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June 2007 Issue
PRRSV Transmission
Porcine reproductive and respiratory syndrome virus (PRRS virus or PRRSV) levels a significant economic impact on the swine industry. It is highly infectious and causes a wide array of reproductive problems, including reproductive failure, abortion, stillbirth, and piglets born weak. In addition, PRRSV causes respiratory and other general clinical symptoms, which often result in confusion with swine influenza and other diseases–yet PRRSV is distinctive in its ability to cause persistent infection, with animals continuing to shed virus while absent other visual symptoms. Dr. Jeffrey Zimmerman’s article below delves into careful detail about the nature of the virus, how it is shed and how it is transmitted. His exploration of the research shows how this mutable virus gains entry to herds from a variety of sources, including the introduction of infected animals, semen, as well as contaminated equipment and clothing. The article also discusses the sanitation procedures and environmental conditions that have been shown to deactivate PRRSV. Zimmerman’s summary of PRRSV research is an important tool that can help direct efforts to control the spread of PRRSV within and among herds.
The basis for understanding the control of porcine reproductive and respiratory syndrome virus (PRRSV)
(Porcine Arterivirus)
J. Zimmerman, DVM, PhD
College of Veterinary Medicine, Iowa State University
About PRRSV
PRRS virus (PRRSV) is a small, positive-strand RNA virus. Together with equine arteritis virus, lactate dehydrogenase-elevating virus and simian hemorrhagic fever virus, PRRSV belongs to the family Arteriviridae, which along with family Coronaviridae form the order Nidovirales (Cavanagh 1997). PRRSV is an enveloped virus with a diameter of 50–65 nm, a relatively smooth surface and a cuboidal, nucleocapsid core with a diameter of 25–35 nm (Benfield et al. 1992).
PRRSV is highly host-restricted, growing primarily in porcine alveolar macrophages and in macrophages of other tissues (Pol et al. 1991). PRRSV can also replicate in testicular germ cells (spermatids, spermatocytes, multinucleated giant cells) in infected boars (Sur et al. 1997). In vitro, PRRSV grows in primary cultures of porcine alveolar macrophages, as well as the MA-104 African green monkey kidney cell line or its derivatives (Benfield et al. 1992, Kim et al. 1993). A cell line derived from cotton rat lung cells has also been reported to be highly susceptible to PRRSV (rat cell line, ATCC PTA-3930).
PRRSV strains or isolates are highly variable based on: (1) variation in the clinical presentation of the disease; (2) experimental evidence of differences in pneumovirulence and/or reproductive losses; (3) antigenic differences as determined by reactivity with polyclonal and monoclonal antibodies; and (4) differences in RNA sequences.
Strain diversity and prevalence
Concomitant with improvements in the diagnostic assays has been an increased awareness of the genetic/antigenic diversity of PRRSV strains in the field (Meng 2000). In many laboratories, genetic sequencing has become routine in the diagnostic assessment of PRRSV infections.
The increased use of genetic sequencing has made it clear that genetically diverse PRRSV strains may coexist on the same farm.
The increased use of genetic sequencing has made it clear that genetically diverse PRRSV strains may coexist on the same farm (Dee et al. 2001; Goldberg et al. 2003). Likewise, sequencing has led to the recognition of European genotype (Type 1) strains in areas previously considered to be exclusively populated by U.S. genotype (Type 2) strains, and vice versa (Ropp et al. 2004). Thus, in North America, Europe and probably elsewhere, two distinct genotypes of PRRSV sharing only partial cross-protection may be found. This has great significance for vaccine strain selection and for the performance of diagnostic assays. Because recombination is likely an important genetic mechanism contributing to PRRSV evolution (Yuan et al. 1999), an additional concern is the possibility that recombination may occur between North American and European viruses coexisting in the same region. However, in vitro (cell culture) conditions indicate that RNA recombination is more likely to occur between two European (Type 1) strains or between two North American (Type 2) strains than between a Type 1 strain and a Type 2 strain (van Vugt et al. 2001).
Accurate estimates of the prevalence of infection with wild-type virus in specific countries or regions are not readily available, but within infected regions, 60–80% of herds are typically infected (Geue 1995; Hirose et al. 1995; Lu et al. 1999; Maes 1997; Mateusen et al. 2002; USDA 1997). The use of modified live virus (MLV) vaccines has made it difficult to estimate prevalence. Antibodies against vaccine virus are not easily differentiated from antibodies against PRRSV field strains. Furthermore, vaccine strain viruses are shed and transmitted in the field, further compounding the problem of identifying infection with wild-type virus (Astrup and Riising 2002; Bøtner et al. 1997; Christopher-Hennings et al. 1996, 1997; Mengeling et al. 1998; Sipos et al. 2002).
Susceptible species
Presumably, PRRSV entered domestic swine from an as-yet-unidentified wildlife species. A number of species have been determined not to be susceptible to PRRSV, including mice, rats (Hooper et al. 1994) and guinea pigs (J. Zimmerman, unpublished data). Wills et al. (2000) found no evidence of PRRSV replication in cats, dogs, mice, opossums, raccoons, rats, skunks, house sparrows or starlings. Zimmerman et al. (1997) reported that mallard ducks (Anas platyrhynchos) were susceptible to PRRSV, but subsequent workers have not replicated these results (Trincado et al. 2004b). Feral swine are susceptible to PRRSV infection, but according to serosurveys, infection in free-ranging feral swine animals is relatively rare (Albina et al. 2000; Lutz and Wurm 1996; Olslage et al. 1994; Saliki et al. 1998). Even so, in areas where feral swine interact with domestic swine, they could conceivably serve as a source of PRRSV. Within superfamily Suoidea (Sus spp., peccaries, warthogs and babirusa), the susceptibility of species, other than Sus scrofa, for PRRSV is unknown.
Routes of shedding
Infected animals shed virus in saliva (Wills et al. 1997a), nasal secretions (Benfield et al. 1994; Christianson et al. 1993; Rossow et al. 1994a), urine (Wills et al. 1997a), semen (Swenson and Zimmerman 1993; Swenson et al. 1994a), and feces (Christianson et al. 1993). Pregnant susceptible females inoculated in late gestation shed virus in mammary secretions (Wagstrom et al. 2001).
Shedding of virus in semen is of particular concern because of the wide-spread use of artificial insemination. The duration of semen shedding varies widely among boars (Christopher-Hennings et al. 1996). Swenson et al. (1994a) found infectious virus in the semen of experimentally infected boars for up to 43 days following exposure. By polymerase chain reaction (PCR), Christopher-Hennings et al. (1995a) detected viral RNA in the semen of experimentally infected boars for up to 92 days post inoculation (DPI) and isolated PRRSV from the bulbourethral gland of a boar euthanized at 101 DPI. Semen shedding of MLV vaccine virus occurred for up to 39 days in one study, but prior vaccination eliminated or reduced shedding upon challenge (Christopher-Hennings et al. 1997).
Christopher-Hennings et al. detected viral RNA in the semen of experimentally infected boars for up to 92 days post inoculation (DPI).
Transmission
Swine are susceptible to PRRSV by several routes of exposure, including intranasal, intramuscular, oral (Magar et al. 1995; Magar and Larochelle 2004; van der Linden et al. 2003), intrauterine (Christianson et al. 1993), and vaginal (Benfield et al. 2000a; Gradil et al. 1996; Yaeger et al. 1993). Pigs are not equally susceptible to PRRSV by all routes of exposure. That is, the probability that a given dose will result in infection differs by route of exposure. Hermann et al. (2005) estimated the infectious dose (ID), i.e., the dose required to infect one-half of the exposed animals, for oral and intranasal routes of exposure to be 10 TCID and 10 TCID, respectively. Based on data from Benfield et al. (2000a), the ID for exposure via artificial insemination is approximately 10 TCID. Yoon et al. (1999) reported that exposure to 20 or fewer PRRSV particles by intramuscular exposure resulted in infection.
Overall, the infectivity data indicate that pigs are extremely susceptible to infection via parenteral exposure (breaks in the skin barrier) and much less susceptible by all other routes investigated to date. In the field, potential parenteral exposures include standard husbandry practices, i.e., ear notching, tail docking, teeth clipping, tattooing and inoculations with medications and biologics. Likewise, because PRRSV is present in saliva for weeks following infection, normal pig behavior commonly results in parenteral exposures through bites, cuts, scrapes and/or abrasions that occur during aggressive interactions among pigs. Bierk et al. (2001) associated transmission with aggressive behavior between carrier sows and susceptible contacts. Other behaviors that result in exchange of blood and saliva, such as tail biting and ear biting, may also function in transmission. The significantly higher ID estimates for oral and intranasal exposures imply that transmission by these routes is less common and more easily prevented.
Pigs are extremely susceptible to infection via parenteral exposure (breaks in the skin barrier) ... In the field, potential parenteral exposures include standard husbandry practices … Likewise, because PRRSV is present in saliva for weeks following infection, normal pig behavior commonly results in parenteral exposures.
Indirect transmission involves transmission by inanimate objects (fomites–e.g., equipment, instruments, clothing), substances (e.g., water, food), living carriers (vectors–e.g. mosquitos, flies), or aerosols. Otake et al. (2002b) confirmed needle-borne transmission of PRRSV under experimental conditions. In addition, Otake et al. (2002a) showed that PRRSV was present on fomites such as workers' coveralls, boots and hands following 60 minutes of contact with acutely infected pigs. Importantly, elementary sanitation procedures, e.g., changing coveralls, changing boots and washing hands, were sufficient to stop transmission (Dee et al. 2004a). Under experimental conditions, Dee et al. (2002, 2003) showed that PRRSV could be moved extensively in the field on fomites under winter conditions, i.e., below 32°F (0°C), but to a much lesser degree during warm weather, i.e., 50-60.8°F (10-16°C), illustrating the importance of temperature in virus survival.
Preliminary reports suggest a possible role for arthropods in PRRSV transmission. PRRSV has been detected in, or on, wild-caught flies and mosquitoes (Otake et al. 2002c; Pringproa et al. 2004; Schurrer et al. 2004). Under experimental conditions, Otake et al. (2002c) demonstrated mechanical transmission of PRRSV by mosquitoes and house flies (Musca domestica) (Otake et al. 2003). Overall, the current research data suggests that flies and mosquitoes might serve as mechanical vectors of PRRSV. However, the available data has not proven that PRRSV is an arthropod-borne infection. Typically, the ecologic relationships among host, infectious agent, arthropod and environment in arthropod-borne infections are complex. Additional data is required to connect the current observations into a cohesive understanding of the role of arthropods in the transmission of PRRSV in the field.
Airborne virus was once considered the primary route of PRRSV transmission. Airborne transmission, along with arthropod-borne transmission, could explain the apparent long-distance transmission (area spread) of PRRSV in the absence of other sources of virus (pigs, inanimate objects, people). However, airborne transmission of PRRSV has been difficult to document. Under experimental conditions, transmission from infected to susceptible pigs over a space of 1.0-2.5 meters has been successful in approximately 50% of the attempts (Lager and Mengeling 2000; Otake et al. 2002d; Torremorell et al. 1997; Wills et al. 1997b). The one exception to this pattern of poor airborne transmissibility is a report by Kristensen et al. (2004). In three trials, approximately 50 acutely infected pigs transmitted PRRSV over a distance of one meter to approximately 50 susceptible pigs when 1%, 10%, or 70% of air was exchanged. In a field setting, airborne transmission did not occur over distances of 15 meters (Trincado et al. 2004a) and 30 meters (Otake et al. 2002d). The role of airborne transmission of PRRSV will not be understood until additional information is available, particularly regarding the quantity of virus excreted by pigs, the source of the virus, the rate of inactivation of aerosolized virus and the infectious dose for pigs by aerosol exposure.
Vertical transmission
PRRSV is transmitted from viremic dams transplacentally to fetuses, resulting in fetal death or birth of infected pigs that are weak or appear normal (Bøtner et al. 1994; Christianson et al. 1992; Terpstra et al. 1991). Some pigs in affected litters may escape infection with PRRSV. PRRSV can replicate in fetuses 14 days of gestational age or older, but infection of fetuses during the first two-thirds of gestation is uncommon because most strains of PRRSV cross the placenta efficiently only in the last trimester of pregnancy (Christianson et al. 1993; Lager and Mengeling 1995; Mengeling et al. 1994; Prieto et al. 1996a,b). The reason for the difference in efficiency of maternal-placental viral transit at different stages of gestation and the mechanism(s) of viral transit are unknown. Transit is independent of the reproductive virulence of the virus strain. Park et al. (1996) showed that PRRSV strains of low and high virulence for fetuses cross the placenta with equal efficiency when sows are inoculated at 90 days of gestation.
Persistent infection
PRRSV produces a chronic, persistent infection in pigs. The virus replicates in susceptible cells of clinically inapparent carrier animals for several months. This is the single most significant epidemiological feature of PRRSV infection. Persistent PRRSV infection has been documented through transmission experiments and by detection of virus in animals. Zimmerman et al. (1992) reported transmission of PRRSV from a sow infected 99 days earlier to susceptible sentinels. Following in utero exposure at day 90 of gestation, Benfield et al. (2000b) isolated virus from the tonsil and lymph nodes of pigs for up to 132 days after farrowing. Wills et al. (1997c) isolated virus from one of four pigs at 157 DPI. Horter et al. (2002) detected infectious PRRSV by virus isolation or swine bioassay in 51 of 59 (84%) animals necropsied between 63 and 105 DPI, including 10 of 11 (91%) of animals euthanized at 105 DPI. Allende et al. (2000) detected infectious virus by bioassay in two of five pigs at 150 DPI.
PRRSV produces a chronic, persistent infection in pigs. The virus replicates in susceptible cells of clinically inapparent carriers for several months. This is the single most significant epidemiological feature of PRRSV infection.
Persistent infection is not a function of pig age at the time of infection. Persistence occurs regardless of whether the pig is exposed in utero (Benfield et al. 1997, 2000b; Rowland et al. 1999), as a young animal, or as an adult (Bierk et al. 2001; Christopher-Hennings et al. 1995a; Fairbanks et al. 2002; Zimmerman et al. 1992). The mechanism(s) by which the virus is able to persevere in the face of an active immune response has not been identified, but probably does not involve evasion of immunity through continual in vivo viral mutation. Chang et al. (2002) found relatively low rates of virus mutation in persistently infected animals.
Stability in the environment
Shedding of virus in saliva, urine and feces results in environmental contamination and creates the potential for transmission via fomites. PRRSV is fragile and quickly inactivated by heat and drying. At 77-80.6°F (25-27ºC), infectious virus was not detected on plastic, stainless steel, rubber, alfalfa, wood shavings, straw, corn, swine starter feed or denim cloth, beyond day zero (Pirtle and Beran 1996).
PRRSV is fragile and quickly inactivated by heat and drying.
PRRSV can remain infectious for an extended time under specific conditions of temperature, moisture and pH. PRRSV is stable for months to years at temperatures of -94°F (-70°C) and -2°F (-20°C). Approximately 90% of PRRSV infectivity is lost within 1 week at 39.2°F (4°C), but low titers of infectious virus can still be detected for at least 30 days. In solution, PRRSV infectivity persists for 1-6 days at 68-69.8°F (20-21°C), 3-24 hours at 98.6°F (37°C), and 6-20 minutes at 132.8°F (56°C). The thermal stability of PRRSV in serum and tissues is similar to that described for virus stored in media. PRRSV was isolated from 47%, 14% and 7% of porcine serum samples stored at 77°F (25°C) for 24, 48 and 72 hours, respectively. When serum was stored at 39.2°F (4°C) or -2°F (-20°C), PRRSV was isolated from 85% of the samples after 72 hours (Van Alstine et al. 1993). PRRSV is stable at pH 6.5-7.5, but infectivity is rapidly lost at pH below 6 and above 7.5 (Benfield et al. 1992; Bloemraad et al. 1994).
Disinfection
PRRSV is inactivated by lipid solvents, e.g., chloroform and ether (Benfield et al. 1992). PRRSV is highly unstable in solutions containing low concentrations of detergents, which disrupt the envelope with concomitant release of the noninfectious core particles and loss of infectivity (Snijder and Meulenberg 2001).
Dee et al. reported that protocols involving cleaning, washing, disinfection and drying were effective at inactivating PRRSV on transport vehicles.
Disinfection first requires removal of all organic material. Thereafter, infectious agents are inactivated in a temperature- and contact time-dependent fashion specific to the agent and the disinfectant. PRRSV is relatively labile in the environment and particularly susceptible to drying (Pirtle and Beran 1996). At “room temperature,” Shirai et al. (2000) reported complete inactivation of PRRSV with chlorine (0.03%) in 10 minutes, iodine (0.0075%) in 1 minute, and a quarternary ammonium compound (0.0063%) in 1 minute. The effects of temperature or pH were not explored. Dee et al. (2004b,c) reported that protocols involving cleaning, washing, disinfection and drying were effective at inactivating PRRSV on transport vehicles.
Transmission within herds
PRRSV tends to circulate within a herd indefinitely. Endemicity appears to be driven by persistent PRRSV infection in carrier animals and the continual availability of susceptible animals either through birth, purchase or loss of protective immunity. The virus is perpetuated by a cycle of transmission from dams to pigs either in utero or post partum, or by commingling susceptible animals with infected animals in later stages of production. Under conditions in which susceptible and infectious pigs are mixed, e.g. at weaning, a large proportion of the population may quickly become infected. Dee and Joo (1994) reported 80–100% of pigs in three swine herds were infected by 8–9 weeks-of-age and Maes (1997) found 96% of market hogs sampled from 50 herds to be positive. However, marked differences in infection rates between groups, pens or rooms of animals may be observed in endemically-infected herds. Houben et al. (1995) even found transmission to vary within litters. Some litter mates seroconverted as early as 6–8 weeks of age, but other individuals reached 12 weeks of age, the end of the monitoring period, still free of PRRSV infection. Likewise, Melnichouk et al. (2005) found differences in the pattern of transmission in farms. In five farms, approximately 50% of the pigs were infected at 5-7.5 weeks of age and at least 90% were infected by 8.5 weeks of age, but on two farms, only 20% to 40% of pigs were infected at 10-12 weeks of age.
Transmission between herds
The role of infected pigs and virus-contaminated semen in herd-to-herd transmission is firmly established (Dee 1992; Mousing et al. 1997; Weigel et al. 2000). In a regional PRRSV control program in France, Le Potier et al. (1997) estimated that 56% (66 of 118) of herds acquired the infection through the introduction of infected pigs, 20% (23 of 118) through infected semen, 21% (25 of 118) through fomites/slurry, and 3% (4 of 118) through unidentified sources. Mortensen et al. (2002) found that PRRSV entered negative herds through the introduction of animals and semen and through area spread from neighboring farms, which they attributed to aerosol transmission. Dee et al. (2002, 2003) have demonstrated the ease with which PRRSV can be moved between farms on commonplace equipment and objects common to swine farms, e.g., insulated semen coolers, metal toolboxes, plastic lunch pails and cardboard boxes, especially when wet and cold. Torremorell et al. (2004) attributed more than 80% of new infections in commercial systems to area spread from neighboring units, the movement of pigs in PRRSV-infected transports, the lack of compliance with the biosecurity protocols, or possibly, introduction via insects.
In a regional PRRSV control program in France, Le Potier et al. estimated that 56% of herds acquired the infection through the introduction of infected pigs, 20% through infected semen, 21% through fomites/slurry…
Proximity to infected herds is a well-recognized risk factor. The risk of a herd becoming PRRSV-positive increased with the density of PRRSV-positive neighboring herds, but decreased with distances (Zhuang et al. 2002). Le Potier et al. (1997) found that 45% of herds suspected to have become infected through area spread were located within 500 meters (0.3 mile) of the postulated source herd and only 2% were one kilometer from the initial outbreak.
Area spread is a major issue in the control and/or elimination of PRRSV. If area spread is to be prevented, it is essential that the mechanisms by which it occurs be firmly established. Area spread is often attributed to aerosols or insects. However, Goldberg et al. (2000) evaluated the ORF 5 gene sequences from 55 field isolates collected in Illinois (United States) and eastern Iowa (United States) and found that the genetic similarity of isolates did not correlate with their geographical distance. On that basis, they concluded that PRRSV was most commonly introduced into herds through animals or semen, as opposed to mechanisms associated with spread from neighboring herds.
Prevention
The objective of PRRSV prevention programs is either to stop the introduction of PRRSV into negative herds or the introduction of new strains into PRRSV-infected herds (Dee et al. 2001). Animals and semen have been considered the primary sources of PRRSV (Le Potier et al. 1997), but the importance of other sources of infection has become evident (Desrosiers 2004). Torremorell et al. (2004) found that more than 80% of new infections occurring in commercial systems in the United States were not due to pigs or semen, but to area spread from neighboring units, the movement of pigs in PRRSV-infected transports, the lack of compliance with the biosecurity protocols, or the possible introduction via insects.
Literature references available upon request by the author or by e-mailing pasweb@idexx.com.
Dual well assays
The PRRS assay is different from most other assays because it has a normal host cell (NHC) well.
Several IDEXX ELISA assays (PRRS, BLVv and PRVv) use a dual well format. Dual well assays require two wells to be used for each sample. One well is coated with virus antigen and the other well is coated with NHC antigens. NHC antigens are derived from non-infected cells.
The NHC well is used to assess the extent of normal host cell contribution to the total signal by relating the reactivity in the viral antigen well to the reactivity in the NHC well. If the NHC well was not present any non-specific reactivity in the antigen well could result in a false positive.
After subtracting the NHC signal from the antigen signal, you will be left with signal directly related to the presence of antibodies. One then follows the specific insert interpretation to classify the result as positive or negative.
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