**3.11.1 Swine Vesicular Disease Virus**

262 A Bird's-Eye View of Veterinary Medicine

infertility in Japanese pigs (Habu et al., 1977). Infection of susceptible boars resulted in edematous, congested testes and semen with numerous abnormal spermatozoa and significantly decreased total and motile sperm counts (Habu et al., 1977). These changes are usually temporary and most boars recover completely. JBEV has been isolated from the testicles of boars with orchitis and also can be shed in the semen for 5 weeks (Habu et al., 1977). JBEV infection can easily be transmitted if gilts are inseminated with infected semen

Porcine circovirus type 2 (PCV2) is a small, single-stranded, ambi-sense DNA virus that belongs to the family *Circoviriade*, genus *Circovirus* (Tischer et al., 1982)*.* PCV2 is a ubiquitous virus and most herds worldwide are seropositive for PCV2 (Dulac & Afshar, 1989; Edwards & Sands, 1994; Segalés et al., 2008; Tischer et al., 1986). When first described in 1998, PCV2 was linked to disease mainly characterized by wasting and generalized lymphadenopathy in growing pigs (Allan et al., 1998; Morozov et al., 1998). Since that time, PCV2 has been associated with several disease manifestations in pigs commonly referred to as PCV-associated disease (PCVAD) which includes systemic disease, respiratory disease, enteric disease, porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure in dams (late term abortions and stillbirths) (Choi & Chae, 2001; Harms et al., 2002; Kim et al., 2004a; Kim et al., 2004b). PCV2-associated reproductive disease under field conditions is rare. The main route of transmission has been postulated to be the fecal-oral route (Segalés et al., 2005); however, due to the rapid spread and the extensive use of AI, semen transmission has been suggested as a potentially significant route of dissemination of PCV2 (Horlen et al., 2007; Lawton et al., 2004; Rose et al., 2003; Sibila et al., 2004; West et al., 1999). Mature boars infected with PCV2 generally lack clinical signs and lesions (Larochelle et al., 2000; Madson et al., 2008). The virus has been detected in semen of naturally and experimentally infected boars, even after the appearance of antibodies in the serum (Larochelle et al., 2000). In the acute phase of infection, PCV2 DNA was detected in serum and semen at two and five days post inoculation, respectively (Larochelle et al., 2000; Madson et al., 2008). Detection of PCV2 viremia commonly precedes the detection of semenassociated virus (Madson et al., 2008), but semen shedding has been reported in the absence of viremia (Larochelle et al., 2000). Anti-PCV2 antibodies typically develop by two weeks post inoculation (Larochelle et al., 2000; Madson et al., 2008). Following intranasal inoculation, intermittent semen shedding of PCV2 DNA was observed during a 47 day observation period (Larochelle et al., 2000). Intermittent semen shedding was confirmed by a different study (Grasland et al., 2008) after testing semen samples for 56 days in four inoculated boars. In contrast, continuous semen shedding was observed following intranasal and intramuscular inoculation following a 90 day observational period (Madson et al., 2008). In addition, naturally infected boars were found to sporadically shed PCV2 DNA in semen for up to 27.3 weeks in a PCV2 positive boar stud (McIntosh et al., 2006). Peak PCV2 shedding in semen occurs between nine and 20 days post inoculation (Grasland et al., 2008; Madson et al., 2008). Changes in morphology, motility, or quantity are not commonly associated with natural or experimental PCV2 infection (Madson et al., 2008; McIntosh et al., 2006). Clinical signs of PCV2 infection in the dam are typically absent or unapparent; however, a low percentage of females may abort due to systemic illness.

(Guerin & Pozzi, 2005; Habu et al., 1977).

**3.10 Porcine Circovirus type 2** 

Swine vesicular disease virus (SVDV) is a small, non-enveloped single-stranded positivesense RNA virus, in the family *Picornaviridae*, genus *Enterovirus* (Nardelli et al., 1968)*.*  Documented outbreaks of this disease have been limited to selected countries in Asia, Europe and Central America. Clinical signs include appearance of vesicles around the coronary bands, snout, tongue and lip (Kanno et al., 1995) making this disease an important differential for FMDV. After natural infection, SVDV has been isolated from infected boar semen for up to four days, but AI with contaminated semen failed to transmit the disease to sows (McVicar et al., 1977).

#### **3.11.2 Non-Classical Swine Fever Virus pestiviruses**

Pigs are also susceptible to non-CSFV pestiviruses, including bovine viral diarrhea virus (BVDV) and border disease virus (BDV), which are associated with disease in cattle and sheep, respectively. Pigs congenitally infected with these viruses may shed large amounts of virus. Previously, BVDV has been isolated from oropharyngeal fluid, urine and semen of a congenitally infected, infertile boar (Terpstra & Wensvoort, 1997).

#### **3.11.3 Porcine retroviruses**

Retroviruses are RNA viruses that exist in two main groups: endogenous or exogenous retroviruses. Endogenous retroviruses are thought to be present in all vertebrates accounting for approximately 8% of their genomes (Tucker et al., 2006). All pigs carry PERV in their genome (Wilson, 2008) and three porcine endogenous retrovirus (PERV) subtypes have been identified based on their envelope sequence and tropism in cell culture (Le et al., 1997; Wilson et al., 2000): PERV-A, PERV-B and PERV-C (Martin et al., 2000a, 2000b; Patience et al., 1997; Specke et al., 2001; Takeuchi et al., 1998). More recently, the possible existence of exogenous porcine retrovirus in pigs was proposed (Scobie et al., 2004; Wood et al., 2004). It was demonstrated that a human-tropic recombination between PERV-A and PERV-C (designated as PERV-A/C) was not a product of *in vitro* recombination; instead PERV-A/C appeared to exist *in vivo* as an exogenous virus (Wood et al., 2004). Furthermore, a PERV-A/C recombinant was isolated from porcine peripheral blood mononuclear cells and was not present in a proviral form in the miniature swine genome (Scobie et al., 2004a; Wood et al., 2004). The PERV A/C virus is a recombination within the *env* region and is thought to arise from exogenous recombination of mRNA (Martin et al., 2006). Shedding via the semen route occurs, since PERVs are embedded in the genome.

Artificial Insemination and Its Role in Transmission of Swine Viruses 265

become a popular sample type in recent years. For blood swab collection, the ear is pricked with a needle during semen collection and a cotton swab is used to catch the resulting blood drop (Reicks et al., 2006). Pathogens that cause systemic disease are typically identified in serum or blood first before they enter the semen and this should be taken into consideration when collecting appropriate samples. A negative result on semen only means that the tested sample does not contain virus, and that the particular semen sample is likely to be virusfree. It does not provide certainty that there will be no risk of contamination in the future. On the other hand, a negative result on serum does not guarantee that the semen is free of

Economic aspects are important to consider. For interpretation of diagnostic results at the boar stud level, in addition to the detection limit of the diagnostic method used, it is extremely important to test a representative number of semen samples, and to include evaluation of more than one parameter, such as presence of antibodies, viremia, or clinical symptoms. Because of the potential high cost of this approach, producers commonly rely on testing pooled samples. This strategy allows testing a larger number of animals while running the same number of tests, thus decreasing the cost per boar stud. Pooling can be successfully applied if the assay utilized has a high analytical sensitivity; however, because of a dilution effect, the sensitivity of the test when run on pooled samples is lower than its

As a very basic and broad approach, in order to avoid spread of disease via contaminated semen, the health status of the animals should be checked daily. In case of any abnormality or clinical disease signs, semen collection should be halted until the animal has recovered. However, as indicated before, clinical examination alone is insufficient for most viral infections, since clinically normal boars can shed pathogens in their semen for extended

For demonstration of viable virus in semen two options exist including virus isolation and conducting bioassays. Virus isolation is often difficult as bacterial contamination of semen can be substantial. Virus isolation can be further complicated by the existence of cytotoxic factors in semen that destroy cell culture systems, and antiviral factors that nonspecifically neutralize virus (Christopher-Hennings et al., 1995b, 1997; Prieto et al., 1996b, 2003). In many cases swine bioassays have been found to be more sensitive than cell culture systems for demonstrating viable viruses in semen. However, animal inoculations cannot be justified to be used routinely

Much progress has been made in recent years to improve the quality of molecular diagnostic assays used on semen. Numerous PCR techniques are now available for detecting

the virus as the viremic stage may have been very short.

sensitivity when run on individual samples (Munoz-Zanzi et al., 2006).

for large numbers of samples and turnaround time for test results is poor.

**4.2 Pooling of samples** 

**4.3 Detection methods** 

periods of time.

**4.3.1 Clinical evaluation of the boar** 

**4.3.2 Detection of viable virus** 

**4.3.3 Direct detection of RNA/DNA** 

#### **3.11.4 Swine Influenza Virus and transmissible gastroenteritis virus**

Swine influenza virus (SIV), a negative-sense, single-stranded RNA virus in the genus *influenzavirus A* of the family *Orthomyxoviridae* (Lamb & Krug, 2001; Wright & Webster, 2001), and transmissible gastroenteritis virus (TGEV), a positive-sense, single-stranded RNA virus in the *Coronaviridae* family (Lai & Cavanagh, 1997) have not been isolated from semen. Contamination of semen and transmission of SIV and TGEV have not been demonstrated to date; however, these viruses are highly contagious and could be easily transmitted via aerosol at the time of collection and preparation of semen. Boars in the acute stages of SIV or TGEV infection are often clinically ill on examination (Guerin & Pozzi, 2005).

#### **3.11.5 Porcine Cytomegalovirus**

Porcine cytomegalovirus (PCVM), a double-stranded DNA virus, is classified in the genus *Cytomegalovirus* in the subfamily *Betaherpesvirinae* of the family *Herpesviridae* (Roizman, 1982). Infection with PCMV is usually subclinical in adults including boars. Following infection in boars, the virus was detected in the testis and the epididymis (Shirai et al., 1985); however, semen shedding of the virus has not been determined.

#### **3.11.6 Porcine Adenovirus**

Porcine adenovirus (PAV) is a double-stranded, linear DNA virus which belongs to the family *Adenoviridae.* PAV is distributed worldwide and associated with gastroenteric disease. As such, its importance in AI is due to the potential of fecal contamination of semen.

#### **4. Diagnostic approaches to prevent virus transmission by the semen route**

The concern of spreading diseases on a global scale via semen has placed restrictions on international movement of semen. From the early 1990s onwards, major improvements in detection methods particularly in terms of sensitivity have better enabled diagnostic laboratories to detect viruses in boar semen. Today, various methods are available for demonstration of viruses in boars and include monitoring for presence of clinical signs, demonstration of virus (virus isolation, bioassay), nucleic acids, and demonstration of antibodies against the virus. However, it needs to be considered that the results of any of these methods may vary substantially, depending on individual sample type and the sensitivity and specificity of a chosen diagnostic method. Accurate diagnosis of semen associated viruses (correctly identified positive or negative status of any given sample) is extremely important as false positive as well as false negative test results can cause substantial economic damage. For example, with PRRSV, any false positive sample out of a boar stud has the following consequences: (1) Immediate hold on all semen samples from the stud to breeding herds resulting in missed cycles and associated production losses in hundreds to thousands of sows supplied by the boar stud. (2) Retesting of boars. (3) Culling of suspect positive boars.

#### **4.1 Importance of sample type**

Samples that are commonly collected in boar studs for routine surveillance include semen (raw or extended), blood swabs, serum and more recently oral fluids. Blood swabs have become a popular sample type in recent years. For blood swab collection, the ear is pricked with a needle during semen collection and a cotton swab is used to catch the resulting blood drop (Reicks et al., 2006). Pathogens that cause systemic disease are typically identified in serum or blood first before they enter the semen and this should be taken into consideration when collecting appropriate samples. A negative result on semen only means that the tested sample does not contain virus, and that the particular semen sample is likely to be virusfree. It does not provide certainty that there will be no risk of contamination in the future. On the other hand, a negative result on serum does not guarantee that the semen is free of the virus as the viremic stage may have been very short.

#### **4.2 Pooling of samples**

264 A Bird's-Eye View of Veterinary Medicine

Swine influenza virus (SIV), a negative-sense, single-stranded RNA virus in the genus *influenzavirus A* of the family *Orthomyxoviridae* (Lamb & Krug, 2001; Wright & Webster, 2001), and transmissible gastroenteritis virus (TGEV), a positive-sense, single-stranded RNA virus in the *Coronaviridae* family (Lai & Cavanagh, 1997) have not been isolated from semen. Contamination of semen and transmission of SIV and TGEV have not been demonstrated to date; however, these viruses are highly contagious and could be easily transmitted via aerosol at the time of collection and preparation of semen. Boars in the acute stages of SIV or

Porcine cytomegalovirus (PCVM), a double-stranded DNA virus, is classified in the genus *Cytomegalovirus* in the subfamily *Betaherpesvirinae* of the family *Herpesviridae* (Roizman, 1982). Infection with PCMV is usually subclinical in adults including boars. Following infection in boars, the virus was detected in the testis and the epididymis (Shirai et al., 1985);

Porcine adenovirus (PAV) is a double-stranded, linear DNA virus which belongs to the family *Adenoviridae.* PAV is distributed worldwide and associated with gastroenteric disease. As such, its importance in AI is due to the potential of fecal contamination of semen.

**4. Diagnostic approaches to prevent virus transmission by the semen route**  The concern of spreading diseases on a global scale via semen has placed restrictions on international movement of semen. From the early 1990s onwards, major improvements in detection methods particularly in terms of sensitivity have better enabled diagnostic laboratories to detect viruses in boar semen. Today, various methods are available for demonstration of viruses in boars and include monitoring for presence of clinical signs, demonstration of virus (virus isolation, bioassay), nucleic acids, and demonstration of antibodies against the virus. However, it needs to be considered that the results of any of these methods may vary substantially, depending on individual sample type and the sensitivity and specificity of a chosen diagnostic method. Accurate diagnosis of semen associated viruses (correctly identified positive or negative status of any given sample) is extremely important as false positive as well as false negative test results can cause substantial economic damage. For example, with PRRSV, any false positive sample out of a boar stud has the following consequences: (1) Immediate hold on all semen samples from the stud to breeding herds resulting in missed cycles and associated production losses in hundreds to thousands of sows supplied by the boar stud. (2) Retesting of boars. (3) Culling

Samples that are commonly collected in boar studs for routine surveillance include semen (raw or extended), blood swabs, serum and more recently oral fluids. Blood swabs have

**3.11.4 Swine Influenza Virus and transmissible gastroenteritis virus** 

TGEV infection are often clinically ill on examination (Guerin & Pozzi, 2005).

however, semen shedding of the virus has not been determined.

**3.11.5 Porcine Cytomegalovirus** 

**3.11.6 Porcine Adenovirus** 

of suspect positive boars.

**4.1 Importance of sample type** 

Economic aspects are important to consider. For interpretation of diagnostic results at the boar stud level, in addition to the detection limit of the diagnostic method used, it is extremely important to test a representative number of semen samples, and to include evaluation of more than one parameter, such as presence of antibodies, viremia, or clinical symptoms. Because of the potential high cost of this approach, producers commonly rely on testing pooled samples. This strategy allows testing a larger number of animals while running the same number of tests, thus decreasing the cost per boar stud. Pooling can be successfully applied if the assay utilized has a high analytical sensitivity; however, because of a dilution effect, the sensitivity of the test when run on pooled samples is lower than its sensitivity when run on individual samples (Munoz-Zanzi et al., 2006).

#### **4.3 Detection methods**

## **4.3.1 Clinical evaluation of the boar**

As a very basic and broad approach, in order to avoid spread of disease via contaminated semen, the health status of the animals should be checked daily. In case of any abnormality or clinical disease signs, semen collection should be halted until the animal has recovered. However, as indicated before, clinical examination alone is insufficient for most viral infections, since clinically normal boars can shed pathogens in their semen for extended periods of time.

#### **4.3.2 Detection of viable virus**

For demonstration of viable virus in semen two options exist including virus isolation and conducting bioassays. Virus isolation is often difficult as bacterial contamination of semen can be substantial. Virus isolation can be further complicated by the existence of cytotoxic factors in semen that destroy cell culture systems, and antiviral factors that nonspecifically neutralize virus (Christopher-Hennings et al., 1995b, 1997; Prieto et al., 1996b, 2003). In many cases swine bioassays have been found to be more sensitive than cell culture systems for demonstrating viable viruses in semen. However, animal inoculations cannot be justified to be used routinely for large numbers of samples and turnaround time for test results is poor.

#### **4.3.3 Direct detection of RNA/DNA**

Much progress has been made in recent years to improve the quality of molecular diagnostic assays used on semen. Numerous PCR techniques are now available for detecting

Artificial Insemination and Its Role in Transmission of Swine Viruses 267

Commercial AI centers need be regularly checked for conformity with specific criteria, assuring that their products are free of certain pathogens and contain a minimal or acceptable number of microorganisms (Guerin & Pozzi, 2005; Prieto et al., 2004). Many veterinary diagnostic laboratories took this need into account and started to offer daily

To reduce the unavoidable presence of bacteria in the ejaculate and to prolong *in vitro* longevity of sperm, use of antimicrobials in semen is a common part of most semen extenders. Apart from a possible dilution effect of pathogens, semen processing and addition of antimicrobials does not eliminate viruses. The use of effective antiviral agents to render semen virus-free has so far not been adopted in the swine AI industry. However, several best practices for handling, storage conditions, and use of fresh semen have been described to reduce the potential for transmission of viral pathogens (Guerin & Pozzi, 2005). For boars, the immediate use of fresh semen increases the risk of pathogen transmission

The use of modified-live and inactivated-virus vaccines in boars can be highly effective in eliminating or decreasing shedding of viruses and with that decreasing the risk of virus transmission by AI. For example, vaccination against PPV may help to reduce shedding of the virus following infection. This has also been shown for PCV2 under experimental conditions (Opriessnig et al., 2011). In many countries, vaccination is done as part of a PRV eradication program (Siegel & Weigel, 1999). In the case of PRRSV, the use of a modified live virus vaccine shortened or eliminated virus shedding in boars challenged with wild-type virus for 50 days after vaccination (Christopher-Hennings et al., 1997; Nielsen et al., 1997). However, based on other studies it appears that PRRSV vaccination provides only partial protection (Nielsen et al., 1997). Moreover, semen shedding has been demonstrated after vaccination with a modified live vaccine virus (Christopher-Hennings et al., 1997). In addition, the modified live PRRSV vaccine virus has been shown to be shed in the semen in low levels (Christopher-Hennings et al., 1995b). In contrast, an inactivated vaccine, did not clearly reduce subsequent shedding of wild-type virus in semen (Christopher-Hennings et al., 1997; Stevenson et al., 1994). The presence of PRRSV in semen was demonstrated by PCR in most of the vaccinated boars during an interval of 7–21 days post-vaccination, although some boars sporadically shed the virus for longer periods of time (Christopher-Hennings et al., 1997). Similarly, when a swine bioassay was used, the presence of infectious virus in semen samples of vaccinated boars was

Virus contamination of boar semen poses a great risk for breeding herds worldwide due the possibility of fast introduction of viruses into large naïve and susceptible populations.

**5.2 Routine surveillance testing** 

**5.3 General semen handling and storage** 

confirmed 14 days post-vaccination (Nielsen et al., 1997).

service for boar studs.

(Prieto et al., 2004).

**5.4 Vaccination** 

**6. Summary** 

pathogens in boar semen. Many of these are highly sensitive, specific and rapid (Christopher-Hennings et al., 1995b). In the case of PRRSV, reverse transcriptase-PCR is today considered to be the most sensitive diagnostic technique. It allows for the detection of as little as 100 TCID50 per seminal dose (Christopher-Hennings et al., 1995b; Gradil et al., 1996), 20 times less virus than what has been shown experimentally to result in transmission of PRRSV in gilts (Shin et al., 1997).
