Evaluation of porcine circovirus type 2 infection in in vitro embryo production using naturally infected oocytes.

In vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) are important breeding techniques for livestock. High-quality MII oocytes produced from in vitro maturation (IVM) are required for the two techniques listed above. The ovaries used for IVM operations are primarily acquired from commercial abattoirs, and the pathogen status of slaughtered animals becomes an unavoidable issue. Our previous monitoring data showed that porcine circovirus type 2 (PCV-2) is the main pathogen present in ovaries from abattoirs. However, the characteristics and effects of PCV-2 infection in oocyte maturation and in vitro production (IVP) of embryos are unclear, and currently there are no relevant studies. Therefore, the aim of this study was to determine the PCV-2 infection pattern and determine whether it affects oocyte in vitro maturation and IVP embryo development. More than five hundred ovaries and five thousand oocytes were utilized in the present study. Polymerase chain reaction (PCR) was used to detect PCV-2 DNA in ovaries, follicular fluid (FF), oocytes, cumulus cells and IVP embryos. The effects of viral infections on the rate of oocyte maturation and IVP embryo development were evaluated. We also analyzed the number of copies of the virus in the IVM and IVP process by absolute quantitative fluorescence PCR. Our study showed that the prevalent virus subgenotype in ovaries was PCV-2a. PCV-2a infection did not significantly affect ovarian/oocyte morphology and maturation. Moreover, virus infection did not have a significant effect on the development of the IVP embryos except for a reduction in IVF blastocyst cell numbers. Further tests showed that the viral copy numbers fluctuated at different stages between the IVP embryos and culture medium. For the first time, this study identified the infection pattern of naturally sourced PCV-2 in the course of oocyte maturation and embryo development.

The relationship between porcine circovirus 2 antigen score and antibody titre and histology of lymph nodes in 375 euthanased sick and healthy pigs from 113 British pig farms with and without postweaning multisystemic wasting syndrome.

Data from a cross-sectional study of 113 British pig herds carried out in 2004 were used to investigate the associations between postweaning multisystemic wasting (PMWS) in pigs and herds and porcine circovirus 2 (PCV2) antigen score and antibody titre, and associated histological signs in lymph nodes. The sensitivity and specificity of published herd definitions for PMWS were tested on the study farms to consider the role of PCV2 in PMWS. Herds were defined as PMWS-affected, -unaffected or -recovered based on current and past postweaning mortality (PWM), grower pigs with clinical signs of rapid wasting, hairiness and pallor and no other known cause of death on the farm. PCV2 antigen and antibody were not used in the definition of PMWS. In each PMWS-affected herd, up to three sick pigs with the clinical signs above and one healthy pig of a similar age were taken for postmortem examination (PME). In all other herds at least one healthy pig was taken for PME. Lymph nodes were analysed for PCV2 antigen and histological changes, and serum samples were analysed for PCV2 antibody. PCV2 antibody was present in all the herds sampled. There was a non-linear association between PCV2 antigen and antibody. There was no association between the presence of high scores of PCV2 antigen in pigs and the presence of high PWM in herds. PCV2 antigen score was significantly higher in sick than healthy pigs within farms, and high PCV2 score was associated with giant cells, coalescence and absence of germinal centres in lymph nodes. These results did not vary by PMWS-affected, -unaffected or -recovered farms. PCV2 antigen was present at high scores in approximately 10% of healthy pigs on all farms. All three herd definitions of PMWS were highly sensitive, defining PMWS-affected herds as affected, but had a specificity ranging from 23% to 43%. We conclude that the current diagnostic tests for PCV2 indicated higher scores of virus in sick pigs but were not useful to define pigs or herds with PMWS. The ubiquity of PCV2 and the lack of specificity of the PCV2 tests indicate that PCV2 may be a necessary but not sufficient cause of PMWS disease. Linking this with the knowledge that the herd breakdowns occurred in a space time epidemic indicates that another infectious co-factor may be necessary for disease to occur.

Effect of a porcine circovirus type 2 infection on embryos during early pregnancy.

The aim of the present study was to assess the effects of porcine circovirus type 2 (PCV2) on porcine embryos and their receptor sows during the first 21 days of pregnancy. Hatched blastocysts exposed to 10(5.0) TCID50 PCV2 per ml (strain 1121, fifth passage PK15) and negative control embryos were transferred to PCV2-immune receptor sows at the 7th day of the cycle. Two weeks after transfer (D21), the receptor sows were euthanized and embryos were recovered. They were assessed macroscopically for viability and examined for viral antigen-positive cells by immunoperoxidase staining. The embryonic survival rate of the PCV2-exposed embryos (6.4%, 7 viable embryos out of 110 transferred) was significantly lower than the survival rate of the negative control embryos (65.4%, 34 viable embryos out of 52 transferred). All of the non-viable PCV2-exposed embryos (n=9) displayed immunohistochemical positive signals for PCV2-antigen in degenerated tissues. In the PCV2-exposed embryos that were categorized as viable at D21, small clusters (n=4) or no PCV2-positive cells (n=3) were detected. The pregnancy results of the receptor sows that received PCV2-exposed embryos (1/5) were considerably different from the negative control receptors (2/2), with 3 out of 5 sows displaying a regular return to oestrus. In conclusion, it can be stated that PCV2 can replicate in embryos and might lead to embryonic death. In a small proportion of embryos, PCV2 exposure does not have a detrimental effect on embryo development before D21.

Porcine circovirus 2 infection in swine foetuses inoculated at different stages of gestation.

The ability of porcine circovirus 2 (PCV2) to replicate and cause pathologic abnormalities in foetuses at selected time points of gestation was examined in this study. Two foetuses were inoculated in utero in each of two sows at 57, 75 and 92 days of gestation, respectively, with PCV2 (1121). The remaining foetuses were left uninoculated to assess whether intra-uterine spread occurred. Twenty-one days after inoculation, the foetuses were collected and examined for gross lesions and for virus and infected cells in different organs. Serum samples from all foetuses were tested for PCV2 antibodies. Virus replication was detected in all inoculated foetuses. Spread to non-inoculated foetuses did not occur. Virus replication was significantly higher in foetuses inoculated at 57 days compared to that inoculated at 75 and 92 days. The heart contained the highest virus titre and highest number of viral antigen positive cells. Gross lesions were observed only in foetuses inoculated at 57 days of age. PCV2 antibodies were detected only in foetuses inoculated at 75 and 92 days. This study shows the ability of PCV2 to replicate in foetuses at different stages of gestation and to cause pathologic abnormalities in foetuses inoculated at 57 gestational days.