The Parvoviridae are small (18–30 nm), nonenveloped, single-stranded, negative-sense DNA viruses with a genome of approximately 5 kb. Productive replication requires cellular factors that are expressed only during cell differentiation and division (Tattersall and Cotmore, 1986; Cotmore and Tattersall, 1987). They account for the predilection of parvoviruses for mitotically active cells and the pathogenicity of several serotypes. Infection begins with binding of virions to cell receptors, internalization by endocytosis, and transport to the nucleus where they replicate and from which they are released during apoptic or lytic cell death. Recent evidence from in vivo studies also suggests that productive replication may not lead irrevocably to cell lysis (Jacoby, et al., 2000) a possibility that requires confirmation. In this context, nonproductive infection (abortive, cryptic, or restrictive) has been demonstrated in vitro for the murine parvovirus, minute virus of mice (MVM) but not for the parvoviruses of rats (Tattersall and Cotmore, 1986; Cotmore and Tattersall, 1987; Jacoby and Ball-Goodrich, 1995; Jacoby et al., 1996).

Current knowledge, based partially on extrapolation from studies of parvoviruses of mice, indicate that parvoviruses of rats replicate autonomously; that is, they do not require a helper virus. They encode two nonstructural regulatory proteins, NS1 and NS2, which are conserved and account for immunologic cross-reactivity among serotypes (Cross and Parker, 1972). They also encode two capsid proteins, VP1 and VP2, which are serotype specific. VP2 is the major capsid protein, although its sequence is contained within the VP1 coding region. A third capsid protein, VP3, results from the proteolytic cleavage of VP2 but is present in only minute quantities in mature virions.

Three established serotypes of parvoviruses infect laboratory rats. The prototype agent is rat virus (RV), which was isolated from a transplantable neoplasm of rats by Kilham and Olivier (Kilham and Olivier, 1959). Therefore, it also has been called Kilham rat virus (KRV). An antigenically distinct virus (H-1 virus) was isolated shortly afterward by Toolan from a human tumor cell line that had been passaged through rats (Toolan et al., 1960). A third serotype, rat parvovirus (RPV), was isolated recently from naturally infected rats (Ball-Goodrich, et al., 1998). A similar agent has been identified by Japanese workers who initially referred to it as “rat orphan parvoviruscenter (Ueno et al., 1996). Most recently, three strains of a putative fourth serotype have been identified by Wan and co-workers (Wan et al, 2002), which they have named, collectively, minute virus of rats. The genomic and amino acid sequences for these isolates are closely related to KRV and H-1 virus but are significantly different from RPV. Because parvovirus infections remain prevalent among laboratory rats and have the potential to distort rat-based research results, efforts to detect, eliminate, and exclude them should receive justifiably high priority.

Many early descriptions of parvoviruses of rats were based on experimentally induced infections (Kilham, 1960; Kilham, 1961; Kilham and Margolis, 1964; Kilham and Margolis, 1966; Kilham and Margolis, 1969), often induced by parenteral inoculation. This reflected interest in using these agents for developing rodent models of virusinduced birth defects. Contemporary studies have emphasized inoculation by natural (oronasal) routes at various ages in both immunocompetent and immunodeficient rats to resemble more closely exposure during natural outbreaks (Jacoby et al., 1987, 1988, 1991; Gaertner et al., 1989, 1993, 1995, 1996).

Agents. The three documented serogroups of parvoviruses infective for rats (RV, H-1 virus, and RPV) each contain multiple isolates. Because they are small, nonenveloped viruses, they are highly resistant to environmental inactivation (Yang et. al., 1995), a characteristic that complicates control and elimination. Early work with RV and H-1 virus showed that they retain infectivity after exposure to lipid solvents and are stable over a pH range of 1 to 12 (Siegl, 1976). They also are highly heat resistant. For example, RV can remain infectious after exposure to 80°C for up to 2 hours and for up to 60 days at 40°C. Rodent parvoviruses are resistant to ultrasonication, treatment with RNAse, DNAse, papain, and trypsin—properties which, however, also facilitate preparation of purified virus from infected cell cultures. RV and H-1 agglutinate guinea pig erythrocytes and aggregate, to variable degrees, erythrocytes of other species (Tattersall and Cotmore, 1986). This property was useful for serotyping viral isolates by hemagglutination inhibition (HAI), a method that has been superseded by more modern serodiagnostic tests, discussed below. There is no evidence that strains within a given serogroup are antigenically distinguishable, even though they may differ in nucleic acid sequence, replication kinetics, and pathogenicity. For example, RV-UMass and RV-Y belong to the same serogroup, but RV-UMass appears to replicate faster in vitro and to be more pathogenic in vivo (Ball-Goodrich et al., 2001).

RV and H-1 virus grow well in primary rat embryo cells and C6 rat glial cells. RV also replicates in continuous cell lines such as 324K (human embryonic kidney) and BHK21 (hamster kidney). H-1 virus also can be propagated in rat nephroma cells (Tattersall and Cotmore, 1986; Paturzo et al., 1987) whereas RPV replicates well in 324K cells (Ball-Goodrich, et al., 1998). Productively infected cells usually develop cytopathic effects (CPE), beginning with intranuclear inclusions and proceeding rapidly to cytolysis. The kinetics and severity of CPE depend on virus strain and dose and the level of confluence of the cell substrate. High concentrations of input virus can induce intranuclear antigen indicative of replication within 12 hours and CPE within several days, whereas low concentrations may not induce measurable effects for a week or more. Cultures that contain actively dividing cells are more susceptible to viral infection and replication than confluent cultures.

Clinical signs. Natural parvoviral infections in rats are usually asymptomatic and first detected by seroconversion or suspected from distorted research results. The prevalence of clinical signs depends on virus strain and dose, host age, and route of exposure. Host genotype is not known to affect susceptibility to infection or disease. In the context of these variables, RV and H-1 serotypes are demonstrably pathogenic after experimental inoculation, whereas RPV is not. Clinical signs of RV or H-1 virus infection are most readily elicited in pregnant or infant rats. Resistance to clinical illness appears to develop by 1 week postpartum (Gaertner et al., 1996). The onset of resistance has been attributed to a decline in mitotic activity among target tissues and postnatal development of immune competence.

Prenatal infection with pathogenic serotypes can cause fetal deaths resulting in partial or complete loss of litters in dams that appear otherwise clinically normal (Kilham and Ferm, 1964; Kilham and Margolis, 1966; Kilham and Margolis, 1969; Jacoby et al., 1988; Gaertner et al., 1996). It is unclear whether intrauterine mortality reduces a dam's subsequent reproductive performance. Infection in late pregnancy or within several days after parturition can cause severe or fatal disease in infants, particularly due to hemorrhage and necrosis in the liver and central nervous system (Kilham and Margolis, 1966; Cole et al., 1970; Margolis and Kilham, 1970). Signs in affected infants can include ataxia, icterus, and diarrhea, or sudden death (Figs. 12-1 and 12-2 ). Rats that survive acute disease may have locomotion deficits or may die later from chronic active hepatitis and progressive hepatic fibrosis. Although clinical signs are rare in rats exposed to virus beyond 1 week after birth, fatal hemorrhagic lesions have been reported in weanlings and immunosuppressed adults (El Dadah et al., 1967; Coleman et al., 1983). They also have been elicited in infected rats with induced increases in hepatic mitotic activity (Ruffolo et al., 1966).Fig. 12-1

Acute rat virus infection. Ataxia (splayed feet) from RV-induced cerebellar hypoplasia.

Rat cytomegalovirus (RCMV) is the only known herpesvirus of rats. It has physical, chemical, and biological characteristics typical of the Herpesviridae, including the capacity for persistent infection of salivary glands (Priscott and Tyrell, 1982; Bruggeman et al., 1983; Bruggeman et al., 1985). Virus can be propagated in primary rat embryo fibroblasts, rat kidney cells, and hamster kidney cells. Infection of immunocompetent rats is asymptomatic. Experimental infection of rats immunosuppressed by whole body irradiation induced systemic disease with high mortality, but no studies of infection in naturally immunodeficient rats have been reported. RCMV appears to be infectious only for rats. There is no published evidence for natural cross-infection of rats with cytomegaloviruses of other species or for infection of other species with RCMV. Although infection has been detected in wild rats, the prevalence of RCMV in contemporary rat colonies is believed to be very low. However, epidemiologic data are inadequate to confirm this perception because serologic surveillance is performed sporadically. Nevertheless, prevention of infection in vivaria should focus on exclusion of wild rats, especially from support areas such as those used for supply and equipment storage.

Although acute infection may be generalized, persistent infection occurs in the salivary glands and may affect the lacrimal glands (Lyon et al., 1959). Virus can be excreted in saliva for at least several months. Typical lesions include cytomegaly with formation of intranuclear inclusions, primarily in ductal epithelium Fig. 12-14 ), accompanied by mild, non-suppurative interstitial inflammation. Infection can be detected from typical histopathologic changes, which are most apparent during the first few weeks of infection. An ELISA is available for serologic testing (Bruggeman et al., 1983). Virus can be isolated by inoculation of or cultivation with rat embryo fibroblasts (Rabson et al., 1969).Fig. 12-14

Rat cytomegalovirus infection. Intranuclear inclusions in acinar epithelial cells of a submandibular salivary gland accompanied by mild interstitial inflammation.

An agent called Turkemia rodent poxvirus, distinct from ectromelia virus, has been reported in rats from Eastern Europe and the former Soviet Union (Iftimovici et al., 1976; Marrenikova and Shelukhina, 1976; Krikun, 1977; Marenikova et al., 1978; Kraft et al., 1982). Although infection can be asymptomatic, if clinical signs occur they resemble mousepox including the development of dermal lesions, tail amputation, and high mortality. Rats also develop lesions of the upper and lower respiratory tract. The latter can include interstitial pneumonia, pulmonary edema and hemorrhage, and pleural effusions.

There is serologic and histologic evidence that rats are susceptible to infection with one or more adenoviruses antigenically related to mouse adenovirus-2 (Ward and Young, 1976). However, the causative agent(s) has not been isolated. Infection, to the best of available knowledge, is asymptomatic. Histologic changes are minimal and resemble those found in adenoviral infections of mice, with the sporadic development of intranuclear inclusions in enterocytes of the small intestine.

A polyoma virus antigenically distinct from polyoma virus of mice has been detected in rats. It was found initially in athymic rats that developed a wasting disease accompanied, in a minority of animals, by pneumonia and sialoadenitis (Ward et al., 1984). Intranuclear inclusions consistent with papovavirus infection occurred in parotid salivary glands, and viral antigen was detected in salivary glands, respiratory airway mucosa, and kidney. Immunocompetent rats did not develop disease. More recent studies in athymic rats have detected intranuclear inclusions in alveolar lining cells and associated with interstitial pneumonia (Percy and Barthold, 2001).

Agents. The Coronaviridae are enveloped, pleomorphic single-stranded RNA viruses with club-like projections (peplomers) radially arranged on their capsids. They replicate in the cytoplasm and are released by budding through the endoplasmic reticulum (Compton et al., 1993). They are widespread among mammals and birds, including laboratory rats and mice, but individual viruses are largely species-specific. Classification of the coronaviruses of rats has been somewhat confused by the context in which the two prototype strains were initially identified. In 1964, Hartley and co-workers detected antibodies in rat sera to a common coronavirus of mice, mouse hepatitis virus (MHV), which suggested that rats were susceptible to an antigenically related agent (Hartley et al., 1964). Parker and associates subsequently isolated a coronavirus from lungs of clinically normal rats which became widely known as Parker's rat coronavirus (Parker et al., 1970). Within several years, a second coronavirus was isolated by Bhatt and co-workers from rats with sialodacryoadenitis and was named sialodacryoadenitis virus (Bhatt et al., 1972). Subsequent studies showed that RCV and SDAV were similar antigenically and physicochemically, and that both induced sialodacryoadenitis and inflammation of the respiratory tract. Therefore, RCV and SDAV should be perceived as different strains of a coronavirus indigenous to rats and called, most inclusively, RCV (Percy and Williams, 1990).

Replication of single-stranded RNA viruses is more error-prone than that of DNA viruses and leads to emergence of divergent strains during natural infections. Thus, additional RCV isolates have been reported since the initial isolations by Parker and Bhatt. These include Japanese isolates—CARS (Kojima et al., 1980; Maru and Sato, 1982)— and U.S. isolates RCV-BCMM, RCV-W (Compton et al., 1999a), and RCV-NJ (Compton et al., 1999b).

RCV has a non-segmented, positive-sense RNA genome (Compton et al., 1993). It is sensitive to lipid solvents and relatively stable at acid pH. It can be stored at −60° C for at least 7 years, but loses infectivity rapidly if stored at −20° C (Bhatt et al., 1972; Jacoby et al., 1979). It replicates in vitro in primary rat kidney (PRK) cells in which most virus strains form multinucleate syncytia typical of coronaviral CPE (Bhatt et al., 1972). Virus can be detected antigenically in infected cells within 12 hours and CPE can develop within 24 hours. Established cell lines such as BHK-21, VERO, Hep-2, and NCTC 1469 resist RCV infection, but mouse L2 fibroblasts and sublines, as well as rat LBC cells and intestinal RCV-9 cells, also can be infected (Hirano et al., 1986; Percy et al., 1989; Percy et al., 1990b; Gaertner et al., 1996; Ohsawa et al., 1996). L2 cell sublines and LBC cells also have been used for plaque assays and plaque purification (Gaertner et al., 1993c).

RCV antigens appear to be similar among all isolates, although some also have a hemagglutinating surface protein (Gaertner et al., 1996a; Gagneten et al., 1996). Because RCV is antigenically related to MHV (Barker et al., 1994), MHV antigens are routinely used in serologic testing to detect RCV infection (Peters and Collins, 1981; Smith and Winograd, 1986; Percy et al., 1991).

Clinical signs. Clinical signs may vary with RCV strain, but their overall expression is characteristic of coronavirus infection. Mild infection can be clinically silent and revealed first by seroconversion of sentinel or index rats, but clinical signs are common in many outbreaks. They can occur in non-immune rats of any age, usually last about a week, and occur in various combinations and with varying severity (Jacoby et al., 1975; Bhatt and Jacoby, 1985). Early signs, especially in sucklings, often include squinting, photophobia, and lacrimation Fig. 12-15 ). Older rats frequently develop audible sneezing and palpable enlargement of cervical salivary glands. Closer examination may also reveal serous nasal discharge and pawing at the external nares resulting in wet forefeet. Red-tinged discharges containing porphyrin (chromodacryorrhea) may stain periorbital and perinasal skin Fig. 12-16 ). Severe ocular inflammation is expressed initially as keratoconjunctivitis, which may resolve quickly or become chronic. Chronic ocular inflammation can lead to corneal opacities and ulcers, pannus, hypopyon, hyphema, and megaioglobus (Lai et al., 1976) (Figs. 12.17 and 12.18 ). Ancillary effects attributable to the discomfort of infection include transient anorexia and weight loss (Sato et al., 2001) and disruption of estrus (Utsumi et al., 1978, 1980). Additionally, birth rates and fertility in breeding colonies have been observed to markedly decline during acute epizootics. Although morbidity may be high, RCV infection rarely causes mortality. Most rats recover, with the most frequent long term sequelae associated with ocular disease.Fig. 12-15

Rat coronavirus infection. Photophobia and lacrimation due to keratoconjunctivitis and secondary to RCV infection of the lacrimal glands.

Agents. Hantaviruses are tri-segmented, negative-sense, single-stranded RNA viruses of the genus Hantavirus in the family Bunyaviridae (Nichol, 2001). There are more than 20 known strains, each of which is closely associated with a rodent or insectivorous reservoir host (Schmaljohn and Hjelle, 1997; Simmons and Riley, 2002). Several hantavirus strains, including Seoul virus, are indigenous to Rattus norvegicus. Black Creek Canal virus, a strain that is highly virulent for humans, has been isolated from cotton rats (Sigmodon hispidus) and implies that extreme caution should be employed if wild-caught cotton rats are used for research. In fact virtually all hantaviruses are known or have the potential to be zoonotic for man with varying pathogenicity ranging from asymptomatic to lethal infection. A newly recognized agent, named rat respiratory virus (RRV), has been detected in laboratory rats with interstitial pneumonia (Simmons and Riley, 2002). Serologic tests suggest that it is related to the hantaviruses.

Clinical signs. Rodent hantavirus infections are typically asymptomatic but persistent, a condition attributed to co-evolution of hantaviruses with their respective hosts.

Epidemiology. Rat-to-rat transmission occurs through direct contact and bite wounds, although indirect contact by fomites contaminated with excreta or secreta is possible. Recent in situ hybridization studies have detected persistent infection in kidney, exocrine pancreas, salivary gland, and skin—sites conducive to secretion/excretion of infectious virus (Compton et al., 2003). Further, one must consider the possibility for transmission by contaminated transplantable rat tumors. Hantavirus infection should be considered persistent in individual rats despite antiviral immunity (Meyer and Schmaljohn, 2000). The mechanism(s) of persistence is not well-understood, but prolongs risks for transmission. The distribution of rat-associated infection is virtually worldwide and has included zoonotic infections among laboratory animal workers (Desmyter et al., 1983; Lloyd et al., 1984; Lloyd and Jones, 1986; LeDuc, 1987; Tsai, 1987). As noted above, human infection has a variety of clinical outcomes ranging from asymptomatic infection to lethal hemorrhagic fevers (Hart and Bennett, 1999). The prevalence of virulent and/or zoonotic hantaviruses in laboratory rats is extremely low. Early assessments of RRV infection indicate a prevalence of approximately 8% among laboratory rats and more than 20% among laboratory mice (Simmons and Riley, 2002).

Pathology. Most studies of hantavirus pathology in rats have utilized experimental infection with Asian isolates such as Seoul virus (Lee et al., 1986; Compton et al., 2003). Acute hantavirus infection has a widespread tissue distribution and a predilection for endothelial cells, as well as glandular and renal epithelium. Infection does not cause significant tissue damage, but can provoke mononuclear cell infiltrations in affected tissues, including lung, salivary gland, pancreas, and kidney, especially during persistent infection. Pancreatic lesions may include insulitis with at least transient elevations in blood glucose (Compton et al., 2003). As noted above, RRV infection appears to cause interstitial pneumonia.

Diagnosis. Diagnosis relies primarily on detection of specific anti-hantavirus antibodies by IFA, with an ELISA serving primarily as a back-up test (Lee et al., 1999). Immunostaining of tissue sections or molecular techniques (in situ hybridization or RT-PCR) also can be used; however, primer sets are not readily available for all strains. Further, RT-PCR is complicated by heterogeneity inherent to hantavirus genomes. Hantaviruses can be propagated in cell culture, with Vero-6 cells being the preferred substrate, but replicate slowly and with low yield.

Attempts at virus isolation should be reserved for only the most experienced and well-equipped laboratories, because of technical and safety concerns.

Differential diagnosis. Interstitial pneumonia, which may be caused be RRV, must be differentiated from other causes of similar lesions including rat coronavirus, Sendai virus, and PVM.

Control and prevention. Serologic monitoring is the key to prevent entry and spread of hantavirus infection. Infected or exposed rats should be placed under strict quarantine, and attempts to speciate the causative agent by a qualified laboratory should be undertaken as a means to assess zoonotic risk. Thus, the Asian hantaviruses present a clear zoonotic hazard, whereas there is currently no evidence for zoonotic transmission of RRV. Confirmation of a zoonotic strain should provoke depopulation of affected and exposed animals and thorough decontamination of facilities, equipment, and supplies. Personnel working in affected or potentially affected areas should don full protective gear, including ventilated hoods. If the potential for infection has been identified, the safest action is to notify and engage institutional safety officers. It also is prudent to assess rat-derived biological materials obtained from countries where hantavirus infection is endemic.

Effects on research. Zoonotic threats from virulent strains are the major impediment to research. As noted above, infection of rats with Seoul virus can cause insulitis and hyperglycemia. There is no evidence for or against the possibility that interstitial pneumonia associated with RRV alters pulmonary function or susceptibility to opportunistic respiratory infections.

A rotavirus has been incriminated as the cause of infectious diarrhea of infant rats (IDIR), a non-lethal condition that resembles rotavirus-caused epidemic diarrhea of infant mice (EDIM) (Vonderfecht et al., 1984; Vonderfecht, 1986). The causative agent is an antigenically distinct group B rotavirus that may be of human origin. Infected suckling rats develop clinical signs within a day or two after initial exposure. These include diarrhea for up to 1 week accompanied by cracking and bleeding of the perineal region, dry, flaky skin, and transient growth retardation. Although rats of all ages are susceptible to infection, resistance to clinical disease develops at about 2 weeks of age. Gross lesions are limited to the gastrointestinal tract. The stomach usually contains milk. The ileum and colon may be distended with yellow-green fluid and gas. Histologic lesions are most prominent in the ileum and are characterized by enterocytic syncytia, which may contain intracytoplasmic inclusions, enterocytic necrosis, and attenuation of villi. Viral antigen and rotaviral particles may be found in affected enterocytes. Diagnosis rests on characteristic clinical and pathologic changes accompanied by detection of rotaviral antigens and/or particles. Serologic assays have not been developed. Differential diagnoses should include clinically apparent infection by pathogenic enterotropic bacteria. The significance of IDIR pertains primarily to transient growth retardation and the possibility that it originated as a human infection.

Margolis and Kilham have induced prenatal reovirus 3 infection in fetal rats (Margolis and Kilham, 1973) and there is anecdotal evidence that rats can develop antibodies to reoviruses, but there are no documented reports of natural reovirus infection. Because reovirus antigens are fairly widespread in nature, it is decidedly unclear whether such seroconversions are due to active infection or exposure to inert reovirus antigens from sources such as plant matter.

Two types of picornavirus infection have been linked to rats. Theiler's mouse encephalomyelitis virus (TMEV) is a member of the cardiovirus genus of the family Picornaviridae, the natural host for which is the mouse (Jacoby et al., 2002). Anecdotal observations indicate that rats can develop antibodies to TMEV, but no associated disease or lesions have been found. In historical perspective, a TMEV-like agent called MHG virus was isolated from rats and infected intestine, lung, and brain after experimental inoculation. A second type of cardiovirus genetically distinct from TMEV has been isolated from rats and designated rat cardiovirus (Ohsawa et al., 2003). It does not appear to be pathogenic after experimental inoculation.

Monitoring for viral infections should cover at least three venues: animals in established breeding and experimental colonies, animals held in entry quarantine, and animal tissues and products destined for in vivo use. For established colonies, monitoring should be tailored to local conditions. Effective monitoring should encompass sampling on a pre-arranged schedule, which can be intensified if evidence or suspicion of viral infection emerges. Although sampling should utilize index animals, such as retired breeders, whenever possible, the most widely accepted approach to assess contemporary conditions in a colony is serologic monitoring of strategically placed and exposed sentinels. Sentinels must be immunocompetent, virus-antibody free, and protected from exposure to infectious agents during shipment. The number of sentinels used should reflect assessment of risks to a colony, as illustrated in Chapter 16. Cages of sentinels should be placed, by rack and room, to facilitate controlled exposure to potential sources of infection. One popular configuration for rats housed in conventional wire-top caging is to place sentinels on the lowest shelf of a rack to maximize natural exposure to bedding expelled from overlying cages. Additionally, samples of soiled bedding collected during routine cage changing (e.g., weekly) can be placed in sentinel cages to increase the sensitivity of detection. This strategy also helps to compensate for the use of isolation and individually ventilated caging, which limits airborne transmission of viral agents and by doing so, also may inhibit early detection of infection. However, limited durability of excreted virus in soiled bedding and the potential that small amounts of virus could be diluted to nonimmunogenic levels in pooled bedding samples also must be considered. Targeted selection of soiled bedding samples also can be helpful to trace infection to a sector of a suspect rack or shelf. For this procedure to work satisfactorily, cage positions should not be changed during the testing interval.

Sampling intervals for sentinel rats generally range from 30 to 90 days, but, minimally, must be long enough to complete desired exposure and allow opportunity for seroconversion, which typically occurs by 2 weeks after an immunogenic exposure. Sentinel animals can be sampled repeatedly if they remain seronegative, but should be replaced if they develop antibodies. As a general rule, sentinels, regardless of serologic status, should be replaced every 6 to 12 months to permit incremental assessment, such as histopathology.

Molecular techniques can be used selectively to confirm serologic results. For example, PCR testing of excreta, soiled bedding, equipment, or surfaces can be used in lieu of virus isolation to help determine if infection initially detected by serology is currently active. Additionally, there is preliminary evidence from studies with mouse coronavirus that PCR sampling of exhaust air manifolds can provide a rapid, “realtime” overview of infection among individually ventilated cage racks (SR Compton, personal communication); however, it remains to be seen whether this technology is applicable to viruses of rats.

Sampling of quarantined arrivals, which is likely to increase with the wider use and exchange of genetically altered rats, should be based on the same sampling principles outlined above. Depending on source, age, gender, animal number and behavioral considerations, sentinel exposures can use soiled bedding or direct placement with index animals. Because cohorts of imported rats are generally small, sentinel exposure for 30 days should be adequate to elicit potential seroconversion. Sampling of animal products destined for in vivo use, such as transplantable tumors or blood products, is essential for a sound monitoring program. It also is important, in this regard, to counsel investigators about the hazards of introducing untested biological products into rats. Aliquots can be tested by inoculation of susceptible cell lines or by molecular methods. The aforementioned RAP test, although apparently sensitive, tends to be costly, time consuming, and likely to be replaced by molecular methods.

Because viral infections of rats can spread insidiously, early detection and epidemiologic “staging” should employ a detection matrix that includes clinical observation, appropriate sampling, and sensitive and specific diagnostic testing. The value of serology to detect infection and determine prevalence and incidence is obvious. As noted in previous sections of this chapter, sensitive and specific tests are available for the most common viruses of laboratory rats. We re-emphasize, however, that the number of test animals should be large enough and the duration of exposure long enough to maximize the likelihood of detecting infection in a room or colony. Further, the clinician/pathologist should have high confidence that the testing laboratory has the testing conditions and expertise to produce incontrovertible results.

Clinical signs are helpful if they are comparatively specific, such as the cervical and ocular manifestations of coronavirus infection. However, non-specific signs, such as inappetence, altered reproduction, problematic anesthesia, distorted immunity, also may signal underlying viral infection.

Exposed rats, and especially those with clinical morbidity, should be subjected to pathologic examination, beginning with a thorough necropsy and including proper preservation of tissues for histopathology, collection of serum for antibody testing, and selection of tissues for potential molecular and/or virologic analysis. Tissue selection and preservation should be guided by a differential diagnosis emerging from clinical and gross pathology examinations complemented by a sound working knowledge of viral infections of rats. Such knowledge should account for the possibility that signs and gross lesions will be absent. Table 12-1 summarizes current recommendations for tissue collection, preservation, and examination. As noted earlier, aldehyde fixation followed by embedding in paraffin wax is generally suitable for routine histopathology. However, we recommend fixation for approximately 16 hours in freshly prepared paraformaldehyde-lysine-periodate (McClean and Nakane, 1974) if immunohistochemistry and/or in situ hybridization are contemplated, because it provides improved stabilization of viral antigens and nucleic acids. We also recommend freezing selected tissues at −60° C. These samples can be used for virus isolation, molecular testing, or antibody production testing, as initial results warrant. Careful attention to minimize contamination during collection of such samples is critical, especially if the option for PCR testing is to be preserved. It also is prudent to complement examinations for potential viral infection with other microbiological testing to assess the possibility for dual infections or emergence of opportunistic bacterial infections secondary to viral infection.

If evidence of viral infection is obtained, the decision to take corrective action should be based on several key questions: Is the infection a threat to animal or human health? Is the infection a threat to research at the affected or collaborating sites? What are the options for control and elimination? Are the options epidemiologically effective, minimally disruptive to research, and fiscally sound? What are the options to prevent reinfection? These questions are best addressed through a thorough understanding of the biology and pathobiology of the offending agent and the functional and structural characteristics of the affected vivarium. Characteristics such as mode of spread, environmental stability, duration, and virulence in immunocompetent compared with immunodeficient hosts and known effects on research can lead to well-established actions for containment and elimination that have been illustrated for specific agents in this chapter. Further, working knowledge of viral infections can facilitate extrapolation of what is known to what may be conjectural due to lack of specific data. For example, the predilection of parvoviruses for mitotically active cells should be a generic factor in considering the potential impact of parvovirus infection on research. However, decision-making is likely to become more problematic as the use of genetically altered rats increases. Rats with either targeted or serendipitous alterations in resistance to infection may have correspondingly altered responses to viral infections. This potential is illustrated by cryptic immunodeficiencies in laboratory mice that can occur as unintended sequelae of genetic manipulation. Therefore, it will be important to consider phenotype as integral to risk assessment, and to broaden clinical, epidemiologic, and pathologic conception of the “textbook” spectra of infection.