Bovine herpesvirus 1

Bovine herpesvirus 1 (synonym infectious bovine rhinotracheitis/infectious pustular vulvovaginitis virus) can cause clinical disease of the male reproductive tract (infectious balanoposthitis) and be shed in seminal plasma due to replication in the mucosa of the prepuce, penis, and urethra (Weiblen et al., Reference Weiblen, Kreutz, Canabarro, Schuch and Rebelatto1992; van Oirschot et al., Reference van Oirschot1995; van Oirschot, Reference Van Der Poel, Parlevliet, Verstraten, Kooi, Hakze-Van Der Honing and Stockhofe1995; Vogel et al., Reference Vogel, Flores, Weiblen, Winkelmann, Moraes and Braganca2004; Wrathall et al., Reference Wrathall, Simmons and Van Soom2006). While BoHV-1 may not affect sperm motility or acrosomal status (Tanghe et al., Reference Tanghe, Vanroose, Van Soom, Duchateau, Ysebaert, Kerkhofs, Thiry, van Drunen Littel-van den Hurk, Van Oostveldt and Nauwynck2005), others have described an effect on semen quality most likely due to generalized illness rather than a direct effect of the virus on sperm (van Oirschot et al., Reference van Oirschot1995; van Oirschot, Reference Van Der Poel, Parlevliet, Verstraten, Kooi, Hakze-Van Der Honing and Stockhofe1995; Vogel et al., Reference Vogel, Flores, Weiblen, Winkelmann, Moraes and Braganca2004). The virus may inhibit sperm-zona binding by interacting with sperm (Tanghe et al., Reference Tanghe, Vanroose, Van Soom, Duchateau, Ysebaert, Kerkhofs, Thiry, van Drunen Littel-van den Hurk, Van Oostveldt and Nauwynck2005). Bovine herpesvirus 1 can be transmitted through semen and result in infection, reduced conception rates, endometritis, abortion and infertility (Bielanski et al., Reference Bielanski, Dubuc and Hare1988; van Oirschot, Reference Van Der Poel, Parlevliet, Verstraten, Kooi, Hakze-Van Der Honing and Stockhofe1995). Once infected, bulls may shed the virus in semen intermittently throughout life though remaining clinically normal (van Oirschot, Reference Van Der Poel, Parlevliet, Verstraten, Kooi, Hakze-Van Der Honing and Stockhofe1995). However, results suggest that BoHV-1 seropositive bulls can be free of virus for long periods of time if bulls are well managed in a low-stress environment (Eaglesome and Garcia, Reference Eaglesome and Garcia1997).

To prevent BoHV-1 from contaminating semen, bulls should be maintained in isolation or in a BoHV-1-free herd or artificial insemination center during collection and blood should be tested negative for antibody to BoHV-1 at least 21 days after the last collection of semen that is to be used for insemination. Alternatively, when a bull is seropositive or of unknown serologic status, an aliquot of semen from each collection should be tested negative for virus using an assay validated for semen samples (virus isolation or PCR; Chapter 11.8 of the Office International des Epizooties (OIE) Terrestrial Animal Health Code) (OIE, 2017a). To prevent BoHV-1 in semen from being transmitted, 0.25% trypsin could be added to semen for 5 min at room temperature before processing to inactivate the enveloped virus (Bielanski et al., Reference Bielanski, Dubuc and Hare1988; Silva et al., Reference Silva, Solana and Castro1999; Bielanski, Reference Bielanski2007). Alternatively, treatment of BoHV-1 contaminated semen with gamma globulins from hyperimmune serum can neutralize the virus and reduce the risk of viral transmission via artificial insemination without affecting fertility (Wrathall et al., Reference Wrathall, Simmons and Van Soom2006).

Bovine viral diarrhea virus

Another important reproductive pathogen, BVDV, can replicate throughout the bull including within the seminal vesicles, prostate gland and epididymis (Kirkland et al., Reference Kirkland, Richards, Rothwell and Stanley1991). In addition, BVDV antigen has been detected in sertoli cells, spermatogonia and epithelial cells of the urethra (Borel et al., Reference Borel, Janett, Teankum, Zlinszky, Iten and Hilbe2007; Newcomer et al., Reference Newcomer, Toohey-Kurth, Zhang, Brodersen, Marley, Joiner, Zhang, Galik, Riddell and Givens2014). Bovine viral diarrhea virus can be present in semen from bulls with persistent infection, acute (transient) infection, persistent testicular infection and prolonged testicular infection (Table 1). Bulls previously exhibiting acute infections (at up to 28 days after initial infection), persistent testicular infections (at up to 42 months of age), and prolonged testicular infections (at up to 33 months after initial infection) may subsequently produce semen that is free of BVDV (Gard et al., Reference Gard, Stringfellow and Givens2007; Givens et al., Reference Givens, Riddell, Edmondson, Walz, Gard, Zhang, Galik, Brodersen, Carson and Stringfellow2009; Newcomer et al., Reference Newcomer, Toohey-Kurth, Zhang, Brodersen, Marley, Joiner, Zhang, Galik, Riddell and Givens2014). Virus has been transmitted to inseminated animals through semen collected from bulls exhibiting persistent infections, acute infections and persistent testicular infections. The defining difference between cases termed ‘prolonged testicular infections’ and those termed ‘persistent testicular infection’ has not been the assessed duration of the testicular infection but the ease with which virus could be isolated from semen and the associated consistency of viral transmission to inseminated cattle (Givens et al., Reference Givens, Riddell, Edmondson, Walz, Gard, Zhang, Galik, Brodersen, Carson and Stringfellow2009; Newcomer et al., Reference Newcomer, Toohey-Kurth, Zhang, Brodersen, Marley, Joiner, Zhang, Galik, Riddell and Givens2014). Extended isolation (quarantine) in association with routine periodic monitoring from presence of detectable virus in semen might be advisable for some valuable bulls diagnosed with persistent testicular infection. Bovine viral diarrhea virus in semen may be associated with sperm and cannot be removed by centrifugation through a percoll gradient, swim-up techniques, glass wool filtration or glass bead filtration (Bielanski et al., Reference Bielanski, Loewen and Hare1992).

Table 1 Characteristics of infectious with bovine viral diarrhea virus that may result in the contamination of bovine semen with virus

The OIE recommended standards (Chapter 4.6 of the Terrestrial Animal Health Code) require bulls to be free of BVDV in blood on entry into artificial insemination centers with ongoing testing for BVDV and anti-BVDV antibodies during semen collection procedures (Gard et al., Reference Gard, Stringfellow and Givens2007). When admitting bulls through an isolation and quarantine procedure, groups of bulls should be assembled, tested and cleared based on all-in and all-out management after assurance that BVDV is not circulating within the group based on serologic evidence. The OIE recommendations necessitate that seropositive bulls produce semen free of BVDV as determined by virus isolation or virus antigen testing. To meet Certified Semen Services (CSS; a subsidiary of the National Association of Animal Breeders to establish industry self-regulation) standards in the United States, bulls must be non-viremic on entry and maintain the non-viremic status. If these non-viremic bulls test seronegative, no semen testing is required (Gard et al., Reference Gard, Stringfellow and Givens2007). If bulls are seropositive, at least one sample of semen that is completely extended, frozen, and transported to the diagnostic lab at cryogenic temperatures must test negative using a PCR assay (CSS, 2014). Research does clearly indicate that straws of semen should be transported to diagnostic laboratories at cryogenic temperatures (i.e. liquid nitrogen or liquid nitrogen vapor tank) to optimize either PCR or virus isolation assays to detect BVDV in semen (Newcomer et al., Reference Newcomer, Toohey-Kurth, Zhang, Brodersen, Marley, Joiner, Zhang, Galik, Riddell and Givens2014).

Bovine leukemia virus

Bovine leukemia virus (BLV; synonym enzootic bovine leucosis virus) produces a benign persistent lymphocytosis and infections may eventually cause lymphatic tumors in some adult cattle. A seropositive status is considered evidence for ongoing infection with this Deltaretrovirus. The virus is rarely found in semen though possibly present in association with virus-infected lymphocytes in the genital tract (Choi et al., Reference Choi, Monke and Stott2002; Wrathall et al., Reference Wrathall, Simmons and Van Soom2006). While substantial evidence demonstrates that BLV is rarely – if ever – transmitted by artificial insemination, transmission was detected when infected lymphocytes were inoculated into the uterus of seronegative cows (Van Der Maaten and Miller, Reference Vanbinst, Vandenbussche, Dernelle and De Clercq1978; Wrathall et al., Reference Wrathall, Simmons and Van Soom2006). To ensure that semen is free of BLV, Chapter 11.6 of the OIE terrestrial animal health code recommends that semen donor bulls be residents of BLV-free herds and come from a serologically negative dam (if less than 2 years of age), or that blood samples from the bull test negative for antibodies against BLV on two occasions, the first being at least 30 days before and the second at least 90 days after collection of semen (OIE, 2017a).

Lumpy skin disease virus

Lumpy skin disease virus, a member of the genus Capripoxvirus, causes acute, subacute or subclinical disease in cattle. The disease is characterized by firm, circumscribed skin nodules, necrotic plaques in the mouth and nares, fever, and generalized lymphadenopathy. This virus is reported to be endemic in sub-Saharan Africa and Egypt with the potential for spread to other parts of Africa, the Middle East, and Europe (Irons et al., Reference Irons, Tuppurainen and Venter2005; Annandale et al., Reference Annandale, Holm, Ebersohn and Venter2014). After experimental infection, LSDV could be isolated from semen up to 42 days after infection while viral DNA was detected in semen using PCR for up to 5 months (Irons et al., Reference Irons, Tuppurainen and Venter2005). Another study involving experimental intravenous inoculation with a large dose of LSDV suggested that the testis and epididymis are sites of viral persistence within bulls that exhibit prolonged viral shedding (Annandale et al., Reference Annandale, Irons, Bagla, Osuagwuh and Venter2010). The potential for viral transmission via semen remains to be clearly demonstrated though semen that was experimentally spiked with a notably high dose of LSDV did result in viral transmission to inseminated heifers (Annandale et al., Reference Annandale, Holm, Ebersohn and Venter2014). To ensure that semen is free of LSDV, Chapter 11.9 of the OIE terrestrial animal health code recommends that semen donor bulls not show clinical signs of lumpy skin disease (LSD) and reside in an LSDV-free country or zone for at least 28 days before semen collection (OIE, 2017a). Alternatively, the code recognizes the assurance of safety of bovine semen collected from bulls in countries or zones not free of LSD if (a) bulls do not show signs of LSD, (b) bulls reside in an artificial insemination center where no case of LSD occurred during 60 days before semen collection, (c) semen to be exported was tested negative for LSDV by PCR, and either (1) bulls were vaccinated according to vaccine manufacturer’s instructions at least 60 days before first semen collection and exhibited antibodies against LSDV at least 30 days after vaccination, or (2) bulls tested negative using PCR for LSDV in blood samples at commencement of, conclusion of, and at least every 28 days during semen collection and tested seronegative for antibodies to LSDV at least every 28 days during semen collection and at 21 days after the final semen collection of the consignment (OIE, 2017a).

Bluetongue virus

Controlled scientific clinical studies and field investigations focused on the excretion of bluetongue virus (BTV) in semen of bulls has significantly influenced regulatory policy regarding the movement (or lack of movement) of both live cattle and semen for many years (Thibier and Guerin, Reference Thibier and Guerin2000; Wrathall et al., Reference Wrathall, Simmons and Van Soom2006). Bluetongue virus is considered the prototype of the genus Orbivirus in the family Reoviridae with the viral genome consisting of ten linear double-stranded RNA segments. Bluetongue viruses can be categorized into various serotypes, of which 27 distinct serotypes are currently proposed. This robust diversity among BTV often leads to contradictory research findings and notable consternation when clear and specific regulatory guidelines pertaining to BTV are pursued (Gu et al., Reference Gu, Davis, Walsh, Melville and Kirkland2014). Epizootic hemorrhagic disease virus is a closely related Orbivirus that cross-reacts with BTV and sometimes causes clinical disease in cattle. These viruses are transmitted between ruminants by biting midges in the Culicoides species. Cattle usually experience a subclinical BTV infection though they serve as a reservoir host due to prolonged, low level cell-associated viremias that can last up to several months (Vanbinst et al., Reference van Oirschot, Rijsewijk, Straver, Ruuls, Quak, Davidse, Westenbrink, Gielkens, van Dijk and Moerman2010). Clinical signs in sheep are more notable than those in cattle in most circumstances and include fever, excessive salivation, nasal discharge and rare cyanosis of the tongue due to vascular compromise. Some sheep may develop respiratory distress from pulmonary edema and notably painful inflammation of the coronary bands. Depending on the strain involved, early embryonic deaths, abortions, malformed calves or lambs, temporary infertility in bulls and rams, and shedding of BTV in semen may occur (Vanbinst et al., Reference van Oirschot, Rijsewijk, Straver, Ruuls, Quak, Davidse, Westenbrink, Gielkens, van Dijk and Moerman2010).

The sporadic detection of BTV in semen of individual bulls has generally been attributed to BTV-associated red blood cells and mononuclear cells leaking into the semen through microvascular injuries due to inflammation within the male reproductive tract (Wrathall et al., Reference Wrathall, Simmons and Van Soom2006). However, with some BTV serotypes, the hypothesis that contamination of semen results from something other than leakage of BTV-carrying blood cells into semen is supported by: (a) the detection of concentrations of BTV in semen that equal concentrations detected in blood, (b) the shedding of BTV in semen of young and old bulls regardless of the perceived propensity for inflammation of the reproductive tract and (c) the absence of detectable blood cells in semen that tested positive for BTV (Vanbinst et al., Reference van Oirschot, Rijsewijk, Straver, Ruuls, Quak, Davidse, Westenbrink, Gielkens, van Dijk and Moerman2010). The presence of live, virulent BTV serotype 8 in extended semen from naturally infected bulls has been clearly demonstrated (Vanbinst et al., Reference van Oirschot, Rijsewijk, Straver, Ruuls, Quak, Davidse, Westenbrink, Gielkens, van Dijk and Moerman2010). Natural infections of bulls with BTV serotype 8 have been demonstrated to precede a transient decrease in the post-thaw motility of sperm and a brief increase in the percentage of sperm exhibiting morphologic abnormalities (Muller et al., Reference Muller, Kemmerling, Straet, Janowitz and Sauerwein2010).

Though scientific claims were published in the late 1970s and early 1980s of transplacental BTV infections resulting in the production of immunotolerant, persistently viremic bulls, a number of attempts to confirm the findings resulted in strong refutation in 1993 of the existence of persistent BTV infection within immunotolerant cattle (Wrathall et al., Reference Wrathall, Simmons and Van Soom2006). More recent research has demonstrated that BTV serotype 8 can cause a transplacental infection of developing fetuses that results in the birth of virus-positive, specific antibody-negative calves (De Clercq et al., Reference De Clercq, De Leeuw, Verheyden, Vandemeulebroucke, Vanbinst, Herr, Meroc, Bertels, Steurbaut, Miry, De Bleecker, Maquet, Bughin, Saulmont, Lebrun, Sustronck, De Deken, Hooyberghs, Houdart, Raemaekers, Mintiens, Kerkhofs, Goris and Vandenbussche2008). However, attempts to follow up on these five detected calves after one month demonstrated that the calves were not maintaining a detectable persistent infection (De Clercq et al., Reference De Clercq, De Leeuw, Verheyden, Vandemeulebroucke, Vanbinst, Herr, Meroc, Bertels, Steurbaut, Miry, De Bleecker, Maquet, Bughin, Saulmont, Lebrun, Sustronck, De Deken, Hooyberghs, Houdart, Raemaekers, Mintiens, Kerkhofs, Goris and Vandenbussche2008). Thus, concerns regarding the persistent infection of a seronegative, post-pubertal bull that consistently or sporadically produces semen contaminated with BTV appear to lack substantial scientific support.

To prevent shipment of semen containing BTV, Chapter 8.3 of the OIE terrestrial animal health code recommends that semen should be collected from bulls which show no clinical signs of bluetongue on the day of semen collection and have been – for at least 60 days before commencement of and during collection of semen – (a) kept outside a restricted zone, (b) protected against viral vectors or (c) kept during the seasonally vector-free period in a BT seasonally-free area. Bulls may also be subjected to diagnostic tests with negative results to provide assurance of uncontaminated semen: (a) lack of detected antibodies to the BTV group at least every 60 days throughout the semen collection period and between 28 and 60 days after the final collection for this consignment, or (b) virus isolation from blood samples collected at commencement, at least every 7 days during, and at conclusion of semen collection for this consignment with negative results or (c) PCR test on blood samples collected at commencement, at least every 28 days during, and at conclusion of semen collection for this consignment with negative results (OIE, 2017a).

Foot-and-mouth disease virus

Foot-and-mouth disease (FMD) is an extremely contagious disease that affects all cloven-hoofed animals. Though the mortality rate of FMD is generally low, the morbidity rate can be very high in naïve populations of cattle (Meyer et al., Reference Meyer, Zamir, Ben Yair Gilboa, Gelman, Pfeiffer and Vergne2017). In acutely infected cattle, this Apthovirus in the family Picornaviridae is shed in all secretions and excretions including expired air, saliva, skin lesions, urine, feces, and semen. After experimental infection, FMDV has been detected in the semen of naïve bulls up to 4 days before the development of oral vesicles (Sellers et al., Reference Sellers, Burrows, Mann and Dawe1968).

Experimental infection studies involving a low number of vaccinated bulls (21 total) have failed to detect FMD virus in semen of vaccinated bulls subsequent to experimental infection (Meyer et al., Reference Meyer, Zamir, Ben Yair Gilboa, Gelman, Pfeiffer and Vergne2017). Infection of cows through artificial insemination with semen collected from FMD-vaccinated bulls has not been reported. In some clinically and subclinically infected cattle, FMDV can be isolated from oropharyngeal fluids and/or tissues >28 days after infection (Hayer et al., Reference Hayer, Ranjan, Biswal, Subramaniam, Mohapatra, Sharma, Rout, Dash, Das, Prusty, Sharma, Stenfeldt, Perez, Rodriguez, Pattnaik, VanderWaal and Arzt2017). This condition is referred to as persistent FMDV infection and these animals are referred to as ‘carriers’. Transmission of FMDV from carrier cattle by direct contact to naïve cattle is extremely difficult to reproduce under experimental conditions and is considered not to be epidemiologically significant (Hayer et al., Reference Hayer, Ranjan, Biswal, Subramaniam, Mohapatra, Sharma, Rout, Dash, Das, Prusty, Sharma, Stenfeldt, Perez, Rodriguez, Pattnaik, VanderWaal and Arzt2017). Accordingly, the potential for seropositive carrier bulls to transmit FMDV via semen has been considered negligible by some authors (Meyer et al., Reference Meyer, Zamir, Ben Yair Gilboa, Gelman, Pfeiffer and Vergne2017). Federal regulations require that all vaccinated donor bulls whose semen shall be imported to the United States exhibit a negative serology test for antibodies against nonstructural proteins of FMDV – vaccination does not induce such antibodies – and a negative test for virus from an esophageal–pharyngeal sample (Callis, Reference Callis1996). To prevent transmission of FMDV in shipped semen, Chapter 8.8 of the OIE terrestrial animal health code recommends that semen donors (a) show no clinical signs of FMD on the day of collection, (b) were kept for at least 3 months before collection in a FMD-free country, zone, or compartment and (c) were kept in an artificial insemination center where no animals had a history of infection with FMDV (OIE, 2017a). If being shipped from areas where vaccination is practiced, bulls should additionally either be vaccinated at least twice with the last vaccination ⩾1 month but ⩽6 months before collection or test free of antibodies against FMDV not <21 days after collection of the semen (OIE, 2017a).

Schmallenberg virus

Schmallenberg virus may cause stillbirths and musculoskeletal and central nervous system malformations of developing fetuses in naïve dams infected during pregnancy (Hoffmann et al., Reference Hoffmann, Scheuch, Hoper, Jungblut, Holsteg, Schirrmeier, Eschbaumer, Goller, Wernike, Fischer, Breithaupt, Mettenleiter and Beer2013). This Orthobunyavirus is transmitted by biting midges of the Culicoides genus and mosquitoes. The viremia in SBV infected cattle is very short (1 to 5 days; Hoffmann et al., Reference Hoffmann, Schulz and Beer2012; OIE, 2017b) though SBV RNA has been detected – sometimes intermittently – up to almost 3 months after seroconversion (Hoffmann et al., Reference Hoffmann, Scheuch, Hoper, Jungblut, Holsteg, Schirrmeier, Eschbaumer, Goller, Wernike, Fischer, Breithaupt, Mettenleiter and Beer2013; Ponsart et al., Reference Ponsart, Pozzi, Breard, Catinot, Viard, Sailleau, Viarouge, Gouzil, Beer, Zientara and Vitour2014). This virus can be detected in seminal plasma early in acute infections (which may clear without serial positive semen samples) and be associated with the seminal cell fraction in serial positive samples weeks after seroconversion (Hoffmann et al., Reference Hoffmann, Scheuch, Hoper, Jungblut, Holsteg, Schirrmeier, Eschbaumer, Goller, Wernike, Fischer, Breithaupt, Mettenleiter and Beer2013). A single insemination dose of semen can contain SBV sufficient to infect naïve cattle through experimental subcutaneous injection though transmission of SBV by natural breeding or artificial insemination remains to be demonstrated (Schulz et al., Reference Schulz, Wernike, Beer and Hoffmann2014). To declare semen free of SBV, testing of semen samples for SBV RNA using PCR is recommended unless the bull tests negative for SBV-specific antibody at least 28 days after semen production (Van Der Poel et al., Reference Van Der Maaten and Miller2014).