netherlands: a case-control study

In this study, 60 pigs with clinical signs of post-weaning multisystemic wasting syndrome (PMWS) from 20 different pig herds and 180 control pigs (without clinical signs of PMWS) were examined to get more insights into the frequencies of porcine circovirus 2 infections and the presence of co-infections in pigs with and without clinical signs of PMWS in the Netherlands. Porcine circovirus type 2 was detected in 100% of the pigs with clinical signs of PMWS by virus isolation and/or PCR and in 50% of the pigs from PMWS-free herds. There was an association between the levels of infectious PCV2 and/or PCV2 DNA load and the severity of clinical signs as described for PMWS. A high variation in PCV2 antibody titres was found in the clinically affected pigs, and 27% of these pigs did not mount PCV2 antibody titres higher than 1:200. A concurrent infection of PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) was found in at least 83% of the pigs with clinical signs of PMWS and in 35% of the pigs from PMWS-free herds. Co-infections of European- and American-type PRRSV were detected only in PMWS herds and in one control herd with a history of PMWS clinical signs.     

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.     

The index herd with PMWS in Sweden: Presence of serum amyloid A,circovirus 2 viral load and antibody levels in healthy and PMWS-affected pigs

Background

Postweaning Multisystemic Wasting Syndrome (PMWS) is an emerging disease in pigs of multifactorial origin, but associated to porcine circovirus type 2 (PCV2) infection. PMWS was first diagnosed in Sweden at a progeny test station that received pigs aged five weeks from 19 different nucleus herds on the day after weaning. The objective of this study was to examine, for the first time in an index outbreak of PMWS, the relationship between PCV2 virus, antibodies to PCV2 and serum amyloid a (SAA) in sequentially collected serum samples from pigs with and without signs of PMWS.

Methods

Forty pigs of the last batch that entered the station at a mean age of 37.5 days were monitored for signs of PMWS during the first 55 days after arrival. Serum was collected on six occasions and analysed for presence of PCV2 DNA and antibodies to PCV2, as well as for levels of SAA.

Results

Four of the pigs (10%) were concluded to have developed PMWS, with necropsy confirmation in three of them. These pigs displayed low levels of maternal antibodies to PCV2, more than 10⁷ PCV2 viral DNA copies per ml serum and failed to mount a serological response to the virus. Starting between day 23 and 34 after arrival, an increase in PCV2 viral load was seen in all pigs, but PCV2 did not induce any SAA-response. Pigs that remained healthy seroconverted to PCV2 as the viral load was increased, regardless of initially having low or high levels of PCV2-antibodies.

Conclusion

In this index case of PMWS in Sweden, pigs affected by PMWS were not able to mount a relevant serum antibody response which contributed to the disease progression. The maximal PCV2 virus load was significantly higher and was also detected at an earlier stage in PMWS-affected pigs than in healthy pigs. However, a viral load above 10⁷ PCV2 DNA copies per ml serum was also recorded in 18 out of 34 pigs without any clinical signs of PMWS, suggesting that these pigs were able to initiate a protective immune response to PCV2.     

The detection of porcine circovirus in the Australian pig herd

OBJECTIVE: To determine if porcine circovirus (PCV) type 1 (PCV1) or type 2 (PCV2) is present in the Australian pig herd, to conduct preliminary genetic characterisation of any viruses detected, and to determine if there is any obvious virological reason why post-weaning multisystemic wasting disease (PMWS), associated with PCV infection in other countries, has not been detected in Australia. DESIGN: Serum samples were collected from 14 randomly selected pig farms in Western Australia and used for detection of PCV antibody. Additional samples from one farm were obtained at 2-week intervals from pigs between 2 and 12 weeks of age to detect any age-associated variations in prevalence of infection. Veterinary practitioners from four Australian states submitted tissues of dead or unthrifty weaned pigs, and these were examined for evidence of PCV1 and PCV2 infection. PROCEDURE: Sera were tested for antibody to PCV using an indirect immunofluorescence assay (IFA). Tissues were tested for PCV1 and PCV2 genomic material using a multiplex PCR. RESULTS: PCV antibody was detected in approximately 30% of Western Australian pigs tested. PCV1 DNA was detected in tissue samples from Western Australia, South Australia and New South Wales and PCV2 DNA was detected in tissue samples from Western Australia, New South Wales and Queensland. Sequence analysis of the PCR products indicated the PCV1 and PCV2 present in Australia were very similar to strains in other countries where PMWS is endemic. CONCLUSION: Both PCV1 and PCV2 are present in Australia and the viruses present appear similar to those in countries with PMWS. The absence of PCV2-associated PMWS in Australia may be due to absence of essential secondary factors required for PCV2 to produce PMWS.     

PCR detection of porcine circovirus type 2 (PCV2) DNA in blood, tonsillar and faecal swabs from experimentally infected pigs

PCV2 infection is now recognized as the major factor in the development of post-weaning multisystemic wasting syndrome (PMWS). In this study we evaluated the use of PCR to detect the presence of PCV2 DNA in blood, faecal and tonsillar swabs collected from 12 pigs experimentally infected with PCV2 and sampled at selected time points post-infection. The PCR results were evaluated together with the presence of PMWS typical histopathological lesions and the presence of PCV2 antigen. PCV2 DNA was present in the blood of all 12 infected pigs at the end of the experiment and faecal and tonsillar swabs of 11 of the 12 pigs. The rate of PCR-positive serum and plasma samples was significantly higher in four pigs that showed virological and pathological evidence of PMWS, than in infected pigs without evidence of disease. In conclusion this study confirms that PCR cannot substitute for the traditional methods used for diagnosis of PMWS, however, PCR amplification of PCV2 DNA from serum or plasma could be a useful tool to support an early diagnosis of PMWS in live animals.     

Pathological findings associated with naturally acquired porcine circovirus type 2 associated disease

Porcine circovirus type 2 (PCV2) is a novel virus of the Circoviridae family which is considered the cause of postweaning multisystemic wasting syndrome (PMWS). PCV2 has also been associated to a number of pathological conditions of pigs, including porcine dermatitis and nephropathy syndrome, reproductive failure, porcine respiratory disease complex, proliferative and necrotising pneumonia and congenital tremor type AII. Pathological studies have been used to describe and characterise PMWS and these emerging conditions associated with PCV2. The objective of this review is to concentrate on the gross, microscopic and ultrastructural pathology associated with natural cases of PCV2 associated disease, along with some speculations on the pathogenesis of naturally occurring PMWS.