Transmission of PCV2 is presently not well characterized, however, detection of PCV2 DNA has been reported in secretions and excretions including feces, urine, saliva, ocular, nasal, colostrum and semen of infected pigs [1
]. Rapid spread of porcine circovirus associated disease (PCVAD) across North America in recent years has raised questions about the risk of transmission of PCV2 in semen; however, epidemiological investigation have not confirmed the relationship between PCV2 positive semen and incidence of PCVAD [4
Semen transmission of certain viruses has been documented and is considered economically important for the swine industry, especially with the wide usage of artificial insemination. A recent review summarized viruses potentially shed in boar semen [11
]. Currently, many North American boar stud operations frequently test for the presence of PRRSV by many methods for surveillance purposes [36
], yet the presence of other viruses associated with reproductive failure including PCV2 are infrequently tested for, although in one report 30% (13/43) of boars tested where found to contain PCV2 DNA in semen [23
]. In other geographical locations, PCV2 DNA was detected in 28 of 60 (47%) and 26 of 98 (27%) semen samples obtained from 30 and 49 Korean swine herds [14
], 18% (86/472) of semen samples from Austrian and German boars [44
], and 22% (67/304) of semen samples collected from 4 Brazilian farms1
. Other European countries have begun to test boar semen for exportation for the presence of PCV2 DNA to prevent the possible spread of PCVAD2
The significance of PCV2 in semen is currently unknown and the purpose of this study was to try to expand the knowledge base on this topic. Due to the cytotoxicity of semen [45
] and the non-cytopathic effect of PCV2 in cell culture, virus isolation techniques can be misleading and unrewarding. Thus, highly sensitive PCR methodologies are currently used to determine if PCV2 is present in semen. Polymerase chain reaction techniques can detect the presence of PCV2 DNA using small amounts of semen; however, positive PCR results do not necessarily correlate with presence of infectious virus. Previously, a swine bioassay model was developed to confirm the infectivity of PRRSV in semen [52
]. In this study, a similar bioassay model was used to test the infectivity of PCV2 DNA present in semen.
The results of the PCV2 swine bioassay confirm that PCV2a and PCV2b present in semen are infectious as evidenced by the detection of PCV2 viremia, seroconversion in the recipients after inoculation, and microscopic lesions compatible with PCV2 infection in growing pigs. In the experimental PCV2 pig model using intranasal and/or intramuscular inoculations, anti-PCV2 antibodies can be detected between 21–28 DPI [27
]. The delayed antibody response to PCV2 observed in pigs intraperitoneally injected with PCV2 DNA-positive semen when compared to pigs inoculated with PCV2 live virus may be dose related. Additional considerations for delayed response in antibody production may be due to natural constituents present in semen [55
] or different antigens present competing for an immunological response [6
]. The peritonitis present at necropsy may have been in part induced by antigens present in the semen or possibly by bacterial contamination during intraperitoneal inoculation and this may have contributed to some of the microscopic lesions observed. Additionally, PCV2 antigen was not detected in tissues from bioassay-PCV2a pigs, or bioassay-PCV2b pigs in this study. This result may due to the detection limits of IHC because it has been previously estimated that 108
PCV2 genomic copies are needed in tissue sections for visualization of IHC signaling [2
Artificial insemination with semen containing PCV2 DNA did not result in dam or fetal infection in this study even with the initial insemination being diluted 1:1 with extender. This may indicate that low exposure to PCV2 in semen may be of lesser consequence. In most field situations, a single semen collection from a boar is typically diluted into many insemination doses and commonly mixed with semen samples from other boars further decreasing the amount of PCV2 in a dose of semen typically used for artificial insemination. However, PCV2 has the ability to infect and replicate in different stages (zona pellucida free morulae, early blastocysts and hatched blastocysts) of in vivo produced embryonic cells with increasing susceptibility of infection in the later stages [22
]. It has been shown that PCV2 with an approximate diameter of 17
nm in size did not infect intact zona pellucida morulae, although 20
nm diameter fluorescent beads could pass through zona pellucida pores. PCV2 is a difficult virus to inactivate, thus PCV2 present in semen may be a risk for infection [22
]. PCV2 is associated with the cell fraction portion of semen [33
]. This may indicate that there is a higher risk of PCV2 infection during fertilization because spermatozoa may be associated with PCV2 DNA.
Differences in virulence and tropism of PCV2 isolates have been suggested as a possible reason for PCV2-associated reproductive failure when compared to PCV2-associated diseases in growing swine [7
]. Two PCV2 isolates were used in this study and both originated from growing pigs with PCVAD. However, it is unknown whether these isolates were also associated with reproductive failure in the field.
In swine, the route of PCV2 exposure is likely fecal-oral [46
] and fetal infection has been associated with viremia of the dam [34
]. It was recently reported that reproductive failure was associated with insemination of PCV2 spiked semen in PCV2 positive dams [39
]. The dams were intranasally inoculated 60 days prior to insemination, and it was unknown whether dams were still viremic at the time of insemination with PCV2 spiked semen. The dams may have been viremic which could have contributed to the reproductive failure [39
]. In support of this, in a different study it has been shown that PCV2 experimentally inoculated gilts had detectable PCV2 DNA in peripheral blood mononuclear cells at 63 DPI [35
The results of our study indicate that PCV2 DNA present in semen ranging from 105.6 to 105.8 genomic copies/mL did not cause reproductive failure, development of dam associated anti-PCV2 antibodies, or fetal infection. In addition, the semen extender used in the study was found to have only a minimal effect on the viability and infectivity of PCV2.
The amount of PCV2 present in semen is likely determining whether PCV2 infection occurs. Previously, non-extended raw semen has been shown to infect PRRS negative gilts [58
], but extended semen, though causing a reduction of pregnancy rates, did not cause seroconversion or infection [53
]. This indicates that the dose of viruses in semen plays a major role in transmissibility. In further support of this, only 20% of negative gilts inseminated with PRRSV at doses of 2
000 and 20
mL semen seroconverted while semen containing PRRSV at ≥ 200
mL semen was infectious in 100% of the negative gilts3
. Alternatively, other possible explanations for not having PCV2 infection in the gilts may be related to the increased numbers of inflammatory cells that are present in the uterus associated with innate immunity during proestrus, estrus, and early diestrus [5
], age related differences in susceptibility, or the influence of altrenogest (Matrix®
) on and uterine defense mechanisms [10
In summary, we determined that PCV2a and PCV2b shed in semen were infectious in a swine bioassay model. We also determined that low doses of PCV2 (105.6–105.8 PCV2 genomic copies per mL) in extended semen when used for artificial insemination does not cause reproductive failure, seroconversion, or PCV2 viremia in naïve gilts and their offspring. The authors acknowledge that the amount of PCV2 present in semen is a possible contributing factor in transmissions of PCV2 via semen to swine herds that utilize artificial insemination. We also are aware of potential PCV2 isolate differences in association with reproductive failure. Further investigations are needed and currently under way.