Porcine semen as a vector for transmission of viral pathogens-Part 3
Dominiek Maesa,*, Ann Van Sooma, Ruth Appeltanta, Ioannis Arsenakisa, Hans Nauwynckb
a Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
b Department of Virology, Immunology and Parasitology, Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
Semen, Pig, Artificial insemination, Virus, Review
Viruses in porcine semen which are not in the OIE list
Nonclassical swine fever pestiviruses
Pigs are also susceptible to non-CSF pestiviruses including BVDV and border disease virus; these are RNA viruses which are associated with disease in cattle and sheep, respectively. Pigs congenitally infected with these viruses may shed large amounts of virus. Terpstra and Wenswoort  isolated BVDV from oropharyngeal fluid, urine, and semen of a congenitally infected and infertile boar.
Bungowannah virus is genetically distinct from CSF virus, but molecular characterization supports its inclusion in the genus Pestivirus. The virus caused a severe disease outbreak characterized by an increase in stillbirths, preweaning mortality, and mummified fetuses in 2003 in two pig herds in Australia . The risk of transmission by embryos or semen has not yet been evalsuated.
Porcine enteric picornaviruses
Porcine enterovirus (PEV) and porcine teschovirus are nonenveloped RNA viruses belonging to the family of Picornaviridae. They are highly resistant in the environment. Infections are transmitted between pigs mainly by oral exposure to contaminated feces. Contamination of semen during collection is possible. Infections with PEV in boars may lead to seminal vesiculitis, sperm abnormalities, and a decreased libido . In the sow, usually no clinical signs are observed, except if infections occur during pregnancy. In the latter case, infections may cause fetal death and mummification, stillbirth, and weak-born piglets .
Porcine teschovirus has also been isolated from the male genital tract , but insemination of gilts with contaminated semen did not affect fertility . Dunne et al.  however suggested that semen contaminated with PEV and porcine teschovirus could cause embryonic and neonatal death. There are no recent reports that confirmed this finding.
Torque teno virus
已經在豬身上發現兩種細環病毒（TTV）：細環病毒1型（TTsuV1）及2型（TTsuV2）。 均為DNA病毒，屬於新創的指環病毒科、細環病毒屬。據估計，豬細環病毒（TTsuVs）在全球豬群中普遍存在 。目前，感染豬細環病毒（TTsuV）無相關臨床症狀出現。但TTsuV與豬圓環病毒（PCV）相關疾病有關[76,77]。細環病毒呈垂直和水平傳播。可在胎兒組織和血液、精液和初乳中檢測到該病毒，由此證明其存在垂直傳播的可能。從人工授精中心收集100頭公豬的獨立精液樣本，用常規PCR檢測TTsuV1和TTsuV2。其中四份樣本呈TTsuV1 PCR陽性，五份呈TTsuV2陽性，合並感染後，兩種TTsuVs均被檢測到。胎兒感染的途徑尚不明確，但相關顯示證明可能是由於精液帶毒或者胎盤傳播。
Two species of TTV have been described in pigs: torque teno sus virus types 1 (TTsuV1) and 2 (TTsuV2). They are DNA viruses that belong to the newly created family anelloviridae, genus Iotatorquevirus. It is considered that TTsuVs occur in pig populations worldwide . At present, no clinical signs are associated with TTsuV infection. However, TTsuVs have been linked to PCV-associated disease [76,77]. Torque teno sus viruses are apparently transmitted both vertically and horizontally. They can be detected in fetal tissues and blood, semen, and colostrum , indicating the potential for vertical transmission. Single semen samples from 100 boars from an AI center were analyzed by conventional PCR for TTsuV1 and TTsuV2 . Four samples were PCR positive for TTsuV1 and five for TTsuV2, whereas in a single coinfection, both TTsuVs were detected. The route of fetal infection is uncertain but may reflect virus-contaminated semen or transplacental transmission.
Viruses in porcine semen which are not in the OIE list
Reston ebolavirus is an enveloped RNA virus, classified in the order mononegavirales, family filoviridae. In 2008, severe disease outbreaks suspected to be caused by PRRSV were reported in swine herds in the Philippines. However, detailed diagnostic work revealed that not PRRSV but REBOV had caused the disease and that REBOV-specific antibodies were found in farm workers . The broader significance of this outbreak of REBOV remains to be determined. Subsequent experimental challenge suggested that REBOV infection in pigs may be asymptomatic despite replication of the virus. The mechanism of transmission of REBOV to pigs or between pigs is unknown. Routes of REBOV shedding by pigs are also not known. In nonhuman primate models, REBOV is shed in all body fluids, including semen . Whether REBOV is shed by boars in semen or transmitted to recipient females is unknown. In contrast with REBOV, Zaire ebolavirus has been shown to cause severe lung pathology in 5- to 6-week old pigs leading to respiratory distress . 2.2.8. 豬內源性反轉錄病毒
Pig endogenous retroviruses
Pig endogenous retroviruses are enveloped RNA viruses of the genus Gammaretrovirus in the family retroviridae. Up to 8% of mammalian genomic DNA is believed to be retroviral in origin . In pigs, three PERV subtypes have been identified: PERV-A, PERV-B, and PERV-C. Subtypes A and B are ubiquitous in all pig breeds, whereas PERV-C is variably distributed between and within breeds .
Exogenous horizontally transmissible retroviruses have not been found in pigs. However, a recombination between PERV-A and PERV-C has been found. The PERV A/C recombinant is not present in proviral form within the host germ line and is therefore considered a candidate exogenous virus . The presence of the full genomic A/C provirus in the somatic cells of some pigs indicates its potential for replication. Little is known regarding the potential for transmission of PERVs between pigs. However, as PERVs are embedded in the genome, they are transmitted via semen.
Rubulavirus (blue eye disease)
Porcine rubulavirus is an enveloped RNA virus that belongs to the genus Rubulavirus in the family paramyxoviridae. The virus is associated with blue eye disease in pigs . It is an important pathogen in Mexico and causes reproductive problems in breeding pigs. Boars, like other adult animals, generally do not show clinical signs, except for epididymitis and orchitis and in severe cases, loss of libido. Transmission of the virus through semen has not been proven experimentally; however, virus has been recovered from semen, the testis, and other tissues of the reproductive tract for up to 49 days after inoculation . More recently, isolation of the virus from semen was achieved between 5 and 48 days post inoculation (DPI) and from the testicles and epididymides between 64 and 142 DPI. Viral RNA was detected in the serum between 2 and 64 DPI and in the semen until 142 DPI . These results confirm that the RNA of the porcine rubulavirus persists in the semen and that this virus remains in the reproductive tract for prolonged periods of infection. Semen evalsuation demonstrated that about 30% of boars in herds infected with the virus showed temporary or permanent infertility, with a decrease in concentration, an increase in abnormalities in morphology, and a decrease in motility and viability of spermatozoa. In some boars, there was azoospermia .
Risk for transmission of viruses by AI to the recipient sow
In general, the risk of virus to be present in semen is the highest during the stage of clinical disease. Under practical circumstances, however, no sperm collection will take place from clinically affected boars, and consequently, the risk of pathogen transmission to the sow is low in this case. Although virus shedding can start before the development of clinical signs, which can be mild or absent, acutely infected boars can remain unnoticed. In these situations, the risk of virus transmission is much higher as no special control measures will be taken.
實驗證明，CSFV  PRRSV 可通過精液傳染母豬，在精液中加入PPV 、ADV 、PRRSV[18,21]和PCV2後，亦可。Habu等 ，Guérin和Pozzi 提出，雖然沒有找到科學的文章證實，但是如果使用含有日本乙型腦炎病毒的精液進行授精，則後備母豬極易被感染。若將被病毒汙染的精液應用於人工授精，母豬乃至母豬群都會有病毒傳播的風險。Nathues等以瑞士引入公豬精液導致PRRSV爆發為例，對此加以說明。
Transmission of viral pathogens by semen to the sow has been clearly proven for CSF virus  and PRRSV  on experimental infection of boars and for PPV , ADV , PRRSV [18,21], and PCV2  after inoculation of the virus in the semen. Habu et al.  and Guérin and Pozzi  reported that Japanese encephalitis virus is easily transmitted to gilts if they are inseminated with contaminated semen, although we did not find a scientific article confirming these statements. Semen contaminated with viral pathogens used for AI does indeed poses a risk for transmission to the recipient sow and by extension the sow herd. This was illustrated by Nathues et al.  who described an outbreak of PRRSV in Switzerland after import of boar semen.
但是，病毒並不總是能夠形成傳播（例如PRRSV）[18,61]。母豬被感染所需的條件很複雜，未能傳播可能是由於母豬免疫、病毒特點以及未達到最小感染劑量。因此，針對精液傳播PRRSV的風險，以及母豬感染病毒所需的最小劑量，開展了更多的研究。那麽，與常規宮頸輸精相比，小劑量精液的深部輸精（40–50 mL對比80–90 mL），可降低病毒傳染母豬的風險。
However, transmission will not always be successful (e.g., PRRSV) [18,61]. The conditions required for establishment of infection in the sow are complex, and lack of transmission might be explained by sow immunity, virus characteristics, and failure to reach the minimum infectious dose. In this regard, much research has been directed toward the risk of transmission of PRRSV by semen and the minimum dose necessary to establish infection in the sow. In this regard, it can be expected that postcervical insemination with smaller semen doses than conventional cervical insemination (40–50 mL compared to 80–90 mL) is associated with a lower risk for pathogen transmission to the sow .
At a population level, also the number of sows inseminated with contaminated semen determines the likelihood of successful transmission. The more sows are inseminated with contaminated semen, the higher the likelihood of successful transmission of the virus. In other animal species such as cattle, AI is mostly practiced with frozen semen and not with fresh semen as in pigs. This allows more time and opportunities to verify whether the bulls and/or semen are free from specific pathogens, and consequently, the risk for pathogen transmission via AI is lower.
Impact of viruses by AI to the recipient sow
The consequences of AI with semen contaminated with viral pathogens for the sow can be quite variable. It can result in reduced conception rates because of reduced semen quality, early embryonic death and/or endometritis, clinical disease in the sow herds, and/or infections with unwanted pathogens leading to reduced health status, stamping out, or regulatory measures . Early embryonic death may result from direct invasion of the embryo by the pathogen after it has hatched from the zona pellucida, and/or by uterine epithelial alterations in response to the pathogen (e.g., with PPV) . Until 6 to 7 days after conception, embryos are surrounded and protected by the zona pellucida, an impervious barrier, which helps the embryo to avoid invasion of pathogens such as PPV, PCV2, ADV, and PRRSV [65,84,85]. After hatching, however, blastocyst stage embryos may become susceptible to the infection as is the case, e.g., with ADV .
The presence of viral infections in boars of AI centers and/or the presence of viral pathogens in semen can be assessed using different diagnostic methods such as demonstration of viable virus, nucleic acid of the virus, or antibodies against the virus. From the early 1990s onward, major improvements have been made in terms of increased sensitivity and specificity, simultaneous testing of different pathogens and speed of testing.
Detection of viable virus
As diluted boar semen is mostly used within a few days after collection, the outcome of analysis should be available within a very short time. Conventional methods for virus detection in semen, such as virus isolation or conducting bioassays, are not very sensitive; they are time-consuming and very expensive. The application of the virus isolation technique is markedly reduced for semen because of cell toxicity, bacterial contamination, resulting in interference with cell culture systems, and antiviral factors that nonspecifically neutralize the virus . Animal inoculations cannot be performed for testing large numbers of samples because of practical reasons and also because of animal welfare reasons . Given these limitations, virus isolation and swine bioassays are not useful techniques for routine diagnosis.
Detection of viral nucleic acid
The PCR technique is known as a sensitive, specific, and rapid tool for the detection of genomic sequences of, e.g., viruses. Different PCR techniques have become available for detecting viral pathogens present in boar semen. Van Rijn et al.  developed quantitative real-time PCR tests for detecting five economically important viruses in semen (ADV, CSFV, FMDV, SVDV, PRRSV). The real-time PCR technique combines amplification and detection of amplified products in one closed tube. Therefore, possible contamination from the environment is strongly reduced. In semen of experimentally infected boars, viruses were detected much earlier after infection and more frequently by real-time PCR tests than by virus isolation. Rovira et al.  investigated the sensitivity of reverse-transcriptase PCR on different biological samples run individually, in pools of 3 and in pools of 5. Twenty-nine boars were inoculated with a low-virulent PRRSV isolate. Serum, blood swab, and semen samples were obtained from each boar every 2–3 days for 2 weeks. Data showed that serum was the best sample to detect PRRSV during acute infection, with the blood swab sample performing almost as well. Semen samples failed to detect PRRSV infection in most of the cases. Pooling samples at pool sizes of 3 and 5 resulted in a decrease in the sensitivity of reverse-transcriptase PCR. Sensitivity was reduced by 6% and 8%, respectively, when serum or blood swab samples were run in pools of 5.
Detection of antibodies
Serology is frequently used to investigate the presence or absence of serum antibodies against different pathogens in boar studs. It is an easy and rather inexpensive method that is suitable for monitoring. The results should be interpreted at group or stud level, not at the individual animal level. An important disadvantage is the time period between pathogen exposure and detectable levels of antibodies. For many serologic tests, this time period ranges from one to several weeks, which implies that during that period, boars may shed the virus although they are not identified as being infected. Puncture of the jugular vein in adult boars has some practical limitations in terms of safety of the handler. Therefore, blood swabs have become popular. For blood swab collection, the ear is pricked with a needle during semen collection and a cotton swab is used to absorb the blood droplet. For PRRSV, increased amounts of PRRSV were shown in the blood swab as compared to semen samples, and infections could be detected earlier .
Interpretation of diagnostic testing
It is clear that categorizing semen as either virus free or virus contaminated is not straightforward. For many viruses, e.g., PRRSV, ADV, PCV2, temporal inconsistencies exist among viremia, the presence of virus in semen, and antibodies in serum. In the case of PRRSV, e.g., viremia in adult boars may be of short duration and end before virus shedding in semen ends . Furthermore, in the initial phases of the infection, serologic results will be negative although the virus can be shed in the semen. Finally, boars may remain serologically positive long after the virus is no longer shed in the semen. Because shedding of virus in semen is often intermittent, especially in the later phases of the infection, a negative test result on a semen sample does not preclude subsequent shedding of the virus. A negative result only means that the tested sample of the ejaculate does not contain virus and that the particular ejaculate is likely to be virus free. It does not guarantee absence of contamination in subsequent ejaculates of a boar. Consequently, a negative semen test from a serologically positive boar should be interpreted with caution.
To be continued…