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 evaluated.
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
已经在猪身上发现两种细环病毒（TTV）：细环病毒1型（TTsuV1）及2型（TTsuV2）。 均为DNA病毒，属于新创的指环病毒科、细环病毒属。据估计，猪细环病毒（TTsuVs）在全球猪群中普遍存在 。目前，感染猪细环病毒（TTsuV）无相关临床症状出现。但TTsuV与猪圆环病毒（PCV）相关疾病有关[76,77]。细环病毒呈垂直和水平传播。可在胎儿组织和血液、精液和初乳中检测到该病毒，由此证明其存在垂直传播的可能。从人工授精中心收集100头公猪的独立精液样本，用常规PCR检测TTsuV1和TTsuV2。其中四份样本呈TTsuV1 PCR阳性，五份呈TTsuV2阳性，合并感染后，两种TTsuVs均被检测到。胎儿感染的途径尚不明确，但相关显示证明可能是由于精液带毒或者胎盘传播。
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 .
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 evaluation 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.
实验证明，CSFV  PRRSV 可通过精液传染母猪，在精液中加入PPV 、ADV 、PRRSV[18,21]和PCV2后，亦可。Habu等 ，Guérin和Pozzi 提出，虽然没有找到科学的文章证实，但是如果使用含有日本乙型脑炎病毒的精液进行授精，则后备母猪极易被感染。若将被病毒污染的精液应用于人工授精，母猪乃至母猪群都会有病毒传播的风险。Nathues等以瑞士引入公猪精液导致PRRSV爆发为例，对此加以说明。
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，病毒并不总是能够形成传播（例如PRRSV）[18,61]。母猪被感染所需的条件很复杂，未能传播可能是由于母猪免疫、病毒特点以及未达到最小感染剂量。因此，针对精液传播PRRSV的风险，以及母猪感染病毒所需的最小剂量，开展了更多的研究。那么，与常规宫颈输精相比，小剂量精液的深部输精（40–50 mL对比80–90 mL），可降低病毒传染母猪的风险。
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
PCR是一种灵敏度高、特异性强，能够快速检测基因组序列（如，病毒）的检测技术。已经有多种PCR技术可用于检测公猪精液中存在的病毒。Van Rijn等研发出的实时荧光定量PCR，可以对精液中5种能够严重影响经济效益的病毒（ADV，CSFV，FMDV，SVDV，PRRSV）进行检测。实时PCR技术是将扩增和扩增产物的检测合二为一。从而来自环境中的污染可能大大降低。通过实验发现，相比病毒分离，采用实时PCR检测带毒精液，可以在感染后更早、更快发现病毒。 Rovira等研究了反转录酶PCR针对不同生物样品的灵敏度，这些样品分别为3个样品为一池和5个样品为一池。用低毒PRRSV分离株接种了29头公猪。2周内，每2到3天从每头公猪获取血清、血液拭子和精液样本。得出的数据显示，急性感染期间，血清是检测PRRSV的最佳样品，血液拭子亦可。但是大多数精液样本中，未能检测到PRRSV感染。以3个和5个样品为一池会造成反转录酶PCR的灵敏度下降。当分别以5个样品为一池检测血清或血液拭子，其灵敏度分别降低6％和8％。
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…