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Infectious Diseases of Mice and Rats (1991)

Chapter: 7. Digestive System

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Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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7 Digestive System

Overview

Diseases of the digestive system, like those of the respiratory tract, are extremely common in both mice and rats. As a group they clearly rival diseases of the respiratory tract in importance, despite the fact that they possibly tend to be even more subtle clinically. This is explained in part by the fact that many pathogens of the digestive system have their most serious effects in the very young, i.e., neonates and sucklings. Clinical signs may no longer be present when they are delivered to the investigator as weanlings or young adults. In addition, even the more obvious clinical signs of digestive tract disease, such as diarrhea and retarded growth, often go completely undetected in mice and rats.

There are approximately 21 infectious agents of the digestive system in mice and rats. Natural infections are usually due to varying combinations of these agents, and if clinical disease is present, careful judgments may be required in deciding which agent(s) have causative role(s). Additive effects are often suspected but are difficult to prove. Where possible, detection methods should identify all possible causes; and pathologic workups, including histopathology of all organs comprising the digestive system, should be done to make definitive diagnoses. Histologic sections should include the salivary and Harderian glands because they are important target organs of sialodacryoadenitis virus as well as other agents.

Table 10 places in perspective the relative importance of the infections of the digestive system as causes of disease. Agents are listed in descending

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

TABLE 10 Agents Grouped According to Importance as Causes of Natural Digestive System Disease

Groupa

Mouseb

Ratb

I

Mouse hepatitis virus

Sialodacryoadenitis virus

 

Spironucleus muris

Spironucleus muris

 

Bacillus piliformis

Bacillus piliformis

 

Salmonella enteritidis

Giardia muris

 

Citrobacter freundii (biotype 4280)

 

 

Giardia muris

 

 

Mouse rotavirus

 

II

Reovirus-3

Salmonella enteritidis

 

Pseudomonas aeruginosa

Rat rotavirus-like agent

 

Hymenolepis nana

Pseudomonas aeruginosa

 

Syphacia spp.

Syphacia spp.

 

Mouse cytomegalovirus

 

III

Mouse thymic virus

Rat cytomegalovirus

 

Adenoviruses

Reovirus-3

 

Aspicularis tetraptera

Adenoviruses

 

Entamoeba muris

Entamoeba muris

 

Tritrichomonas muris

Tritrichomonas muris

a Group Key: I = Agents that are unquestionably important digestive tract pathogens. II = Agents of questionable importance or pathogenicity. except in special circumstances. III = Agents not considered significant, natural pathogens in laboratory animals of the species indicated.

b Reading down each list of agents for the mouse or rat, agents are listed approximately in descending order of importance as digestive system pathogens for that rodent species.

order of importance for mice and rats. Agents listed in group I are undisputed important pathogens, but because of their high prevalence and known effects on research, mouse hepatitis virus and Spironucleus muris in the mouse and sialodacryoadenitis virus and S. muris in the rat are of greatest concern. Bacillus piliformis, Salmonella enteritidis, and Citrobacter freundii (biotype 4280) are important, but far less common, pathogens. Giardia muris is common but probably less important. Mouse rotavirus is pathogenic only in neonates.

Those agents listed in group II of Table 10 appear to be of little significance except possibly in rare circumstances. For example, reovirus-3 is a rare contaminant of transplantable tumors, S. enteritidis is found very rarely in rats, rat rotavirus-like agent has been found in rats on only one occasion, and mouse cytomegalovirus is probably very rare in laboratory mice (but common in wild mice).

The agents in group III of Table 10 are of doubtful importance as pathogens in laboratory mice and rats. Rat cytomegalovirus has been found only in wild rats.

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Mouse Cytomegalovirus
Significance

Low.

Perspective

Mouse cytomegalovirus (MCMV)* is a common subclinical infection of the submaxillary salivary glands and other tissues in wild mice. Although it has been found in laboratory mice on occasion (Mannini and Medearis, 1961; Rowe et al., 1962), its chief importance is that it provides a variety of laboratory models for the study of phenomena similar to those in human cytomegalovirus infections (Osborn, 1982).

Agent

MCMV is an enveloped, double-stranded DNA virus, family Herpesviridae, subfamily Betaherpesvirinae, cytomegalovirus group. Like other herpesviruses, the virion is 120-200 nm in diameter. The capsid is 100-110 nm in diameter, is icosahedral in symmetry, and has 162 capsomeres (Matthews, 1982).

MCMV differs from most herpesviruses in that it has a larger genome (Misra et al., 1978). Several strains of MCMV are known, but the Smith strain and its substrains have been studied most. All strains are antigenically and biophysically similar and presently are considered the same virus (Mosmann and Hudson, 1973, 1974).

Cytomegaloviruses are relatively unstable, sensitive to freezing and thawing, acid pH, and heating at 56°C for 30 minutes. They can be stored for prolonged periods at -90°C in the presence of 35% sorbitol or dimethyl sulfoxide (Buxton and Fraser, 1977a; Osborn, 1982).

MCMV produces permissive infections in cultures of mouse embryo fibroblasts and 3T3 cells (Osborn, 1982).

Hosts

Wild mice (Mus musculus) are considered the natural hosts (Mannini and Medearis, 1961; Rowe et al., 1962; Gardner et al., 1974). A similar virus

*  

This agent is most often referred to as murine cytomegalovirus in the literature. In this report the designation mouse cytomegalovirus is used to distinguish it from those cytomegaloviruses that have been isolated from rats and other members of the family Muridae.

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

has been isolated from the field mouse Apodemus sylvaticus (Kim et al., 1975), but its relationship to MCMV is uncertain.

Epizootiology

MCMV has been reported to occur in approximately 65% of adult wild mice and in only 0.5-3% of laboratory mice (Mannini and Medearis, 1961; Rowe et al., 1962). More recent surveys of laboratory mice have given conflicting results. One study reported 55% of 256 mice from 9 commercial sources (and mice from 8 of the 9 sources) in the United States to be positive for MCMV by enzyme-linked immunosorbent assay (ELISA), while no positives were found using complement fixation (CF) and immunofluorescent antibody (IFA) test (Anderson et al., 1986) In another study of mice from commercial sources in the United States, no ELISA positive mice were found among mice from 4 commercial sources (Classen et al., 1987). Further studies are needed to clarify the prevalence of MCMV in contemporary laboratory mice.

In natural infections of wild mice, the salivary glands (particularly the submaxillary glands) and possibly the pancreas are persistently infected with MCMV. Virus is transmitted via saliva, and infection can persist for life. Since infection is more frequently seen in adult than in young mice, it appears that infection can be acquired throughout extrauterine life. Latent infections can occur in submaxillary glands, B cells, T cells, the prostate, and the testicles (Osborn, 1982). In spleen the cells that become infected are predominantly sinusoidal-lining cells (Mercer et al., 1988).

Vertical transmission can occur but has not been fully explained. Direct passage of the virus across the placenta to the fetus and/or transmission via germ cells have been suggested (Chantler et al., 1979; Brautigam and Oldstone, 1980; Osborn, 1982).

Clinical

Natural infections of MCMV are subclinical (Osborn, 1982).

Pathology

In naturally infected mice large acidophilic intranuclear inclusions can be seen in salivary gland acinar and duct cells. Affected cells are typically enlarged three or four times their normal size (cytomegaly). The submaxillary glands are affected most, the sublinguals less, and the parotids least (Mims and Gould, 1979; Osborn, 1982);

Pathogenesis of experimental MCMV infection in laboratory mice is very complex because it is markedly influenced by virus strain, passage history,

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

dose, route of inoculation, and mouse strain and age. Newborn mice are most susceptible, and resistance increases greatly at 21-28 days of age. Mice of the H-2k haplotype (C3H and CBA) have been found to be relatively more resistant to intraperitoneal inoculation of the virus than are mice of the H-2d (BALB/c) and H-2b (C57BL/6 and C57BL/10) haplotypes, and resistance is thought to be controlled by two loci in the H-2 region (Chalmer et al., 1977; Osborn, 1982; Price et al., 1987; Quinnan and Manischewitz, 1987).

Numerous models of human cytomegalovirus have been produced by varying the strain, dose, and route of MCMV inoculation into laboratory mice of different ages and strains. These include models of subclinical infection (Osborn and Medearis, 1967; Osborn et al., 1968; Osborn and Shahidi, 1973), intrauterine infection (Medearis, 1964; Johnson, 1969; Basker et al., 1987), fetal and neonatal ear disease (Davis and Hawrisiak, 1977; Davis et al., 1979; Baskar et al., 1983), encephalitis (Lussier, 1973, 1975), glomerulonephritis (Lussier, 1975; Wehner and Smith, 1983), and interstitial pneumonitis (Jordan, 1978; Rose et al., 1982; Shanley et al., 1982).

Athymic (nu/nu) mice (Starr and Allison, 1977) and beige (bg/bg) mice (Shellam et al., 1981; Papadimitriou et al., 1982) are far more susceptible than their immunocompetent counterparts. The induction of natural killer cells and interferon early after infection are thought to be most important in non-specific defense against MCMV infection (Griffiths and Grundy, 1987). Cellular immunity is thought to be of pivotal importance in MCMV infection, particularly MCMV-specific cytotoxic T lymphocytes, nonspecific natural killer cells, and antibody-dependent killer cells (Quinnan et al., 1978, 1980; Quinnan and Manischewitz, 1979; Bukowski et al., 1984). CD8+ CD4- T lymphocytes against early MCMV antigens are thought to be most important in specific defense (Reddehase et al., 1987, 1988; Mutter et al., 1988). Subclinical or latent infections have been activated by many regimens of immunosuppression, most notably antilymphocyte serum alone or in combination with cortisone or cyclophosphamide (Brody and Craighead, 1974; Gardner et al., 1974; Lussier, 1976; Howard et al., 1979; Shanley et al., 1979; Jordan et al., 1982).

Diagnosis

The prevalence of MCMV in contemporary laboratory mice is generally thought to be negligible except in instances in which stocks may have been contaminated by contact with wild mice. Enzyme-linked immunosorbent assays (ELISAs) for MCMV have been developed and compared with the CF and IFA tests for detection of antibodies in mice experimentally infected with the virus (Anderson et al., 1983; Classen et al., 1987; Lussier et al., 1987b). The IFA and CF tests were found to be more sensitive for detecting acute infection, and the ELISAs were more sensitive for detecting

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

persistent infection (Anderson et al., 1983; Lussier et al., 1987b). These methods may prove useful for monitoring laboratory or wild mice for MCMV infection in selected situations (Anderson et al., 1986; Classen et al., 1987; Lussier et al., 1987).

Virus isolation can be accomplished by using mouse embryo fibroblasts and other tissue culture systems (Osborn, 1982).

Control

Complete exclusion of wild mice from rodent facilities is essential. In experimental infections of mice, the virus is readily transmitted between cage mates but not from one cage to another. Therefore, with appropriate measures of containment (such as use of filtered cages, use of a hood while cages are changed, and handling the mice by sterile procedures), studies with this agent can be conducted in most facilities without the risk of contaminating other stocks (Osborn, 1982).

Interference with Research

Natural MCMV infection in laboratory mice has not been reported to interfere with research. However, acute MCMV infection in laboratory mice due to experimental inoculation of the virus is known to cause severe disturbances in immune responses.

Experimental MCMV infection results in depression of the following:

  1. Antibody production (Osborn and Medearis, 1967; Osborn et al., 1968; Howard and Najarian, 1974; Tinghitella and Booss, 1979; Schilt, 1987).
  2. Interferon induction (Osborn and Medearis, 1967; Kelsey et al., 1977).
  3. Lymphocyte proliferation responses to mitogens (Howard et al., 1974; Selgrade et al., 1976; Booss and Wheelock, 1977a,b; Kelsey et al., 1977; Loh and Hudson, 1981, 1982; Allan et al., 1982) and in mixed lymphocyte cultures (Howard et al., 1974, 1977).
  4. Allogeneic skin graft rejection (Howard et al., 1974, 1977; Lang et al., 1976; Dowling et al., 1977).
  5. Reduced litter size and increased fetal abnormalities (Baskar et al., 1987).

Experimental MCMV infection can depress (Ho, 1980) or augment (Quinnan et al., 1982) cytotoxic lymphocyte responses.

Experimental MCMV infection increases susceptibility of mice to infection with Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, or Candida albicans (Hamilton et al., 1976, 1977; Shanley and Pesanti, 1980; Leung and Hashimoto, 1986; Kournikakis and Babiuk, 1987) or Newcastle disease virus (Osborn and Medearis, 1967).

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

Experimental MCMV infection in mice can cause thrombocytopenia (Osborn and Shahidi, 1973).

Experimental MCMV infection has been reported to exacerbate the naturally occuring dystrophic cardiac calcification in BALB/c mice (Gang et al., 1986).

Rat Cytomegalovirus
Significance

Low

Perspective

There are no known reports of natural cytomegalovirus (CMV) infections in laboratory rats. Acidophilic intranuclear inclusions thought to be due to CMV infection have been observed from time to time in the submaxillary salivary glands or kidneys of wild rats (Thompson, 1932; Kuttner and Wang, 1934; Syverton and Larson, 1947). More recently, three different CMV isolates have been obtained from wild rats: Rattus norvegicus in The Netherlands (Bruggemen et al., 1982) and England (Priscott and Tyrrell, 1982) and Rattus rattus in Panama (Rabson et al., 1969). These agents may have importance as potential contaminants of laboratory rats and as model infections for the study of CMV host-parasite relationships.

Agent

Enveloped, double-stranded DNA virus, family Herpesviridae, subfamily Bethaherpesvirinae, CMV group. The rat CMV (RCMV) of Bruggeman et al. (1982) is the most extensively studied of the cytomegaloviruses isolated from R. norvegicus. Its morphologic and cultural characteristics are typical of the CMV group and comparable to those of mouse CMV (MCMV). However, RCMV appears to be distinctly different from MCMV based on results of DNA homology studies (Meijer et al., 1984).

Host

RCMV strains have been reported to occur naturally only in wild rats, R. norvegicus (Bruggeman et al., 1982; Priscott and Tynell, 1982) and R. rattus (Rabson et al., 1969).

Epizootiology

RCMV was originally isolated from the salivary glands of eight of ten wild rats (Bruggeman et al., 1982). After experimental infection of rats

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

with RCMV by either subcutaneous or intraperitoneal inoculation, the virus appears at low titer in many organs, including brown fat (Bruggeman et al., 1987), during the first few weeks, and subsequently is found only in salivary glands (at high titer) where it persists for at least several months. The virus is shed in saliva and urine (Bruggeman et al., 1985). Although the salivary gland is the primary site of infection, persistent, latent infection is thought to occur in many organs (Bruning et al., 1986).

Clinical

Natural infections of RCMV are subclinical (Bruggeman et al., 1982). Clinical illness does not occur even after the inoculation of rats with high doses of the virus (Bruggeman et al., 1985).

Pathology

Experimental infections of RCMV were studied in LEW and BN rats by Bruggeman et al. (1983a). Four- to five-week-old rats were inoculated intraperitoneally with salivary gland suspensions containing the virus. Subsequently, rats were killed at intervals for attempted isolation of virus from salivary glands, spleens, and kidneys. Maximal virus titers were reached in salivary glands at 1 month post infection. Intranuclear inclusions typical of CMV infection were present in duct cells of the salivary glands, and clusters of virions typical of CMV were demonstrated in these cells by electron microscopy. Culture methods successfully detected virus in salivary glands of LEW rats for up to 12 months but in BN rats for only 5 months. Immunosuppression with cyclophosphamide or x-irradiation successfully reactivated the infection. Cocultivation of spleen cells from latently or chronically infected rats also was successful in recovering the virus. The intraperitoneal administration of interferon was shown to reduce the amount of virus recovered after experimental infection (Bruggeman et al., 1983b).

For purposes of differential diagnosis, it is imperative that morphologic peculiarities of the lacrimal glands in the rat be discussed here. Lyon et al. (1959) observed intranuclear inclusions and cytomegaly in the exorbital and intraorbital lacrimal glands of male rats from eight colonies and attributed them to CMV, although no virus was isolated. Meier (1960) observed cytomegalic inclusion body disease in the lacrimal glands of adult male and female SD (Sprague-Dawley®) rats from a cesarean-derived colony and claimed that passage of cell-free filtrates of these glands to newborn rats resulted in 20% of deaths due to systemic cytomegalic inclusion body disease. Meier's (1960) report, however, is probably best discounted altogether because it lacked both a meaningful morphologic description of the lesions and supporting evidence that a virus was involved.

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

The lesions described in the exorbital and intraorbital lacrimal glands of male rats by Lyon et al. (1959) are entirely compatible with the normal sexual dimorphism that occurs in these glands in the rat. The lacrimal glands of prepubertal rats are composed of small, closely packed acini of serous cells with small (5-7 µm), round to oval nuclei located in the basal position. At sexual maturity the lacrimal glands of male rats undergo dramatic changes. The acini and some of the nuclei enlarge, and the nuclei become more randomly distributed in the cytoplasm. Some of the acinar cells appear to be hypertrophic (cytomegalic) and contain markedly enlarged nuclei. The larger nuclei are often 20 µm or more in diameter; often irregular and lobulated in shape; and may contain inclusions or vacuoles of varying size, shape, and staining affinity. Under the light microscope these inclusions appear to be bounded by discrete basophilic membranes rather than halos, as are characteristically seen surrounding viral inclusions. The electron microscope, however, reveals that the discrete membrane is actually nuclear membrane; the inclusions, which contain varying amounts of cytoplasmic organelles, are formed by herniation of the nuclear membrane into the nucleus (Cordier and de Harven, 1960; Gaertner et al., 1988). In older animals patches of acini have uniformly small nuclei, foamy (vesicular-mucus) cytoplasm, and dilated acinar lumens, and thus resemble the Harderian gland. Similar but relatively very mild changes can be seen in the lacrimal glands of female rats (Walker, 1958; Baquiche, 1959; Cavallero, 1967; Klinge and Siveke, 1971; Paulini et al., 1972a, b; Paulini and Mohr, 1975; Gaertner et al., 1988).

Diagnosis

An enzyme-linked immunosorbent assay has been found to be very sensitive for the detection of antibodies to RCMV after experimental infection (Bruggeman et al., 1983a), but is not generally available for routine use. In active infections, the virus can be isolated from tissues such as the salivary gland and the spleen by using the rat embryo fibroblast culture technique. Light and electron microscopy of salivary glands for inclusions and virions of CMV would also be of value. Latent infections can be activated by x-irradiation or cyclophosphamide-induced immunosuppression, followed by attempted virus isolation by using rat embryo fibroblasts (Bruggeman et al., 1983a).

Control

Exclusion of wild rats from laboratory rodent facilities is apparently the only measure necessary to control RCMV infection.

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×
Interference with Research

Natural infections of RCMV have not been reported in laboratory rats. Experimental RCMV infection has been reported to alter peritoneal macrophage functions (Hendrix et al., 1986) and transiently suppressed humoral response to sheep red blood cells during the weeks following infection (Bruggeman et al., 1985). Experimental infection with RCMV (strain of Priscott and Tyrrell, 1982) exacerbates the arthritis in type II collagen-induced arthritis in rats (Smith et al., 1986). RCMV infection can be transferred to recipient rats in organs transplanted from latently infected rats (Bruning et al., 1986).

Mouse Thymic Virus
Significance

Uncertain, probably low.

Perspective

Rowe and Capps (1961) discovered this virus during studies in which specimens from mice suspected of containing mouse mammary tumor virus were being passaged in newborn mice. The only observed effect in the recipient mice was severe necrosis of the thymus, and the virus was named mouse thymic virus (MTV). Subsequent investigations of this agent have been severely hampered because no cell culture system has been found that will support its growth (Osborn, 1982).

Agent

MTV is considered a herpesvirus because of its ultrastructural features and its properties of heat and ether lability (Rowe and Capps, 1961; Parker et al., 1973). Intranuclear particles have a complete nucleoid and measure approximately 100 nm in diameter compared to cytoplasmic and extracellular particles, which are 135 nm in diameter. Large accumulations of intranuclear filaments measuring 10 nm in diameter also occur in the thymus of infected infant mice (Parker et al., 1973). Infectivity is destroyed by treatment with 20% ether for 2 hours at 2°C or by heating at 50°C for 30 minutes and is greatly reduced by storage at -60°C for short periods (Rowe and Capps, 1961).

MTV has been shown to be antigenically distinct from mouse

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

cytomegalovirus, another herpesvirus, by immunofluorescent antibody (IFA), complement fixation (CF) and neutralization tests (Cross et al., 1979).

MTV has not been grown successfully in cell cultures (Osborn, 1982).

Hosts

Wild and laboratory mice (Rowe and Capps, 1961: Cross et al., 1979).

Epizootiology

The prevalence of MTV in contemporary mouse stocks is unknown. However, the limited data available suggest that natural infections may be common in both wild and laboratory mice (Rowe and Capps, 1961; Cross, 1973; Lussier et al., 1988a). On the basis of virus isolations from saliva and the use of IFA and CF tests for serum antibody, Cross (1973) found 4 of 15 colonies of laboratory mice and 3 of 4 individual wild mice to be infected with MTV. In a more recent survey using an enzyme-linked immunosorbent assay (ELISA), Lussier et al. (1988a) reported finding 1 of 8 colonies of laboratory mice to be serologically positive for MTV. Wild mice frequently have dual infections of MTV and mouse cytomegalovirus (Rowe and Capps, 1961; Cross, 1973).

Information on the epizootiology of MTV in naturally infected mouse populations is very meager. The virus apparently occurs as a persistent, subclinical infection in the salivary glands with virus shedding in saliva (Cross et al., 1973; Osborn, 1982). MTV has been isolated on one occasion from mammary tissue of a lactating mouse, suggesting that it can be transmitted in milk (Morse, 1987).

Although of questionable applicability to the epizootiology of natural MTV infection, transmission of the virus following intraperitoneal inoculation has been studied (St.-Pierre et al., 1987). Under these conditions, low transmissibility of the virus occurred between cagemates, but not by the transplacental route. Thus, the weight of evidence presently suggests that horizontal transmission is most important, although vertical transmission cannot be ruled out.

Clinical

Natural infections are subclinical (Rowe and Capps, 1961).

Pathology

Although necrosis presumably occurs in the thymus, lymph nodes, and spleen of mice naturally infected with MTV as neonates (Cross, 1973; Wood et al., 1981), it usually goes unnoticed (Rowe and Capps, 1961).

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

Disease due to MTV infection is age-dependent, i.e., experimental inoculation of the virus causes morphologic lesions and depression of immunologic functions only in mice infected as neonates (before 72 hours of age). The characteristic lesion is lymphoid necrosis in the thymus, lymph nodes, and spleen, with the thymus being most severely affected (Cross, 1973; Wood et al., 1981). Following intraperitoneal inoculation of virus into 24 hour-old mice, virus is first detectable in the thymus on day 3, reaches peak titer on day 7, and disappears by day 14. Macroscopic necrosis of the thymus begins on day 7 and is most severe between days 10 and 14. Intranuclear inclusions are present in thymocytes on days 5 through 10. The weight of the thymus is reduced to only 25% of normal. Subsequently, the necrosis is superseded by a diffuse "granulomatous" response with giant cells. The thymus regains normal histology around day 21 and normal weight by day 42. Necrosis and repair follow similar patterns in the lymph nodes and spleen. After neonatal infection, persistent infection of the salivary glands occurs, but the mice do not produce serum antibody (Cross et al., 1979). In contrast, adult mice infected with the virus develop persistent infections of the salivary glands and produce serum antibody, without the occurrence of lymphoid necrosis (Cross, 1973, 1979; Wood et al., 1981).

The severe lymphoid necrosis seen in mice neonatally infected with MTV involves mainly helper T lymphocytes (Cohen et al., 1975; Mosier et al., 1977) and cytotoxic T lymphocytes (Cohen et al., 1975). There is reduced responsiveness to the T cell mitogens concanavalin A and phytohemagglutinin (Cohen et al., 1975), and reduced graft-versus-host response (Cross et al., 1976). Immunosuppression peaks at about 4 weeks and appears to return to normal by about 12 weeks after infection (Cohen et al., 1975). B lymphocyte functions appear to be unimpaired (Cohen et al., 1975; Morse et al., 1976), except that neonatally infected mice do not produce antibody to the virus (Cross et al., 1979).

Diagnosis

Mice infected postnatally produce serum antibodies that can be detected by ELISA, IFA or CF tests, with the ELISA test being the most sensitive (Lussier et al., 1988 a,b). In cases where neonatal infection is suspected, evidence of MTV infection can be obtained by inoculating pathogen free neonatal mice with salivary gland homogenate, saliva, or other test material followed by histologic examination of their thymuses, lymph nodes, and spleens for lymphoid necrosis and intranuclear inclusions 10-14 days later (Cross, 1973; Wood et al., 1981). In vitro isolation of the virus is impossible as no cell culture system is known to support the growth of MTV (Osborn, 1982).

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×
Control

Wild mice presumably serve as the main reservoir of infection and must be excluded from laboratory animal facilities (Lussier et al., 1988a). Periodic health surveillance testing of mouse stocks by ELISA is recommended (Lussier et al., 1988a,b). Elimination of the virus from infected mouse stocks might be possible through isolation of breeding pairs, with selection of progeny by ELISA testing (Lussier et al., 1988a,b) and passage of tissues in neonatal mice to test for development of lymphoid necrosis in thymus, lymph nodes, and spleen (Morse, 1987).

Interference with Research

The passage of tissues from mice subclinically infected with MTV in neonatal mice may be inadvertently complicated by the effects of this virus (Rose and Capps, 1961).

Sialodacryoadenitis Virus
Significance

High.

Perspective

1961: Innes and Stanton (1961) reported two epizootics in which weanling rats had swollen, thickened necks and red porphyrin pigment along the eyelid margins. Microscopically, there was severe inflammation and edema of the submaxillary salivary glands and Harderian glands (other lacrimal glands were not examined), and the disease was named sialodacryoadenitis. A viral etiology was suspected but virus isolation was not attempted.

1963: Hunt (1963) reported a disease in young rats from two shipments. Ten days after receipt, many of the rats had suborbital swelling and conjunctivitis, and a few had keratitis with corneal ulceration. The Harderian and intraorbital lacrimal glands were twice the normal size and were histologically characterized by loss of glandular tissue, hyperplasia and squamous metaplasia of the ductal epithelium, intense histiocytic inflammation, and acidophilic intranuclear inclusions in occasional epithelial cells. The disease was called dacryoadenitis.

1969: Jonas et al. (1969) reproduced the disease of salivary and lacrimal glands by intranasally inoculating gnotobiotic rats with an ultrafiltrate of the submaxillary gland from a rat with sialodacryoadenitis.

1970: Parker et al. (1970c) isolated and characterized a virus from the lungs of laboratory rats with mild interstitial pneumonia, carried out experi-

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

mental infections in suckling and weanling F344 and WI (Wistar) rats, and named the virus rat coronavirus (RCV). [This virus is here considered a strain of sialodacryoadenitis virus (SDAV), the best studied of this group of closely related coronaviruses (Jacoby, 1986)].

1972: Bhatt et al. (1972) used primary rat kidney cells to isolate a virus from rats with sialodacryoadenitis, characterized the virus, identified it as a coronavirus, and named it SDAV.

1977: Weisbroth and Peress (1977) investigated an epizootic of SDAV infection in which lesions were limited to orbital tissues. They proposed the hypothesis that the variability in clinical and morphological expression of the disease due to SDAV was attributable to the occurrence of viral mutants with different tissue tropisms.

1982: Maru and Sato (1982) isolated and characterized a strain of SDAV from rats with sialoadenitis in Japan, and called it the causative agent of rat sialoadenitis (CARS). Unlike previous strains, it could be grown in 3T3 cells but not in primary rat kidney cells.

1986: Wojcinski and Percy (1986) demonstrated that SDAV produces significant albeit transient disease throughout the respiratory tract, and suggested that the respiratory disease due to SDAV had generally been overlooked except in the studies of RCV.

1987: Schoeb and Lindsey (1987) reported experimental evidence that SDAV can exacerbate murine respiratory mycoplasmosis in rats.

Agent

SDAV is an RNA virus, family Coronaviridae, genus Coronavirus. It is antigenically related to many other coronaviruses, including mouse hepatitis virus (MHV) (Machii et al., 1988) and human coronavirus (strain OG38). SDAV, RCV, CARS, and other similar coronavirus isolates from rats are considered different strains of the same virus (Bhatt et al., 1977; Jacoby, 1986).

Virions of SDAV measure approximately 114 nm in diameter and have characteristic projections from the surface, giving a crown-like appearance. The virus is relatively unstable. Infectivity is quickly lost at room temperature, freezing to -20°C, heating to 56°C, and exposure to lipid solvents. It has been stored at -60°C for at least 7 years (Jacoby et al., 1979).

SDAV and RCV can be propagated in primary rat kidney and LBC cells (Parker et al., 1970c; Bhatt et al., 1972; Hirano et al., 1985, 1986). CARS grows in 3T3 cells (Maru and Sato, 1982).

Hosts

Rats. Mice have been shown to be susceptible experimentally (Bhatt et al., 1977; Percy et al., 1986, 1988a), but natural infection in mice has not

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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been reported. [A naturally occurring disease similar to SDAV in rats has been reported for mice (Wagner et al., 1969; Maronpot and Chavannes, 1977), but a causative agent was not identified.]

Epizootiology

SDAV is one of the most common viruses in laboratory rats (Parker et al., 1970c). It is highly contagious, spreading rapidly within rooms of susceptible rats by contact and aerosol. It is not transmitted vertically. The virus is present in tissues of infected rats for only about 7 days, and there is no carrier state (Hanna et al., 1984; Wojcinski and Percy, 1986). Tissues affected by SDAV infection are mixed (submaxillary) and serous (parotid) salivary glands, lacrimal glands (Harderian, intraorbital, and exorbital), cervical lymph nodes, thymus, and the mucosa of the respiratory tract (Jacoby et al., 1979; Wojcinski and Percy, 1986).

LEW, WAG/Rij, and SHR rats are more susceptible than other strains (Tuchi et al., 1977: Weisbroth and Peress, 1977; Jacoby et al., 1979: Carthew and Slinger, 1981). Less susceptible rat strains include WI (Wistar), SD (Sprague-Dawley®), LE (Long Evans), and F344; they are approximately equal in susceptibility (Percy et al., 1984).

Clinical

Natural infections usually take one of two forms:

  1. Enzootic infection in breeding colonies. Adults are immune due to previous infection. Suckling rats have transient (1 week or less) conjunctivitis characterized by winking and blinking. Eyelids may adhere together due to exudate. Signs of this form of disease usually are mild and subtle, escaping detection by most observers. All clinical signs usually have disappeared by the time the investigator receives weanlings or older animals (Jacoby et al., 1979).
  2. Epizootic disease in fully susceptible weanlings or adults. The incubation period is often less than 1 week. Sudden high prevalence of overt disease heralds an explosive outbreak. Signs can include any or all of the following: cervical edema, sneezing, photophobia, nasal and ocular discharge (serous to seropurulent, often porphyrin stained), corneal ulceration, and keratoconus. Characteristically, there is high morbidity and no mortality. Most clinical signs disappear in about a week, but the eyes may be more prominent than normal for 1 or 2 weeks due to inflammation of retroorbital tissues (Jacoby et al., 1979).

The possibility has been raised that some outbreaks of SDAV infection

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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may be completely asymptomatic (Eisenbrandt et al., 1982). This may be true, but one must remember that enzootic infections can be very mild and transient, and easily overlooked. Severe clinical disease characterized by cervical edema and eye lesions may be the exception rather than the rule.

Pathology

SDAV has a tissue tropism for tubuloalveolar glands of the serous or mixed serous-mucous types, with the submaxillary and parotid salivary glands, exorbital lacrimal, Harderian, and intraorbital lacrimal glands being the major target organs. The cervical lymph nodes, thymus, and respiratory tract also are affected but usually by relatively mild changes (Jacoby et al., 1979; Wojcinski and Percy, 1986; Schoeb and Lindsey, 1987).

SDAV strains from different natural outbreaks also differ in organ tropism; i.e., they vary greatly in relative incidence and severity of disease produced in three organ systems: salivary glands, lacrimal glands, and the respiratory tract. Presumably, such differences in organ tropism explain, at least in part, the variations in pathological (and clinical) expression seen in the natural disease. There is no clear evidence that virus mutation has a role (Weisbroth and Peress, 1977), although this is still an interesting hypothesis. Differences in virulence of SDAV strains and host factors may be important (Nunoya et al., 1977; Tuchi et al., 1977; Utsumi et al., 1978; Jacoby et al., 1979).

The histopathologic changes in salivary and lacrimal glands are characteristic. Diffuse necrosis of alveolar and ductal epithelium occurs about 5 days post infection. Rapid infiltration of polymorphonuclear leukocytes into the necrotic debris and interstitium is accompanied by varying degrees of interstitial edema. Repair of ductal epithelium quickly ensues, becoming hyperplastic and squamous in appearance by 10 days post infection. Intranuclear inclusions are occasionally observed in the ductal epithelium. Continued glandular repair and lymphohistiocytic inflammation follow, with complete restoration of normal glandular architecture by about 30 days post infection (Jonas et al., 1969; Jacoby et al., 1975; Doi et al., 1980).

Eye lesions can include interstitial keratitis, corneal ulceration, keratoconus, synechia, hypopyon, hyphema, and conjunctivitis. Sequelae of the infection can include megaloglobus with lenticular and retinal degeneration (Innes and Stanton, 1961; Hunt, 1963; Jonas et al., 1969; Jacoby et al., 1975, 1979; Lai et al., 1976; Weisbroth and Peress, 1977).

Thymic lesions are limited to focal necrosis of the cortex and medulla with some widening of interlobular septae. Focal necrosis and lymphoid hyperplasia occur in the cervical lymph nodes. Mild interstitial pneumonia has been seen in experimental cases (Jacoby et al., 1975; Bhatt and Jacoby, 1977; Wojcinski and Percy, 1986; Schoeb and Lindsey, 1987).

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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It has been suggested that SDAV can act as a copathogen in murine respiratory mycoplasmosis due to Mycoplasma pulmonis (Jacoby et al., 1975; Bhatt and Jacoby, 1977; Wojcinski and Percy, 1986). Schoeb and Lindsey (1987) have shown experimentally that SDAV infection can exacerbate Mycoplasma pulmonis infection in rats.

Diagnosis

Serologic tests are invaluable because subclinical infection is very common. The complement fixation (CF) test was the standard procedure for many years. The enzyme-linked immunosorbent assay (Peters and Collins, 1981, 1983) and indirect immunofluorescence test (Smith, 1983) are more sensitive than the CF test and are now standard in most laboratories. It must be emphasized, however, that a positive result from any or all of these tests is merely indicative of anti-coronavirus antibodies, not prior infection by a specific coronavirus (e.g., SDAV, RCV, CARS, or MHV). Experimental SDAV infections in mice have been shown to result in positive CF tests for SDAV and MHV (Bhatt et al., 1977).

In routine health monitoring it is recommended that histologic sections be taken of both Harderian glands and the submaxillary and parotid salivary glands of each animal. A presumptive diagnosis of SDAV infection often can be based on the characteristic histologic changes in these glands. Lesions may be bilateral or unilateral and are frequently found in animals with negative serologic tests for coronavirus antibody (i.e., lesions appear before seroconversion).

SDAV virus can be isolated by culture methods using primary rat kidney, 3T3 or LBC cells, or by intracerebral inoculation of neonatal mice (Bhatt et al., 1972; Kojima et al., 1980, 1983; Maru and Sato, 1982; Hirano et al., 1985, 1986). The virus can be demonstrated in affected tissues by immunofluorescence for about 7 days postinoculation (Jacoby et al., 1975).

Control

The key to effective control in infected colonies is recognition that SDAV spreads rapidly through rat populations, infected rats shed virus for only about 7 days, and latent infections do not occur. Thus, infections in breeding colonies can be eliminated by quarantining the room, suspending breeding completely, and destroying all newborn pups for 6 to 8 weeks. The same result is achieved in nonbreeding populations through a 6- to 8-week quarantine period during which no new animals are introduced. Recovered rats are considered free of virus. However, there is some question as to the possibility of later reinfection with the same strain or a different strain of SDAV (Jacoby et al., 1979).

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Since SDAV is not transmitted vertically, cesarean derivation is a very effective method of control; however, the disease runs its course so rapidly that this technique is usually impractical.

SDAV resists environmental conditions poorly, so fomites probably do not play an important role in transmission (Jacoby et al., 1979).

Interference with Research

SDAV infection can seriously complicate studies involving the eyes, salivary glands, lacrimal glands, or the respiratory tract of rats (Jacoby, 1986). It causes depletion of epidermal growth factor in submaxillary salivary glands that could affect carcinogenesis studies (Percy et al., 1988b). It reduces interleukin-1 production by alveolar macrophages (Boschert et al., 1988) and exacerbates Mycoplasma pulmonis infection (Schoeb and Lindsey, 1987). Additional effects of infection that have been reported include reduced food consumption and weight loss (Nunoya et al., 1977; Utsumi et al., 1978, 1980), and reduced breeding performance and slowing of growth rate in young rats (Utsumi et al., 1978, 1980).

Mouse Hepatitis Virus
Significance

Very high.

Perspective

1949: Cheever et al. (1949) at Harvard reported discovery of the JHM strain of mouse hepatitis virus (MHV), and Bailey et al. (1949) associated it with naturally occurring encephalomyelitis in mice.

1951: Gledhill and Andrewes (1951) in England discovered a viral agent (later designated MHV-1) that caused fatal hepatitis when passaged in young mice. Subsequently, Gledhill and associates (Gledhill and Dick, 1955; Gledhill and Niven, 1955; Gledhill et al., 1955) showed that the fatal hepatitis was actually due to combined MHV-1 and Eperythrozoon coccoides infections, and that several mouse strains differed in susceptibility to MHV-1 infection.

1960: Bang and Warwick (1960) showed that the inherited capacity of the mouse macrophage to restrict virus growth was important in the resistance of certain strains of mice to MHV.

1962: Kraft (1962a) described a new viral agent that caused diarrhea in suckling mice and named it the lethal intestinal virus of infant mice (LIVIM).

1963: Rowe et al. (1963) used a variety of methods to survey seven

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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mouse colonies (including one germfree colony) for MHV and found that five had the infection. They also compared the effectiveness of Trexlertype plastic isolators, filter-covered cages, and open cages in controlling the spread of MHV within their experimental mouse room. Two of the most important conclusions from their work were that MHV was recognized as "a highly contagious, prevalent, enteric infection of mice," and that "precise animal experimentation with these viruses cannot be done without strict isolation facilities and known virus-free mice."

1963: East et al. (1963) reported that neonatal thymectomy of mice led to a wasting syndrome resulting from increased susceptibility to MHV.

1974: Sebesteny and Hill (1974) reported that MHV was responsible for the wasting syndrome in athymic (nu/nu) mice.

1976: Broderson et al. (1976b) reported that LIVIM was actually MHV, a concept later confirmed by Carthew (1977).

1979: Peters et al. (1979) described a highly sensitive serologic test for MHV, the enzyme-linked immunosorbent assay (ELISA), bringing to an end the long era of reliance on a very insensitive test, complement fixation (CF). The practical result was that MHV soon became recognized as being ubiquitous in contemporary mouse stocks.

1984: Barthold and Smith (1984) presented evidence supporting two basic patterns of natural MHV pathogenesis depending on the tropism of virus strains: respiratory and enteric.

Agent

The term mouse hepatitis virus (MHV) is used to designate a large group of single-stranded RNA viruses belonging to the family Coronaviridae, genus Coronavirus. Like other coronaviruses, they are surrounded by an envelope with a corona of surface projections called peplomers. The virions are pleomorphic and measure 80-160 nm in diameter. Each peplomer is about 20 nm long by 7 nm wide at the tip (Robb and Bond, 1979; Matthews, 1982; Sturman and Holmes, 1983; Holmes et at., 1986a). Approximately 25 different strains or isolates of MHV have been described. Of that number, six have been studied most extensively and are generally considered the prototype strains: MHV-1, MHV-2, MHV-3, JHM (MHV-4), A59, and S. The other strains and new isolates of MHV are often compared to the prototype strains by cross-neutralization tests, but antigenic relatedness, as determined by that method, correlates poorly with pathogenicity and has limited usefulness for epizootiologic studies. Other coronaviruses that are antigenically related to MHV include sialodacryoadenitis virus, human coronavirus OC43, hemagglutinating encephalomyelitis virus of pigs, and neonatal calf diarrhea coronavirus (Robb and Bond, 1979; Sturman and Holmes, 1983; Barthold, 1986a,b).

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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MHV is relatively resistant to repeated freezing and thawing, heating (56°C for 30 minutes), and acid pH but is sensitive to lipid solvents, drying, and disinfectants. It can be stored for 30 days at 4°C and indefinitely at -70°C (Hirano et al., 1978; Robb and Bond, 1979). MHV is commonly grown in cell cultures, but some strains grow better in certain cell lines than others. Cell lines that have been found to be most useful are NCTC 1469, 17C1-1, DBT, BALB/c-3T3, and CMT-93 (Barthold et al., 1985).

Hosts

Mice (Mus musculus) are considered the natural hosts. Suckling rats have been found to be modestly susceptible to experimental intranasal infection; the virus replicated briefly in the nasal mucosa and there was seroconversion but no clinical signs (Taguchi et al., 1979b). When given the virus intranasally, deer mice (Peromyscus maniculatus) developed positive antibody titers but failed to develop clinical illness or transmit the virus to sentinel laboratory mice (Silverman et al., 1982).

Epizootiology

MHV is extremely contagious. Infection of the majority of mice housed under conventional conditions in multipurpose facilities is the norm. It is one of the most ubiquitous infections of laboratory mice worldwide, with reported prevalence rates frequently exceeding 80% (Parker, 1980; Lindsey, 1986).

The epizootiology of natural MHV infections has not been studied well because sensitive detection methods were not available until very recently. Numerous factors such as virus strain and mouse strain are known to influence the pathogenesis of MHV infection and may be important determinants of its epizootiology. However, current evidence indicates that in immunocompetent mice the infection runs its course within 2-3 weeks, and there is no carrier state (Barthold, 1986a,b). During active infection virus is shed in the feces and by aerosol. Direct contact, fomites, and airborne particles are all probably very important in transmission (Rowe et al., 1963; Robb and Bond, 1979; Barthold, 1986b). Transplacental transmission is of doubtful importance in natural infections (Piccinino et al., 1966), although it has been achieved by the intravenous inoculation of MHV into pregnant dams (Katami et al., 1978).

MHV has been a frequent contaminant of transplantable tumors (Braunsteiner and Friend, 1954; Nelson, 1959; Manaker et al., 1961; Collins and Parker, 1972; Fox et al., 1977a) and cell lines (Sabesin, 1972; Stohlman and Weiner, 1978; Yoshikura and Taguchi, 1979; Holmes, 1986a).

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Clinical

MHV infections in immunocompetent mice are usually subclinical. However, inasmuch as numerous factors related to the virus (e.g., virulence and organotropism) and the host (e.g., age, genotype, pathogen status, and experimental modifications) affect disease expression due to MHV, a range of clinical diseases can be anticipated (Barthold, 1986a,b). Most infections follow one of the following clinical patterns:

  1. Enzootic (subclinical) infection. This has been the most common pattern in the United States because of the high prevalence of MHV in breeding populations. Adults are immune due to prior infection. Newborn mice are protected by maternally derived passive immunity that wanes by weaning age. Infection is perpetuated among partially protected weanlings with little or no clinical disease (unless they are compromised immunologically such as by experimental procedures) (Manaker et al., 1961; Hierholzer et al., 1979; Ishida and Fujiwara, 1982; Barthold, 1986a,b).
  2. Epizootic (clinically apparent) infection. This is the pattern usually seen in infant mice of naive breeding populations housed in open cages. The infection spreads rapidly through the entire population. In infections due to the more virulent enterotropic strains, diarrhea with high (up to 100%) mortality can be seen in infant mice, whereas only moderate losses of infant mice are usually incurred due to the nonenterotropic strains. Infections in naive adults are usually subclinical (Rowe et al., 1963; Barthold et al., 1982; Barthold, 1986a,b).
  3. Wasting syndrome in athymic (nulnu) mice. Athymic mice develop severe generalized disease characterized clinically by progressive emaciation (wasting) leading ultimately to debility and death (Sebesteny and Hill, 1974; Hirano et al., 1975b; Fujiwara et al., 1977; Ward et al., 1977). Jaundice may be observed in some cases. Diarrhea may be a leading sign in cases with enterotropic MHV infection (Barthold et al., 1985).
Pathology

Much of the vast literature on MHV infection is not helpful in understanding the pathogenesis of natural MHV infections. In many published studies, inordinately high doses of the virus were inoculated by unnatural routes, mice of unknown (or unstated) pathogen status were used, and few target organs were examined for lesions (Barthold and Smith, 1984). Nevertheless, much has been learned about MHV infection. Strains of MHV differ greatly in virulence and tissue tropism (Taguchi et al., 1983; Barthold and Smith, 1984; Boyle et al., 1987), mouse strains differ greatly in susceptibility to MHV (Knobler et al., 1981; Smith et al., 1984; Barthold, 1986a,b), and these factors interact with host age, and route and dose of

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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virus inoculation to determine the outcome of infection (Barthold, 1986a; Holmes et al., 1986b; Barthold, 1987). Because disease expression depends on interaction of host genotype with virus strain, it is not possible to strictly categorize mouse strains as susceptible or resistant (Barthold, 1986b).

The presence (or absence) of virus receptors on host cells appears to be very important in pathogenesis. SJL/J mice appear to be completely resistant to infection with MHV strain A59 because they lack a specific receptor that is present on plasma membranes of target cells from genetically susceptible mice (Boyle et al., 1987). Such receptors also may explain tissue tropisms of the virus. Barthold and Associates (Barthold et al., 1982, 1985; Barthold and Smith, 1984; Barthold, 1986a,b) have shown that there are two major disease patterns depending on the tropism of virus strains:

  1. Respiratory pattern. MHV infection consistently involves the nasal passages and lungs with dissemination to other organs by the blood vascular system; intestinal involvement, if present, is minimal. After experimental infection, the virus appears sequentially in nasal mucosa; then lungs; and then other organs including lymph nodes, thymus, spleen, bone marrow, brain, liver, and intestine. The majority of MHV strains are thought to follow this pattern, but the evidence is best for MHV-1, MHV-2, MHV-3, A59, S, JHM, Tettnang, and wt-1 (Barthold and Smith, 1984; Barthold, 1986a,b).
  2. Enteric pattern. MHV infection is primarily restricted to the nasal passages and bowel, with variable spread to other abdominal organs such as the liver and abdominal lymph nodes but usually not to the lungs, although a few strains are known to spread to other sites such as brain. MHV strains that follow this pattern include LIVIM, MHV-S/CDC, MHV-D, DVIM, MHVY, and wt-2 (Barthold and Smith, 1984; Barthold, 1986a,b).

The respiratory and enteric patterns of disease are considered basic, relative patterns of pathogenesis because some overlap between the two patterns is known to occur in infections caused by some strains of MHV. Variations in these patterns also can occur because of other factors such as host age, genotype, and immune status and concurrent infections (Barthold and Smith, 1983, 1984; Barthold, 1986a,b).

In immunocompetent mice, not immunosuppressed by an experimental regimen or by another infection, the lesions of MHV infection are present for only a brief period (7-10 days) following infection. Also, they are usually nonspecific and subtle, particularly those that follow the respiratory pattern. For example, the following lesions have been observed in 3-week-old mice following the intranasal inoculation of MHV-S, a strain with respiratory tropism: mild olfactory mucosal necrosis, neuronal necrosis of olfactory bulbs and tracts, lymphoplasmacytic infiltrates and vacuolation in the brain, multifocal interstitial pneumonia with mild perivascular lymphoid

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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infiltrates, and multifocal necrotizing hepatitis (Taguchi et al., 1979a; Barthold and Smith, 1983). Lesions caused by the enterotropic strains of MHV are mainly confined to the intestinal tract and are most severe in neonatal mice because of their relatively slow kinetics of mucosal epithelium turnover (or replacement). Varying degrees of epithelial lysis and blunting of villi occur in the small intestine. Numerous multinucleate syncytial giant cells (balloon cells) may occur on the villi as well as in the crypts. Ulcerations of the mucosa may be seen in the more severe cases. A similar lytic process with the presence of syncytial giant cells occurs in the cecum and ascending colon. Severe typhlocolitis has been observed in some outbreaks. On occasion cases may be seen with multifocal necrotizing hepatitis and/or encephalitis (Biggers et al., 1964; Ishida et al., 1978; Hierholzer et al., 1979; Ishida and Fujiwara, 1979; Sugiyama and Amano, 1981; Barthold et al., 1982; Perlman et al., 1987, 1988).

The pathogenesis of MHV infection in athymic (nu/nu) and neonatally thymectomized mice also appears to follow the basic respiratory and enteric patterns. However, in these T-cell-deficient mice the infection and resulting disease tend to become progressively more generalized, severe, and chronic, with involvement of many organs, including brain, liver, lungs, bone marrow, lymphoreticular organs, vascular endothelium, and intestine. In the liver areas of multifocal necrosis appear with a zone of acute inflammatory cells and variable numbers of multinucleate giant cells at their periphery, followed by partial replacement of hepatocytes and continuing necrosis, leading to chronic active hepatitis if the animal survives long enough. Grossly, the liver appears shrunken, with the darker pits and grooves representing areas of hepatocyte loss and the lighter ridges representing areas of relatively normal hepatocytes. Small proliferative lesions with few to several syncytial cells may be seen on endothelial surfaces of many organs, particularly lungs and brain. Syncytial giant cells can occur in lymph nodes, on mesothelial surfaces, and at other sites. Splenomegaly may occur because of compensatory myelopoiesis, and large numbers of myelopoietic cells may appear in the liver (Hirano et al., 1975b; Fujiwara et al., 1977; Tamura et al., 1977; Ward et al., 1977; Ishida et al., 1978; Furuta et al., 1979; Barthold, 1986a). One outbreak has been reported in which athymic (nu/nu) mice were found to be infected with an enterotropic strain of MHV and had chronic hyperplastic typhlocolitis as the predominant lesion (Barthold et al., 1985).

The mechanisms of host resistance to MHV infection are poorly understood. There is strong evidence that mice are fully susceptible to the virus as neonates, but some strains acquire resistance at 2-3 weeks of age as lymphoreticular function matures, with the result that older mice have a spectrum of relative susceptibility ranging from susceptible to highly resistant (Shif and Bang, 1970; Hirano et al., 1975a; Taguchi et al., 1977, 1979c). Humoral immunity is considered relatively unimportant. Cell-mediated

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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immunity is clearly important because of the high susceptibility of athymic (nu/nu) mice. Also, there is evidence that macrophages (Bang and Warwick, 1960; Weiser and Bang, 1977), interferon (Robb and Bond, 1979; Garlinghouse et al., 1984; Garlinghouse and Smith, 1985), and natural killer cells (Bukowski et al., 1983; Pereira et al., 1984) have important roles. Infection of mice with one strain of virus confers strong resistance to that strain, but not necessarily to other strains (Barthold and Beck, 1987).

Diagnosis

The enzyme-linked immunosorbent assay (ELISA) is the test of choice for routine serologic monitoring because it is far more sensitive than the CF or serum neutralization tests (Peters et al., 1979; Peters and Collins, 1983). An immunofluorescent antibody (IFA) test also is available and is about equal to the ELISA in sensitivity (Smith, 1983). The heterozygotes of nude mice or sentinel mice should be used in testing nude mouse stocks; nude mice do not develop CF antibody in response to MHV and only weak and variable ELISA or serum neutralization antibody responses (Barthold, 1986a).

A presumptive necropsy diagnosis of active enterotropic MHV infection in either clinical or subclinical infections is relatively easy to make based on typical lesions in the small and/or large intestine. The blunting of intestinal villi due to the loss of villous epithelium plus the presence of large, multinucleate syncytial giant cells in the mucosal epithelium of the small intestine, cecum, and/or ascending colon are virtually diagnostic. Lesions of MHV infection in other organs are usually nonspecific and often very subtle. An immunofluorescence method has been developed for identifying MHV antigen in formalin-fixed, paraffin-embedded tissue, but it is not practical for routine diagnostic purposes (Brownstein and Barthold, 1982).

MHV can often be isolated by cell culture methods by using NCTC 1469, 17C1-1, DBT, BALB/c-3T3, or CMT-93 cells; but all strains do not grow equally well in all cell lines (Barthold et al., 1985). It also is essential that tissues with a high titer of virus be used for attempted virus isolations. Frequently, the best approach is to expose pathogen-free athymic (nu/nu) mice to animals with suspected infection and use the livers from the nude mice with clinical disease for virus isolation (Barthold, 1986b). Cross-neutralization testing may be used for determining the antigenic relatedness of wild-type isolates to prototype strains (Barthold and Smith, 1984).

Transplantable tumors and other biologic materials from mice may be screened for MHV by isolating the virus in cell cultures and/or by the mouse antibody production (MAP) test (Rowe et al., 1959a, 1962).

Control

MHV is both highly contagious and highly prevalent in breeding

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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populations of mice. Therefore, the strictest adherence to systematic measures of pathogen exclusion is required to prevent entrance of this agent into research facilities. MHV-free mice must be identified by regular health monitoring of supplier subpopulations, transported to the user facility in filter-protected cartons to prevent infection en route, quarantined in a barrier room at the receiving institution until tested and shown to be free of infection, and subsequently maintained by strict barrier protocol with regular health surveillance testing to check the effectiveness of the barrier. In addition, all biologic materials from mice such as transplantable tumors coming into the institution must be screened and shown to be free of infectious agents before experimental use in animals (Collins and Parker, 1972).

Once MHV infection has been diagnosed in a facility, the affected population should be either promptly eliminated or quarantined in an area or facility completely away from pathogen-free mice as MHV is highly contagious. Cesarean derivation followed by barrier maintenance has traditionally been recommended for rederivation of breeding stocks. However, recent evidence suggests that MHV infections in immunocompetent mice may have an acute course, with complete elimination of the virus in about 2 weeks (Weir et al., 1987; Barthold, 1986b). Thus, a practical alternative to cesarean derivation is the isolation of individual breeding pairs of mice from MHV-infected populations in separate containment devices such as filter-top cage systems (Sedlacek et al., 1981), with subsequent selection of seronegative progeny as breeders (Weir et al., 1987).

Interference with Research

An extremely large number of effects of MHV on mice and their biologic responses to experimental treatments have been observed. Some occurred as a result of natural infections or conditions simulating natural infections, while others were observed under somewhat artificial experimental circumstances (e.g., large doses of virus inoculated by unnatural routes such as intraperitoneally). Both types of examples are included in the following extensive (albeit incomplete) lists.

In athymic (nu/nu) mice, MHV infection:

  1. Can cause severe destructive lesions in many organs, including intestine, liver, brain, lungs, spleen, lymph nodes, and bone marrow (Sebesteny and Hill, 1974; Fujiwara et al., 1977; Ward et al., 1977; Ishida et al., 1978).
  2. Causes spontaneous differentiation of lymphocytes bearing T-cell markers (Scheid et al., 1975; Tamura et al., 1978b).
  3. Alters IgM and IgG responses to sheep erythrocytes (Tamura et al., 1978b; Tamura and Fujiwara, 1979).
  4. Enhances phagocytic activity of macrophages (Tamura et al., 1980).
Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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  1. Is made more severe by experimental insults, such as injection of silica dust, that interfere with macrophage function (Tamura et al., 1979).
  2. Causes rejection of xenograft tumors (Kyriazis et al., 1979; Akimaru et al., 1981).
  3. Impairs liver regeneration after partial hepatectomy (Carthew, 1981).
  4. Causes hepatosplenic myelopoiesis (Ishida et al., 1978).

In immunocompetent mice, MHV infection:

  1. Causes immunosuppression or immunostimulation during acute infection and chronic immunodepression in persistent infection (Virelizier et al., 1976).
  2. Inhibits lymphocyte proliferative responses in mixed lymphocyte cultures and mitogen-stimulated cells (Krzystyniak and Dupuy, 1983).
  3. Inhibits immunoglobulin secretion by Peyer's patch B cells (Casebolt et al., 1987).
  4. Depresses phagocytic activity (Gledhill et al., 1965; Williams and DiLuzzio, 1980).
  5. Increases the number and tumoricidal activity of peritoneal macrophages in infected mice (Boorman et al., 1982).
  6. Increases hepatic uptake of injected iron (Tiensiwakul and Husain, 1979).
  7. Increases susceptibility to other indigenous mouse pathogens (and vice versa), including Eperythrozoon coccoides (Niven et al., 1952; Gledhill et al., 1965: Lavelle and Bang, 1973), K virus (Tisdale, 1963), leukoviruses (Nelson, 1952a,b; Braunsteiner and Friend, 1954; Gledhill, 1961; Manaker et al., 1961), and Schistosoma mansoni (Warren et al., 1969).
  8. Activates natural killer cells and production of local and circulating interferon (Mallucci, 1964; Tardieu et al., 1980; Schindler et al., 1982).
  9. Diminishes the production of interferon in response to infection with Sendai virus (Virelizier et al., 1976).
  10. Alters the course of concurrent viral infections due to pneumonia virus of mice or Sendai virus (Carrano et al., 1984).
  11. Alters the course of experimental ascites myeloma (Nelson, 1959; Fox et al., 1977a).
  12. Alters hepatic enzyme activity (Ruebner and Hirano, 1965; Cacciatore and Antoniello, 1971; Budillon et al., 1972, 1973; Paradisi et al., 1972; Carter et al., 1977).
  13. Slows liver regeneration after partial hepatectomy (Carthew, 1981) and increases proliferative activity of liver and bowel during the recovery phase of infection (Carthew, 1981; Barthold et al., 1982).
  14. Induces production of serum a-fetoprotein (Piazza et al., 1965).
  15. Induces macrophage procoagulant activity (Levy et al., 1981).
Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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  1. Causes anemia, leukopenia, and thrombocytopenia (Piazza et al., 1965; Hunstein et al., 1969; Namiki et al., 1977).
  2. Delays the increase in plasma lactic dehydrogenase activity following infection with lactic dehydrogenase virus (Dillberger et al., 1987).
  3. Subclinical infections (of MHV) are transformed into severe disease with mortality by thymectomy (East et al., 1963; Dupuy et al., 1975; Sheets et al., 1978), cortisone (Starr and Pollard, 1958; Gallily et al., 1964; Vella and Starr, 1965; Taylor et al., 1981), cyclophosphamide (Willenborg et al., 1973), whole body irradiation (Vella and Starr, 1965; Dupuy et al., 1975), anti-lymphocyte serum (Riet et al., 1973; Dupuy et al., 1975; Levy-Leblond and Dupuy, 1977), reticuloendothelial blockade by iron salts (Warren et al., 1968), chemotherapeutic agents (Braunsteiner and Friend, 1954), and halothane anesthesia (Moudgil, 1973).
  4. Resistance (to MHV) in mice is increased by giving glucan, a macrophage stimulant (Williams and DiLuzzio, 1980); concanavalin A, a mitogen (Weiser and Bang, 1977); triolein (Lavelle and Starr, 1969); Corynebacterium parvum (Schindler et al., 1981); and silica (Schindler et al., 1984).

MHV is notorious as a contaminant of transplantable tumors (Braunsteiner and Friend, 1954; Nelson, 1959; Manaker et al., 1961; Collins and Parker, 1972; Fox et al., 1977a) and cell lines (Sabesin, 1972; Stohlman and Weiner, 1978; Yoshikura and Taguchi, 1979), including hybridomas (Holmes, 1986a).

Mouse Rotavirus
Significance

Low.

Perspective

There are three main phases in the work concerned with mouse rotavirus (MRV) and the disease it causes:

1947-1956: Discovery of MRV infection and other early work of Cheever, Papenheimer, and colleagues at Harvard (Cheever and Mueller, 1947, 1948; Papenheimer and Enders, 1947; Papenheimer and Cheever, 1948; Cheever, 1956). The disease was called epidemic diarrheal disease of suckling mice. The infectious nature of the disease, its clinical manifestations, and influence of parity of dams on survival of young were emphasized.

1957-1967: Work of Kraft and associates (Kraft, 1957, 1958, 1961, 1962a, 1966; Adams and Kraft, 1963, 1967; Biggers et al., 1964; Kraft et al., 1964). The disease name was changed to epizootic diarrhea of infant mice (EDIM), and the causative agent was shown to be a virus. The clinical

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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syndrome was described and contrasted with a second diarrheal syndrome caused by lethal intestinal virus of infant mice (LIVIM). Filter-top cages were introduced for control of the two diarrheal syndromes.

1968-1988: Period of improving diagnostic methodology for distinguishing MRV and mouse hepatitis virus (MHV) infections, and recognizing the relatively low importance of MRV as a natural infection of mice. LIVIM was shown to be MHV (Broderson et al., 1976; Carthew, 1977a). No natural epizootic of clinical disease due to MRV was reported in mice during this period.

Agent

MRV is a double-stranded RNA virus, family Reoviridae, genus Rotavirus, group A. Although MRV shares a common antigen(s) with the group A rotaviruses, it is serotypically distinct from them. Also, serotypic and genomic differences have been found among isolates of MRV (Greenberg et al., 1986). Spherical virus particles 65-80 nm in diameter occur in the cytoplasm of the intestinal epithelium, and tubular structures about 60 nm in diameter occur in both the cytoplasm and the nucleus. It is unstable at -24, 4, and 37°C. It is not resistant to environmental conditions (Banfield et al., 1968; Woode et al., 1976; Flewett and Woode, 1978; Much and Zajac, 1978; Wolf et al., 1981; Kraft, 1982; Greenberg et al., 1983).

MRV has been grown successfully in trypsinized primary monkey kidney cells and in the continuous rhesus monkey kidney cell line MA 104 (Tajima et al., 1984; Greenberg et al., 1986).

Hosts

Mice (Carthew, 1977b; Flewett and Woode, 1978; Kraft, 1982). Other rotaviruses are recognized as important causes of neonatal diarrhea in children, rabbits, piglets, calves, lambs, foals, and many other young animals.

Epizootiology

MRV is generally held to be a widely prevalent, important pathogen of mice, but this has not been documented for contemporary mouse stocks. Carthew (1977b) reported a small survey in England in which complement-fixing antibodies to rotavirus were found in 47% of mouse colonies and 16% of rat colonies.

Transmission is by airborne infection in which contaminated dust and bedding from adjacent cages probably play key roles (Kraft, 1957), hence the reason that filter-top cages are useful. Mice are most susceptible to infection from birth to about 17 days of age. Infected neonates shed high

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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concentrations of virus in the feces from about 2 days to 8-10 days post infection. Transient viremia and viruria can occur. Mice infected after about 17 days of age shed lower concentrations of virus in the feces for 2-4 days. It is not known whether there is persistent infection or whether very low concentrations of virus are shed in the feces beyond these time points. There is no evidence of transplacental transmission (Malherbe, 1978; Kraft, 1982; Little and Shadduck, 1982; Eydelloth et al., 1984; Eiden et al., 1986a; Starkey et al., 1986; Riepenhoff-Talty et al., 1987; Osborne et al., 1988).

Clinical

Diarrhea during the first two weeks of life is considered the only consistent sign of disease. Watery, yellow stools usually begin around 48 hours post infection and persist for about one week. Varying amounts of stool accumulate around the anus and base of the tail, and soil the coats of neonates and dams. Affected neonates may appear lethargic and have distended abdomens. Usually there is no mortality. Mice infected with MRV as adults show no clinical signs (Kraft, 1982; Little and Shadduck, 1982).

Some clinical manifestations, particularly the mortality sometimes attributed to MRV in the earlier literature, do not fit with current knowledge. For example, Cheever and Mueller (1948) observed high infant mortality. Survival of young increased with increasing parity of the dams: approximately one-third of infants survived in first litters, one-half in second and third litters, and three-quarters in fourth and fifth litters, presumably because of increasing maternal antibody in the milk. This high mortality was more likely due to some other intercurrent infection(s), possibly mouse hepatitis virus (Kraft, 1982). The observation that increasing age and parity of dams in infected colonies was associated with protection of young can not be fully explained although it has now been shown that some degree of protection against infection (not disease) is afforded infected neonates suckled by dams with pre-existing antibody to MRV (Runner and Palm, 1953; Little and Shadduck, 1982).

Pathology

Susceptibility to infection and disease (diarrhea) due to MRV is age dependent, and occurs from birth to about 17 days of age with the peak period of susceptibility being from four days to 14 days of age (Wolf et al., 1981; Eydelloth et al., 1984). During this time enterocytes in the small intestine are particularly susceptible to infection and support maximal cytoplasmic replication of the virus, possibly due in part to the high pinocytotic activity of enterocytes (Clark, 1959; Wolf et al., 1981) or availability of viral receptors on enterocytes (Reipenhoff-Talty et al., 1982).

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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The occurrence of diarrhea and histopathologic changes in the small intestine parallel virus concentrations in the epithelium. In animals older than about 18 days of age, small numbers of enterocytes become infected, there is little replication of virus and diarrhea does not occur (Little and Shadduck, 1982; Eydelloth et al., 1984; Starkey et al., 1986; Osborne et al., 1988).

Infection progresses from proximal to distal parts of the intestine, involving sequentially the duodenum, jejunum, ileum, and colon. Virus infects primarily enterocytes in the apical one-fourth to one-third of intestinal villi, causing degeneration and sloughing of these cells into the lumen. Thus, the major pathologic change is mild villous atrophy. Since the crypt epithelium is spared, regeneration occurs rapidly. Grossly, the intestines usually contain watery fluid and gas bubbles during the period of clinical diarrhea (Moon, 1978: Little and Shadduck, 1982).

Although the precise mechanisms are unknown, both passively and actively acquired humoral immunity are thought to be important in host defense. Infant mice that nurse rotavirus seropositive dams have partial resistance to experimental MRV infection for three to four days after birth, presumably due to specific IgG and/or IgA in colostrum (Little and Shadduck, 1982; Sheridan et al., 1983). Also, diarrhea and shedding of virus in the feces occur 2 or 3 days longer in infant mice nursing seronegative dams (Little and Shadduck, 1982). In neonates nursed on seronegative dams antirotavirus IgA appears in the intestine by seven days post infection and specific IgG appears around 14 days post infection (Sheridan et al., 1983). Neonatal athymic (nu/nu) mice experimentally infected with MRV experience a self-limiting infection identical to that seen in age-matched immunocompetent mice (Eiden et al., 1986a). In contrast, severe combined immunodeficient (scid/scid) mice have higher percentages of enterocytes infected, achieve greater concentrations of virus in intestinal epithelium, shed higher concentrations of virus for longer periods of time in the feces, and remain persistently infected (Riepenhoff-Talty et al., 1987a,b). Vacuolation (lipid droplets) in the epithelium of the small intestine has been emphasized as a response to MRV (Adams and Kraft, 1967). In MRV infection numerous cytoplasmic vacuoles of varying size and distribution occur in the mucosal epithelium, near the tips of villi (Starkey et al., 1986; Osborne et al., 1988), a change that should not be confused with the orderly distension of the cytoplasm by very large vacuoles seen in the intestinal epithelium of entire villi in normal neonatal animals (Moon, 1972), including mice (T. R. Schoeb and J. R. Lindsey, Department of Comparative Medicine, University of Alabama at Birmingham, unpublished). Papenheimer and Enders (1947) described intranuclear inclusions in the intestinal epithelium of infected mice that are compatible with inclusions now recognized as being due to mouse adenovirus-2 (Van der Veen and Mes, 1974; Luethans and Wagner, 1983; Hamelin et al., 1988).

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Diagnosis

Detection of anti-rotaviral antibodies can be accomplished by complement fixation (CF) (Blackwell, 1966; Wilsnack et al., 1969; Carthew, 1977b), radioimmunoassay (Wolf et al., 1981), immunofluorescent antibody (IFA) test (Smith et al., 1983), enzyme-linked immunosorbent assay (ELISA) (Ghose et al., 1978; Sheridan et al., 1983; Smith et al., 1983), and numerous other methods (Kraft, 1982; Ferner et al., 1987). A homologous enzyme immunoassay inhibition method utilizing MRV-derived reagents has been considered the most efficient (Ferner et al., 1987) The ELISA and IFA are available commercially.

Definitive diagnosis of diarrheal disease due to MRV can be made by demonstration of lesions compatible with MRV infection in the small intestine, demonstration of MRV antigen in the intestine or feces, and isolation of the virus by using trypsinized primary monkey kidney cells (Tajima et al., 1984; Greenberg et al., 1986). Radioimmunoassay (Cukor et al., 1978; Wolf et al., 1981), immunofluorescence (Wilsnack et al., 1969), electron microscopy (Wolf et al., 1981), and polyacrylamide gel electrophoresis of the viral genome (Smith et al., 1983) have been used to demonstrate rotaviruses in the intestinal tract. A commercially available ELISA (Rotazyme II, Abbott Laboratories, North Chicago, III.) for this purpose has been found to give a high proportion of false positives in testing fecal specimens from mice, but the problem could be eliminated by pretreatment of test beads with 0.1% bovine serum albumin (Jure et al., 1987).

Control

Cesarean derivation followed by barrier maintenance has traditionally been recommended for rederivation of breeding stocks, but it is not known whether this approach is necessary to eliminate MRV. It probably would be necessary for scid/scid mice that are known to become persistently infected and to shed the virus in feces for many months, perhaps for life (Riepenhoff-Talty et al., 1987a,b). For immunocompetent and athymic (nu/nu) mice, it may be that virus is shed for only a few weeks after the acute infection. If so, this would permit the isolation and quarantine of individual breeding pairs with subsequent selection of MRV seronegative progeny for breeding, as has been achieved with certain other agents (Lipman et al., 1987; Weir et al., 1987).

Use of filter-top cage systems can be beneficial in controlling transmission of infection between subpopulations in the same room. To be most effective these systems require that rooms be closed to the entry of outside animals, that filter covers be removed from one cage at a time (and only while the cage is inside a transfer cabinet within the room), and that measures be taken to avoid cross-contamination between cages during handling

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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of mice (Kraft, 1958; Jennings and Rumpf, 1965; Schneider and Collins, 1966; Poiley, 1967).

Vaccination may be useful in the control of MRV in some instances. Neonatal mice born to dams that have been vaccinated with purified empty capsids of simian rotavirus SA-11 have been found to be protected from diarrheal disease when challenged with MRV (Sheridan et al., 1984).

Interference with Research

Infection can alter results of studies with infant mice. Protein-calorie deprivation of nursing dams has been found to result in increased severity of diarrhea, increased mortality, and reduced weight gains of their infant pups infected with MRV (Noble et al., 1983; Offor et al., 1985). Folic acid deficiency also has been reported to increase the severity of diarrheal disease due to MRV (Morrey et al., 1984). Infant mice with MRV infection have increased mortality when challenged with enterotoxigenic Escherichia coli (Newsome and Coney, 1985). MRV infection alters intestinal absorption (Ijaz et al., 1987) and intestinal enzyme profiles (Collins et al., 1988).

Rat Rotavirus-Like Agent
Significance

Uncertain.

Perspective

Rat rotavirus-like agent (RVLA) was recently discovered as the cause of a spontaneous outbreak of diarrhea in suckling rats in Baltimore, Maryland (Vonderfecht et al., 1984). A similar, if not identical, virus was subsequently found to be a prevalent infection associated with diarrhea in human adults and children in that city (Eiden et al., 1985).

Agent

RVLA is a double-stranded RNA virus, family Reoviridae, genus Rotavirus, group B (tentative classification). Virions are spherical, 65-80 nm in diameter, and composed of capsomeres with cubic symmetry. It is antigenically and genetically related to group B rotaviruses from pigs and calves with diarrhea (Eiden et al., 1986b) and Chinese group B rotavirus from diarrheic human patients (Hung et al., 1984). RVLA resists treatment with ether or acidification to pH 5 but is inactivated by pH 3 or heating at 56°C for 30 minutes. It has not been grown in cell cultures (Vonderfecht et al., 1984, 1985).

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Hosts

Rats and man (Vonderfecht et al., 1984; Eiden et al., 1985).

Epizootiology

Unknown. During the diarrheal phase of the infection, the feces contain a high titer of virus, and fecal-oral transmission is undoubtedly most important. Presumably, transmission between rat cages involves airborne infection as in mouse rotavirus infection (Kraft, 1982). Personnel may be the major source of infection for rat colonies (Eiden et al., 1985; Vonderfecht et al., 1985).

Clinical

The disease syndrome in rats has been called infectious diarrhea of infant rats (IDIR). Diarrhea occurs in suckling rats 1-11 days old. The feces of affected rats consist of poorly formed pellets, liquid, and gas. Diarrhea persists for 5-6 days, during which there is erythema, cracking, and bleeding of the perianal skin. Growth retardation and drying and flaking of the perianal skin are apparent for several more days. There is no mortality (Vonderfecht et al., 1984).

Pathology

At necropsy affected suckling rats always have stomachs filled with milk. The contents of the proximal small intestine are watery and tan to green in color. The distal small intestine and colon contain fluid, poorly formed pellets, gas, and mucinous material (Vonderfecht et al., 1984).

In histologic sections lesions are restricted to the small intestine and consist of villous epithelial necrosis, formation of epithelial syncytial cells, and villous atrophy. Epithelial necrosis involves the luminal one-third of the villi. Syncytial cells are found on the villi but never in the crypts. The cytoplasm of the syncytial cells often contain an abundance of 1- to 2-µm eosinophilic inclusions. By electron microscopy, the cytoplasm of epithelial syncytial cells contains large numbers of 80-nm viral particles associated with reticular and amorphous aggregates of electron-dense material (Vonderfecht et al., 1984).

Diagnosis

The specialized methods for definitive diagnosis of RVLA are not available in most rodent diagnostic laboratories. The preferred methods presently are the indirect enzyme immunoassays (Vonderfecht et al., 1984) and the enzyme

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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immunoassay inhibition test (Vonderfecht et al., 1985) for detection of the virus in intestinal and fecal specimens. Although the enzyme immunoassay inhibition test has been found to be excellent for this purpose, its usefulness may be somewhat limited in diagnosing RVLA infection as fecal shedding of the virus occurs for only a few days after infection and the timing of sample collection is of critical importance (Vonderfecht et al., 1988). Light and electron microscopic methods provide important supporting data for diagnosis (Vonderfecht et al., 1984).

Control

Uncertain. Infected personnel may be an important source of infection.

Interference with Research

RVLA could interfere with studies involving the intestinal tract, but no examples have been reported. Presumably, infected rats can serve as a source of infection for personnel.

Reovirus-3
Significance

Natural infections due to this virus have little significance for most studies with mice and rats but can interfere with studies involving transplantable tumors and in vitro test systems that use cells from these animals. The chief importance of reovirus-3 is that experimental infections with this agent, particularly in mice, provide a large number of models of human disease and test systems for studying the molecular biology of the virus.

Perspective

The literature on reovirus-3 infections in rodents is so dominated by studies of mice experimentally infected with the virus that experimental and natural infections are best considered separately.

Experimental Infections and Models

1953: Stanley et al. (1953) carried out animal inoculations using a virus isolated from the feces of a child in Australia. Suckling mice inoculated with the virus developed hepatitis, encephalitis, diarrhea, oily coats, alopecia, and jaundice; and the agent was called the hepato-encephalomyelitis virus or HEV. It was later identified as reovirus-3 (Stanley, 1961).

1954-1986: During this period further studies of experimental infections

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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in mice were conducted by Stanley and colleagues (Stanley et al., 1954, 1964). Subsequently, experimental infections of reovirus-3 were proposed as models of many diseases in humans, including acute and chronic hepatitis, chronic biliary obstruction, pancreatitis, and lymphoma (reviewed by Stanley, 1974). Also, experimental infection models using mice were used extensively in studies relating the molecular biology of reovirus-1 and -3 to viral pathogenesis (reviewed by Sharpe and Fields, 1985).

Natural Infections and Disease

1960: Bennette (1960) and Nelson and Tarnowski (1960) reported that a viral contaminant was oncolytic for their transplantable ascites tumors in mice; the causative agent was later identified as reovirus-3 (Hartley et al.,

1961; Bennette et al., 1967). Nelson and Tarnowski (1960) also observed that some of the uninoculated suckling mice in their colony had "oily coats and yellow foci in the liver," and concluded that this was probably a natural occurrence of the disease produced earlier (Stanley et al., 1953) by the inoculation of reovirus-3 into suckling mice.

1961: Hartley et al. (1961) isolated reovirus-3 from two populations of laboratory mice and five of seven mouse leukemia cell lines.

1963: In Australia, Cook (1963) described two epizootics in suckling mice characterized by runting, diarrhea, oily coats, alopecia, focal hepatic necrosis, and jaundice. Reovirus-3 was isolated and considered the causative agent, but additional diagnostic procedures were not done.

1964: Nelson (1964) was unable to reproduce the experimental disease syndrome described by Stanley et al. (1953) by inoculating the reovirus-3 isolate of Nelson and Tarnowski (1960) into infant mice. Later, Bennette et al. (1967) reported similar failures using their isolate of the virus (Bennette, 1960).

1972: Collins and Parker (1972) reported reovirus-3 to be a contaminant in 2% of 465 murine leukemia cell lines and transplantable tumors.

Thus, the literature contains only two instances that seem to implicate reovirus-3 as the cause of spontaneous disease in mice (i.e., mice not inoculated with the virus), those reported by Cook (1963) and Nelson and Tarnowski (1960). Also, there are only two reported instances in which reovirus-3 was found to interfere with research involving transplantable tumors in mice (Bennette, 1960; Nelson and Tarnowski, 1960).

Agent

The reoviruses are double-stranded RNA viruses, family Reoviridae, genus Reovirus. Other genera of the Reoviridae are Orbivirus and Rotavirus. The

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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three genera are morphologically similar but differ in structure, antigenicity (i.e., are not immunologically related), stability, and preferred hosts. The reoviruses have a wide host range and are considered ubiquitous in nature. The orbiviruses multiply in insects and include blue tongue virus of ruminants and Colorado tick virus of people. The rotaviruses cause mainly infantile diarrheas of humans and animals (Davis et al., 1980).

The genus Reovirus contains mammalian and avian serotypes. The mammalian reoviruses are divided into serotypes 1, 2, and 3, which are distinguished by the neutralization and hemagglutination tests (Rosen. 1960). The major or type-specific antigen is the sigma 1 outer capsid protein. Serologic types 1, 2, and 3 share a common group antigen and cross-react in the complement fixation (CF) test. Types 1 and 2 agglutinate human erythrocytes, and type 3 agglutinates bovine erythrocytes. There are five serotypes of avian reoviruses; they are not serologically related to the mammalian reoviruses (Jackson and Muldoon, 1973; Ramig and Fields, 1977; Matthews, 1982; Sharpe and Fields, 1985).

Reovirus virions are spherical particles 75-80 nm in diameter, consisting of an outer and an inner capsid, or core, measuring about 52 nm in diameter. There is no lipoprotein envelope. Reoviruses are highly resistant to lipid solvents, acid pH, heating (56°C for two hours or 60°C for 30 minutes), and many disinfectants (Stanley et al., 1953; Fenner et al., 1974; Buxton and Fraser, 1977a).

Mammalian reoviruses have been grown in a wide range of cells but most commonly in L cells and primary monkey kidney cells (Kraft, 1982).

Hosts

Mice, rats, hamsters, and guinea pigs (Carthew and Verstraete, 1978; Parker, 1980; Suzuki et al., 1982). Reovirus-2 has been isolated from wild mice (Hartley et al., 1961). Mammalian reoviruses have also been found in many other species because they have a broad host range (Buxton and Fraser, 1977a; Davis et al., 1980).

Epizootiology

Based on recent serologic surveys using either the CF or hemagglutination inhibition (HAI) test, one or more of the reoviruses may be quite prevalent in contemporary rodents. The combined data from Canada, England, Japan, and the United States give the following ranges in percentages of colonies found infected: mice, 8-100%; rats, 6-44%; hamsters, 30-33%; and guinea pigs, 33-77% (Descoteaux et al., 1977; Carthew and Verstraete, 1978; Parker, 1980; Suzuki et al., 1982; Lindsey, 1986). Prevalence values based on the HAI test may have overstated the true prevalences of reovirus-3 infection

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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because of false positive results (Kraft and Meyer, 1986; Van Der Logt, 1986).

Natural infections caused by reoviruses in rodents are assumed to involve mainly the respiratory and gastrointestinal tracts as they do in people. Therefore, transmission probably involves the aerosol as well as the fecal-oral route. Infected fomites may have an important role because reoviruses resist environmental conditions moderately well (Davis et al., 1980).

Clinical

Natural infections of mice and rats due to reoviruses are almost always subclinical.

The following clinical manifestations were observed in the colony studied by Cook (1963). For more than 9 years litters 10-14 days old frequently showed stunting, diarrheic yellow stools, oily coats, abdominal alopecia, and jaundice. Signs usually were confined to first litters of parents mated at 7-8 weeks of age. At least 1 and up to 5 per litter were stunted (i.e., weights of 3 grams instead of the expected 10 grams). Jaundice persisted in some mice to 5 or 6 weeks of age.

Pathology

The mammalian reoviruses are not considered important pathogens because they are commonly isolated from the feces or respiratory tracts of healthy humans and animals. However, on occasion they have been associated with minor respiratory and gastrointestinal illnesses (Davis et al., 1980).

In the natural outbreak studied by Cook (1963), stunted mice had enlarged black protruding gall bladders, yellow necrotic areas in the livers, and yellow kidneys. Unfortunately, no efforts were made to detect other infectious agents, and no histopathologic studies were done.

Nelson (1964) and Nelson and Tarnowski (1960) described a few gross lesions associated with the transplantation of reovirus-3-contaminated ascites tumors. On the seventh day after intraperitoneal inoculation of infant or weanling mice with uninfected tumors, copious amounts of ascitic fluid and large numbers of tumor cells were present. In contrast, after inoculation of reovirus-3-contaminated tumors the ascitic fluid was slightly turbid and contained many inflammatory cells but reduced numbers of tumor cells. Fibrinous exudate covered the liver, and chalky foci of necrosis were present in peritoneal fat deposits. A few animals had multifocal hepatic necrosis and jaundice.

Numerous morphologic lesions have been observed in mice inoculated experimentally with reovirus-3, including emaciation, stunting, encephalitis, pneumonia, hepatitis, cholangitis, pancreatitis, adrenalitis, myocarditis,

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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ascites, and many others (Stanley et al., 1953, 1954, 1964; Walters et al., 1963, 1973; Jenson et al., 1965; Papadimitriou, 1966, 1968; Papadimitriou and Walters, 1967; Stanley and Joske, 1975a,b; Papadimitriou and Robertson, 1976: Ondera et al., 1978; Bangaru et al., 1980).

A large research effort during the past decade has focused on relating the molecular components of reovirus, particularly types 1 and 3, to pathogenesis in mice. Among the major findings has been the fact that each of the three outer capsid proteins has a distinct role in pathogenesis. The sigma 1 protein is the determinant of cell and tissue tropism, and both humoral and cellular immune responses to the reoviruses. Protein mu 1C determines viral sensitivity to intestinal proteases. Sigma 3 protein is an inhibitor of host cell RNA and protein synthesis and participates in mutations related to lytic versus persistent infections by the virus. The mechanisms of protective immunity are not well understood; however, it is known that athymic (nu/ nu) mice resist reovirus infections as well as immunocompetent mice (Sharpe and Fields, 1984, 1985).

Diagnosis

The CF and HAI tests for antibodies to reovirus-3 have been the standards for routine health monitoring for many years, but the more sensitive enzymelinked immunosorbent assay (ELISA) is now used by most laboratories (London et al., 1983; Parker, 1983). The CF test is not reovirus type specific, as is probably true of most ELISAs currently in use. Also, the HAI test is prone to give false positive results (Kraft and Meyer, 1986; Van Der Logt, 1986).

Virus isolations can be performed with L cells or embryonic kidney cells (Kraft, 1982). Transplantable tumors and cell lines can be screened for reoviruses by using tissue culture methods or the mouse antibody production test (Rowe et al., 1962).

Control

Cesarean derivation and barrier maintenance have proven effective. However, the common occurrence of reoviruses in man suggests that personnel may be a source of contamination for reovirus-free rodent stocks.

Interference with Research

There are relatively few published examples in which natural infections of reovirus in mice and rats were found to interfere with research, i.e., only one alleged clinical outbreak in mice that was incompletely studied and of totally unknown pathogen status (Cook, 1963), and two instances of

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

interference with transplantable ascites tumor studies in mice (Bennette, 1960; Nelson and Tarnowski, 1960). More recently, reovirus-3 has been reported to be an occasional contaminant of transplantable tumors and cell lines (Collins and Parker, 1972).

Experimental infections of reovirus-3 have been reported to result in the following altered biological responses:

  1. Reduced pulmonary clearance of Staphylococcus aureus (Klein et al., 1969).
  2. Suppression of pulmonary carcinogenesis due to urethan (Theiss et al., 1978).
  3. Enhancement of tumor-specific immunity (Kollmorgen et al., 1976: Sansing et al., 1977).
Adenoviruses
Significance

Low.

Perspective

Two distinct strains of so-called "mouse" adenovirus have been recognized in mice. The FL strain (MAd-1) )was first isolated by Hartley and Rowe (1960) in the United States during attempts to establish the Friend leukemia virus in tissue culture. The K87 strain (MAd-2) was first isolated in Japan by Hashimoto et al. (1966) while searching for cytopathic viruses in the feces of healthy mice. Neither agent appears to be an important pathogen in contemporary rodents.

Agent

These agents are DNA viruses, family Adenoviridae, genus Mastadenovirus. Adenovirus virions measure 70-90 nm in diameter and have icosahedral symmetry. There is no envelope surrounding the nucleocapsid. More than 80 different adenovirus species have been isolated from mammalian hosts. Adenoviruses generally have strong host specificity (Wigand et al., 1977; Matthews, 1982; Otten and Tennant, 1982).

Determination of the species (formerly, type designation) of adenoviruses traditionally has been based on immunological distinctiveness as determined by quantitative neutralization with animal antisera (Wigand and Adrian, 1986). Conflicting reports have been published on the antigenic relationship between the FL and K87 strains (Van der Veen and Mes 1974; Wigand et al., 1977; Smith et al., 1986; Wigand and Adrian, 1986; Lussier et al., 1987a). Nevertheless, there is general agreement that the two agents represent

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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distinct species; the FL strain is designated mouse adenovirus-1 (MAd-1) and the K87 strain as mouse adenovirus-2 (MAd-2), as originally proposed by Van der Veen and Mes (1974). More recently, restriction endonuclease cleavage studies of DNA from the two strains have confirmed their distinctiveness and separation into two species (Hamelin and Lussier, 1988; Hamelin et al., 1988).

Adenoviruses are resistant to ether and acid pH. Stability is retained for over 2 months at 4°C, 2 weeks at room temperature, and 1 week at 37°C, but the viruses are inactivated by 50°C for 15 minutes (Buxton and Fraser, 1977b).

Hosts

Mice and rats. Inclusion of the rat as a host is based on serologic (Jacoby et al., 1979; Parker, 1980; Suzuki et al., 1982) and morphologic (Ward and Young, 1976) evidence, but rats have been reported to be refractory to experimental infection with either MAd-1 or MAd-2 (Smith and Barthold, 1987).

Epizootiology

The prevalence of these viruses in contemporary mouse and rat stocks is not well understood. Recent data obtained by using the time-honored complement fixation (CF) test for antibody to the MAd-1 (alone) suggest that the prevalence of this agent in mice is quite low. Using this test, Parker (1980) found all of 21 mouse colonies to be negative in the United States, and Suzuki et al. (1982) reported that all of 196 mouse colonies in Japan were negative. For rats, Parker (1980) reported 36% of 25 colonies in the United States to be positive, and Suzuki et al. (1982) found 8% of colonies in Japan to be positive. More recently, Smith et al. (1986), using an immunofluorescent antibody (IFA) test for both MAd-1 and MAd-2, reported serological evidence of MAd-2 in mice from 1 of 6 commercial sources, MAd-1 in rats from 2 of 6 sources, and MAd-2 in rats from 3 of 6 sources surveyed in the United States.

Few surveys have been done using a serological test known to be reliable for detecting antibodies to MAd-2. However, that agent may be more prevalent than presently suspected as intestinal inclusions compatible with MAd-2 infection have been observed infrequently in mice in The Netherlands (Cohen and de Groot, 1976) and the United States (Takeuchi and Hashimoto, 1976; Luethans and Wagner, 1983), and in rats in the United States (Ward and Young, 1976).

MAd-1 is said to be shed in the urine. Viruria has been reported to persist for periods ranging from 2 weeks to as long as 2 years post infection (Van der Veen and Mes, 1973).

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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MAd-2 infects the intestinal tract and is shed for only about 3 weeks post infection in immunocompetent mice (Hashimoto et al., 1970). Athymic (nu/nu) mice shed the virus for about 6 weeks post infection and thereafter intermittently for at least 6 months (Umehara et al., 1984). Thus, fecal-oral transmission is most important.

Clinical

Neither MAd-1 nor MAd-2 is known to cause clinical disease in naturally infected immunocompetent rodents. MAd-1 (Winters and Brown, 1980) and MAd-2 (Cohen and deGroot, 1976) have been associated with wasting in athymic (nu/nu) mice.

Pathology

All reports of pathology caused by MAd-1 have been the result of experimental infections. Intraperitoneal, intracranial, or intranasal inoculation can produce a systemic fatal disease in suckling mice born to mothers without antibody (Hartley and Rowe, 1960; Heck et al., 1972; Wigand, 1980). In 28-day-old mice inoculated intraperitoneally, most animals showed no clinical disease, although some became ill and 10 of 250 animals died (Van der Veen and Mes, 1973). MAd-1 has tropisms for adrenal gland, myocardium, endocardium, heart valves, brown fat, kidney, salivary gland, and brain. Thus, experimental infections of mice with this virus have been proposed as models of human diseases such as Addison's disease (Margolis et al., 1974) and nonrheumatic endocarditis (Blailock et al., 1967, 1968). Intranuclear inclusions are frequently observed in infected tissues (Heck et al., 1972; Hoenig et al., 1974; Margolis et al., 1974).

The proclivity of MAd-1 to produce systemic infection contrasts sharply with the strict intestinal tropism of MAd-2. Inoculation of MAd-2 into mice, including neonates and athymic (nu/nu) mice, by any route results in subclinical infection during which virus replication occurs exclusively in the intestine (Sugiyama et al., 1967; Umehara et al., 1984). Viral antigen appears in the feces of immunocompetent mice about three days post infection and peaks between seven and 14 days post infection. At that time large numbers of amphophilic, intranuclear inclusions can be found in the mucosal epithelium of both the small and large intestine, but particularly in the small intestine, involving both crypt and villous cells. Little inflammatory response occurs. Ultrastructurally, the inclusions contain densely packed crystalline arrays of angular-shaped, homogeneous virions, each measuring about 85 nm (Takeuchi and Hashimoto, 1976). Host resistance has been attributed to locally produced neutralizing antibody of the IgA class (Hashimoto and Umehara, 1977). Cyclophosphamide administration delays the onset of resistance (Hashimoto et al., 1973).

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Diagnosis

The CF test for detection of antibodies to only MAd-1 was the standard test used in health surveillance testing until very recently. With the recognition of two distinct adenovirus species in mice and rats, it is now standard practice to test sera against antigens of both MAd-1 and MAd-2 (Smith et al., 1986; Lussier et al., 1987a). The prefered methods are IFA (Smith et al., 1986; Lussier et al., 1987a) and enzyme-linked immunosorbent assay. The IFA has been shown to give antibody titers to MAd that are approximately 10-fold higher than those determined by CF test (Lussier et al., 1987a).

No histologic lesions have been reported for natural infections of MAd-1 in mice and rats. Presumptive diagnosis of MAd-2 infection can be made by finding the characteristic intranuclear inclusions in histologic sections of intestinal epithelium (Cohen and de Groot, 1976; Ward and Young, 1976; Luethans and Wagner, 1983). A fluorescent antibody method has been used for the detection of MAd-2 antigen in the intestine (Takeuchi and Hashimoto, 1976).

Definitive diagnosis of mouse adenovirus infections requires virus isolation. MAd-1 can be grown in primary mouse kidney cells, L 929 murine fibroblasts, and CMT-93 murine rectal carcinoma cells (Wigand et al., 1977; Otten and Tennant, 1982; Smith et al., 1986). MAd-2 grows in mouse kidney and CMT-93 cells (Hashimoto et al., 1966; Smith et al., 1986).

Control

Definitive information is lacking. Cesarean derivation and barrier maintenance apparently have been very effective in eliminating these agents from infected stocks in the past.

Interference with Research

MAd-1 has been reported to produce extensive persistent lesions in the kidneys of adult mice and to render them more susceptible to experimental Escherichia coli-induced pyelonephritis (Ginder, 1964). MAd (strain not given) infection has been reported to accelerate experimental scrapie in mice (Ehresmann and Hogan, 1986).

Bacillus piliformis
Significance

Unknown. Outbreaks of disease caused by this agent have occurred occasionally, but the actual prevalence of the infection in contemporary mice and rats remains unknown.

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Perspective

Clinical disease caused by B. piliformis, commonly called Tyzzer's disease, was first recognized by Tyzzer (1917) at Harvard in a colony of Mus bactrianus. Since then several outbreaks of the disease in laboratory mice and rats have been reported, and a few occurrences of the disease have been seen in other species. Unfortunately, little progress has been made in understanding the natural history and prevalence of the infection (Ganaway et al., 1971; Weisbroth, 1979; Ganaway, 1982).

Agent

B. piliformis is an unclassified bacterium. It occurs in vegetative and spore forms. The vegetative form is a Gram negative, motile, pleomorphic. slender rod measuring 0.5 x 8-10 µm. Also, it is silver positive, weakly periodic acid-Schiff positive, and forms subterminal spores (Ganaway, 1980).

The vegetative form is an obligate intracellular parasite. It loses infectivity in host tissues quickly after the host's death and is very unstable to environmental conditions. The spores survive at room temperature for a year or longer (Allen et al., 1965; Craigie, 1966a) and at 60°C for 30 minutes (Ganaway, 1980). Spores are inactivated by 70°C for 30 minutes, 80°C for 15 minutes, and treatment with 0.3% sodium hypochlorite or 2% peracetic acid (with 0.025% sodium alkylarylsulfonate as the wetting agent) for 5 minutes. Solutions of formaldehyde, iodophor, benzalkonium chloride, ethanol, or phenolic disinfectant have little or no sporicidal effect (Ganaway, 1980).

B. piliformis is generally considered not cultivable in cell-free media, although there have been reports to the contrary (Kanazawa and Imai, 1959; Simon, 1977). It can be cultivated in the yolk sac of embryonating hen's eggs (Craigie, 1966a; Ganaway et al., 1971; Fries, 1977b) and in primary cultures of hepatocytes (Kawamura et al., 1983; Thunert, 1984).

A number of methods have been found useful for the laboratory maintenance of B. piliformis, including the following:

  1. infected tissues such as liver from a diseased animal can be harvested shortly after the animal's death and frozen at -70°C;
  2. the organism can be passaged in the yolk sac of 6- to 9-day-old embryonated hen's eggs and harvested at about the time of embryo death, and the yolk sac suspensions can be frozen at -70°C;
  3. suspensions of infected tissue can be passaged in immunocompetent mice given a concurrent dose of 100-200 mg/kg of cortisone acetate; and
  4. suspensions of infected tissue can be passaged in immunodeficient CBA/N-xid or C3.CBA/N-xid homozygous female or hemizygous male mice, with or without concurrent administration of cortisone (Waggie et al., 1981).
Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Hosts

Mice, rats, gerbils, hamsters, guinea pigs, rabbits, cats, dogs, nonhuman primates, horses, and others (Ganaway et al., 1971; Weisbroth, 1979).

Epizootiology

Tyzzer's disease has been reported in Europe, North America, and Asia (Ganaway et al., 1971), suggesting that B. piliformis has a worldwide distribution. Most of the outbreaks in laboratory mice and rats have occurred in conventional colonies, but a few have been seen in cesarean-derived, barrier-maintained colonies (Mullink, 1968; Hunter, 1971; Tsuchitani et al., 1983; Thunert et al., 1985; Gibson et al., 1987).

Surveys based on serologic tests suggest that subclinical infection is common even in cesarean-derived, barrier-maintained rodents. Fujiwara (1980) reported that 5% of 80 mouse colonies and 47% of 83 rat colonies in Japan were seropositive by the complement fixation (CF) test for B. piliformis. Using an indirect immunofluorescent antibody (IFA) test for serum antibody, Fries (1980) found seven of eight mouse colonies and all of six rat colonies in Europe, representing both conventional and specific-pathogen-free colonies, to be serologically positive. Both of these investigators have confirmed the presence of B. piliformis in representative seropositive mouse and rat colonies by use of cortisone administration to provoke the expression of Tyzzer's disease or by other methods (Fujiwara, 1967; Fries, 1977a, 1979; Fries and Svendsen, 1978; Fujiwara et al., 1981).

The spores are passed in the feces and retain infectivity at room temperature for 1 year or more (Allen et al., 1965; Craigie, 1966a). Natural infection is thought to be by ingestion of spore-contaminated food or bedding. Thus, a high concentration of spores in the animal environment, due to poor sanitation practices and crowding of animals, provides an ideal setting for the occurrence of clinical disease (Ganaway et al., 1971).

Very little is known about the epizootiology and natural history of B. piliformis in nature. However, the occurrence of Tyzzer's disease in wild animals (e.g., muskrats in Iowa and Wisconsin and cottontail rabbits in Maryland) and domestic animals (e.g., foals in at least seven states in the United States, as well as in Canada and Europe) from numerous geographic locations strongly suggests that the organism is widely distributed in nature (Ganaway et al., 1976; Turk et al., 1981). Thus, it is possible that laboratory animal diets could contain spores of B. piliformis when made from ingredients such as grains and alfalfa hay that have been contaminated by the feces of infected rodents or other animals (Ganaway et al., 1976). If so, inadequate sterilization of spore-contaminated diet or bedding may be a major cause of B. piliformis infection in cesarean-derived, barrier-maintained rodents.

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Clinical

Based on the results of serologic surveys, subclinical infection is probably far more common than clinical disease (Fujiwara, 1967, 1980; Fries and Svendsen, 1978; Fries, 1979, 1980; Fujiwara et al., 1979, 1981).

Numerous environmental and host factors have been recognized as important contributors to the expression of disease caused by B. piliformis, including poor sanitation, overcrowding, transportation stress, food deprivation, dietary modifications, and altered host immune status (Tuffery, 1956; Takagaki et al., 1966; Fujiwara et al., 1973; Weisbroth, 1979; Ganaway, 1982).

Clinical disease occurs most frequently in sucklings and weanlings, but animals of any age can be affected. Unexpected deaths, watery diarrhea, pasting of feces around the perineum, ruffled fur, and inactivity are the most common signs. Morbidity and mortality can vary from low to high (Tyzzer, 1917; Gard, 1944; Rights et al., 1947; Mullink, 1968; Jonas et al., 1970; Stedham and Bucci, 1970; Tsuchitani et al., 1983; Thunert et al., 1985; Gibson et al., 1987).

Pathology

There are three main phases in the evolution of Tyzzer's disease: the establishment of primary infection in the ileum and cecum, the ascension of organisms by the portal vein to the liver, and bacteremic spread to other tissues, most notably the myocardium. The organism preferentially replicates in intestinal epithelium, intestinal smooth muscle, hepatocytes, and myocardium; but the degree of replication (and lesions) in the three major organs involved varies considerably from case to case and between species. Intestinal lesions are usually more severe in rats (and in gerbils, hamsters, and rabbits) than in mice. Myocardial lesions occur inconsistently in all species except for, possibly, the gerbil, which is considered the most susceptible of the common laboratory animal species (Allen et al., 1965; Takagaki and Fujiwara, 1968; Ganaway, 1971; Fujiwara et al., 1973; Weisbroth, 1979; Tsuchitani et al., 1983; Waggie et al., 1984).

Gross lesions range from none to severe involvement of the intestine, liver, and/or heart. In mice the most consistent finding is multiple pale to yellow foci in the liver. Infrequently, the ileum and cecum may appear thickened, edematous, and hyperemic; and the myocardium may contain circumscribed pale gray areas. Lesions in rats are similar, except that the ileum often appears dilated, atonic, and edematous (megaloileitis). The mesenteric lymph nodes usually are enlarged (Yamada et al., 1969; Jonas et al., 1970; Weisbroth, 1979; Ganaway et al., 1982; Tsuchitani et al., 1983).

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Microscopically, intestinal lesions are found in the ileum, cecum, and sometimes the proximal colon. There is mild to severe loss of the mucosal epithelium, with blunting of villi in the ileum, thinning of the surface epithelium, and even severe ulceration and hemorrhage. In the more advanced stages there is hyperplasia of the crypt epithelium. In areas of severe epithelial loss there is transmural acute to subacute inflammation. In the liver there are multiple discrete foci of coagulative necrosis that are rapidly converted to microabscesses but that may contain varying numbers of macrophages and lymphocytes in the more advanced stages. If the myocardium is affected, there is focal to diffuse myocardial necrosis with intense acute to subacute inflammation. In each of the affected tissues, the characteristic large, filamentous bacilli are best demonstrated in the cytoplasm of viable cells along the margin of the necrotic tissues. Silver stains (e.g., the Warthin-Starry and methenamine silver methods) are preferred (Jonas et al., 1970; Stedham and Bucci, 1970; Ganaway et al., 1971; Weisbroth, 1979; Tsuchitani et al., 1983).

B-cell deficient CBA/N-xid and C3.CBA/N-xid homozygous female and hemizygous male mice are highly susceptible, whereas T-cell deficient athymic (nu/nu) male and female mice are as resistant as immunocompetent mice. Thus, resistance to B. piliformis infection in the mouse appears to be mainly dependent on B-cell function (Waggie et al., 1981). The passive transfer of immune serum to mice protects against experimental infection (Fujiwara et al., 1969). Athymic rats are reported to be highly susceptible (Thunert et al., 1985).

Diagnosis

The diagnosis of Tyzzer's disease is made by necropsy and is based on the finding of characteristic gross and microscopic lesions and the demonstration of the characteristic organisms in silver-stained histologic sections (Allen et al., 1965; Jonas et al., 1970; Stedham and Bucci, 1970; Weisbroth, 1979).

IFA (Fries, 1977a, 1980), CF (Fujiwara, 1967, 1980) and enzyme-linked immunosorbent assay (Toriumi et al., 1986) tests have been used for the diagnosis of subclinical infections, but none of these tests is currently available commercially in the United States. Alternatively, weanling animals can be immunosuppressed by the administration of cortisone acetate (100200 mg/kg) to provoke active disease, and killed 7 days later for demonstration of characteristic lesions and organisms (Kaneko et al., 1960; Fujiwara et al., 1963, 1973; Takagaki et al., 1966). Tissues suspected of containing B. piliformis can be passaged in gerbils or in homozygous female or hemizygous

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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male xid mice (CBA/N or C3.CBA/N), which are killed 5 to 7 days later for the demonstration of lesions and organisms (Waggie et al., 1981, 1984).

Control

The use of cesarean-derivation and barrier-maintenance procedures appears to have minimized the occurrence of clinical disease, but the true prevalence of subclinical B. piliformis infection in contemporary rodent colonies in the United States is unknown. If subclinical infections are found to be widespread, major revisions in current control methods might be required.

A number of practical approaches have been recommended for reducing losses due to Tyzzer's disease in conventional colonies. Good sanitation practices, avoidance of crowding, autoclaving of food and bedding, and the use of 0.3% sodium hypochlorite for disinfecting room surfaces are recommended for reducing spore contamination in the animal environment (Ganaway et al., 1971; Ganaway, 1980). The oral administration of tetracycline can be helpful in controlling losses during outbreaks (Hunter, 1971; Yokoiyama and Fujiwara, 1971).

Interference with Research

Tyzzer's disease has been reported to cause high mortality in breeding colonies of mice (Gard, 1944) and in mice used in long-term carcinogenesis studies (Hunter, 1971). However, it appears that both the provocation of overt disease from subclinical disease and the exacerbation of clinical disease by immunosuppressive treatments have been particularly troublesome.

The administration of cortisone or adrenocorticotropic hormone to animals with subclinical infection has provoked severe clinical disease (Kaneko et al., 1960; Fujiwara et al., 1963, 1973; Takagaki et al., 1966; Yamada et al., 1969).

Whole body x-irradiation has provoked severe clinical disease with high mortality (Tuffery, 1956; Takagaki et al., 1966; Taffs, 1974).

Transplantation of ascites tumors has provoked Tyzzer's disease in recipient mice (Craigie, 1966b).

Tyzzer's disease has been reported to alter the pharmacokinetics of warfarin and trimethoprim (Fries and Ladefoged, 1979), and the activity of hepatic transaminases (Naiki et al., 1965) in mice.

A high-protein diet has exacerbated Tyzzer's disease in mice (Maejima et al., 1965).

Experimental Tyzzer's disease in weanling mice was exacerbated by treatment of the mice with carbon tetrachloride (Takenaka and Fujiwara, 1975).

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Salmonella enteritidis
Significance

Uncertain because the prevalence of subclinical infection is unknown.

Perspective

Salmonella enteritidis serotypes enteritidis and typhimurium ranked among the most important causes of epizootic and enzootic disease in laboratory mice and rats during the first half of the twentieth century (Edwards et al., 1948; Habermann and Williams, 1958; Weisbroth, 1979). More recently, clinical disease caused by these serotypes has virtually disappeared, while subclinical infections caused by weakly pathogenic S. enteritidis of many other serotypes have been recognized as relatively common (Werner, 1957; Wetmore and Hoag, 1960; Margard and Litchfield, 1963; Brennan et al., 1965; Morello et al., 1965; Franklin and Richter, 1968; Kirchner et al., 1982; Steffen and Wagner, 1983).

Agent

Salmonellosis in rodents is caused by a bacterium, family Enterobacteriaceae, tribe Salmonellae, Salmonella enteritidis (with more than 1,500 serotypes, see below). Members of the genus Salmonella are Gram-negative, non-spore-forming, usually motile, facultatively anaerobic, straight rods, measuring 0.7-1.5 x 2.0-5.0 µm. They reduce nitrites, produce gas from glucose, usually produce hydrogen sulfide on triple sugar iron agar, and usually utilize citrate as a sole source of carbon. They are indole and urease negative (LeMinor, 1984; Farmer et al., 1985).

According to the classification system of the Centers for Disease Control (Ewing, 1972; Farmer et al., 1985), all salmonellae belong to three species: Salmonella choleraesuis, Salmonella typhi, and Salmonella enteritidis. S. choleraesuis and S. typhi are distinct serotypes. All other serotypes, of which there are over 1,500, are defined as serotypes of S. enteritidis. Thus, organisms previously designated S. enteritidis are technically "S. enteritidis serotype enteritidis"; S. typhimurium is "S. enteritidis serotype typhimurium;" and so on. However, this system proved to be confusing and cumbersome, and is commonly simplified in laboratory reports so that "S. enteritidis serotype typhimurium" is reported as "Salmonella serotype typhimurium". In scientific articles the designations are often simplified further by artificially treating Salmonella serotypes as if they are species. Thus, "S. enteritidis serotype typhimurium" becomes simply "S. typhimurium" (Farmer et al., 1985). In the following paragraphs S. enteritidis refers exclusively to genus and species, and serotypes are always so designated.

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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S. enteritidis serotypes typhimurium and enteritidis were the serotypes most commonly isolated from laboratory mice and rats during the first half of the twentieth century (Edwards et al., 1948). Serotypes most commonly isolated from these hosts in recent years have included agona, amsterdam, anatum, binza, blockley, bredeney, california, cerro, infantis, kentucky, livingstone, montevideo, oranienburg, poona, senftenberg, tennessee, and others (Werner, 1957; Brennan et al., 1965; Morello et al., 1965; Weisbroth, 1979; Simmons and Simpson, 1980; Simpson and Simmons, 1981; Ganaway, 1982; Kirchner et al., 1982; Lentsch et al., 1983).

Hosts

Mice, rats, humans, and many others (Edwards et al., 1948; LeMinor, 1984).

Epizootiology

The prevalence of S. enteritidis in contemporary stocks of mice and rats is not well known, but there is evidence that subclinical infections due to weakly virulent strains of S. enteritidis may be common in the United States. Kirchner et al. (1982) isolated S. enteritidis serotypes agona, anatum, or oranienburg from the feces of mice at 11 of 22 breeding and research facilities. These isolates were found to be only mildly pathogenic when inoculated into mice. Steffen and Wagner (1983) reported the occurrence of subclinical S. enteritidis serotype amsterdam infection in rats of a large commercial breeding facility. However, Casebolt and Schoeb (1988) recently have reported an outbreak in mice of severe disease with high mortality due to S. enteritidis serotype enteritidis.

Salmonella infections are acquired primarily by ingestion. Contaminated food, water, and bedding are usually considered the major sources of infection for barrier-maintained rodent stocks. The inadvertent use of contaminated animal products in the preparation of rodent diets traditionally has been very troublesome (Hoag et al., 1964). Stott et al. (1975) found 19% of animal diets in the United Kingdom to be contaminated with Salmonella spp., the source being contaminated meat and bone meal. Although the pasteurization procedures currently used for rodent diets in the United States are thought to be highly effective, they may not eliminate all salmonellas. Where sources other than municipal water supplies are used for rodent facilities, rigorous measures must be taken to assure Salmonella-free water by filtration or other methods (Steffen and Wagner, 1983). Only sterilized bedding should be used in order to avoid the use of bedding contaminated by wild rodents or other hosts.

Infected hosts shed large numbers of salmonellas in the feces during

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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early phases of their infections but may subsequently become chronic carriers, shedding organisms continuously or intermittently for many months. Thus, infected rodent and other hosts, including personnel, may be sources of infection for rodents. Such risks are especially great in multipurpose research facilities that house animals of varying pathogen status, including wild or random source animals (Fox and Beaucage, 1979).

Clinical

The majority of S. enteritidis infections in mice and rats are subclinical. Clinical infections are rare in contemporary rodent stocks (Margard et al., 1963; Morello et al., 1965; Weisbroth, 1979). According to Weisbroth (1979), no natural outbreak of clinical disease caused by S. enteritidis has been reported for laboratory rats in the United States since 1939.

Lentsch et al. (1983) have reported an outbreak caused by serotype oranienburg in a breeding colony of mice that had reduced weaning rate as the leading clinical sign. Two outbreaks in which enteritis and deaths of mice up to 3 weeks of age were allegedly due to either serotype montevideo (Simmons and Simpson, 1980) or serotype livingstone (Simpson and Simmons, 1981) must be questioned because diagnostic procedures were not done to rule out other causes or contributing agents such as mouse hepatitis virus infection.

Following ingestion of an infectious dose of the organism, clinical signs may appear after an incubation period of 2-6 days. The signs are nonspecific and may include ruffled fur, hunched posture, reduced activity, weight loss, conjunctivitis, diarrhea, and variable mortality. Diarrhea is inconstant, usually occurring in no more than 20% of animals. Following the acute phase of the infection, 5% or more may become asymptomatic chronic carriers, shedding the organisms in the feces for many months. Enzootically infected breeding colonies may have alternating periods of inapparent infection and overt clinical disease with mortality. Litter sizes and birth weights may be reduced (Ratcliffe, 1949; Habermann and Williams, 1958; Rabstein, 1958; Margard et al., 1963; Jenkin et al., 1964; Morello et al., 1965; Lentsch et al., 1983).

Pathology

Many factors are known to affect the expression of disease caused by S. enteritidis infection, including virulence and dose of the organism, host age and genotype, intestinal microflora, nutritional state, immune status, and intercurrent infections. Weanling mice are more susceptible than adults (Tannock and Smith, 1971). The normal intestinal microflora has an inhibitory effect on the establishment of infection by the oral route. Whereas more than 106 organisms are required to establish infection in 50% of mice

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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with a normal flora, less than 10 organisms are required in axenic mice or mice pretreated with oral antibiotics (Bohnhoff and Miller, 1962; Collins and Carter, 1978). Susceptibility to infection is increased by food and water deprivation (Miller and Bohnhoff, 1962; Maenza et al., 1970; Collins, 1972; Tannock and Smith, 1972), nutritional iron deficiency in rats (Newberne et al., 1968; Baggs and Miller, 1974), nutritional iron overload in mice (Jones et al., 1977), pretreatment with sodium bicarbonate by gavage (Collins, 1972), and administration of morphine sulfate to slow gastrointestinal motility (Miller and Bohnhoff, 1962).

Inbred strains of mice have a wide range of susceptibility to S. enteritidis. Based on the results of subcutaneous, intravenous, or intraperitoneal inoculations of virulent S. enteritidis serotype typhimurium, O'Brien et al. (1980) separated mouse strains into two groups. Relatively resistant strains were those having an LD50 (the dose required to cause the deaths of 50% of the animals) of greater than 1 x 103 organisms, including C3H/HeN, C3H/ St, C3H/Bi, CBA/Ca, BRVR, A/J, A/HeN, SWR/J, and DBA/2. Relatively susceptible strains were those having an LD50 of less than 2 x 101 organisms, including BSVS, DBA/1, BALB/c, C57BL/6, C3H/HeJ, and CBA/N. Three distinct genetic loci that affect susceptibility to virulent strains of serotype typhimurium have been identified as follows:

  1. Ity (immunity to S. typhimurium) locus on chromosome 1. This locus is thought to influence control of initial (less than 10 days post infection) replication of the organism in the spleen and liver, possibly through modulation of uptake and/or killing by macrophages. There are two alleles, Ityr (resistant) in A/J, BRVR, C3H/HeJ, and DBA/2 and Itys (susceptible) in BSVS and C57BL/6 (O'Brien et al., 1980; Briles et al., 1981).
  2. Lps (lipopolysaccharide response) locus on chromosome 4. This locus has to do with response to lipopolysaccharide and also appears to influence control of the initial (less than 10 days post infection) replication of serotype typhimurium in the spleen and liver. The allele designated Lpsn (endotoxin sensitive) confers normal responsiveness and is found in most mouse strains. Lpsd (endotoxin insensitive) is a mutant gene that results in the inability of the host's macrophages and B cells to be stimulated by bacterial endotoxin. It occurs in strains C3H/HeJ and C57BL/10/ScCr. This gene explains the relative susceptibility of C3H/HeJ, although that strain is Ityr (O'Brien et al., 1980; Briles, 1981).
  3. xid (X-linked immune deficiency) locus. The mutant allele xid confers a B lymphocyte functional defect on CBA/N mice, making them susceptible. It controls antibody production and is thought to influence control of late (more than 10 days post infection) replication of salmonellae (O'Brien et al., 1980).
Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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The pathogenesis of natural salmonellosis has been simulated by experimental studies in which S. enteritidis was inoculated intragastrically into mice (Collins, 1972; Carter and Collins, 1974). Following the inoculation of virulent organisms, more than 99% of the original dose is inactivated or voided from the gastrointestinal tract within 48 hours. The cecum and large intestine are exposed to large numbers of organisms for longer time periods than the small intestine, but the mucosa and Peyer's patches in the distal ileum are the primary sites of invasion. Organisms reach the mesenteric lymph nodes that drain this region in 24 hours, and the liver and spleen in 48 hours. Bacteremia occurs by 72 hours. Acute inflammation begins in the ileum and cecum by 24 hours, but the gut becomes heavily infected and develops severe lesions only as a result of the generalized septicemia. The cervical lymph nodes may be infected in some animals by 36 hours following ingestion or intragastric inoculation of organisms (Carter et al., 1975).

Gross lesions are extremely variable, depending on the stage of disease. Animals that die of acute infection may have only hyperemia and congestion of visceral organs, as seen in any septicemia. Animals that survive a week or longer often appear emaciated, have hyperemia and thickening of the ileal and cecal walls, an empty or fluid-filled large intestine, multiple white or yellow foci in the liver, splenomegaly, enlarged mesenteric lymph nodes, and scanty fibrinous exudate in the peritoneal cavity. Chronic carriers usually do not have gross lesions (Buchbinder et al., 1935; Ratcliffe, 1949; Habermann and Williams, 1958; Maenza et al., 1970; Casebolt and Schoeb, 1988).

The predominant microscopic lesions of salmonellosis are ileocecitis, mesenteric lymphadenitis, and multifocal inflammation in the liver and spleen, each varying in character depending on the stage of disease. There is multifocal to diffuse destruction of villous epithelium in the ileum, with blunting of villi and hyperplasia of crypt epithelium and purulent to pyogranulomatous inflammation in the lamina propria. Similar changes occur in the cecum. The cecum is more severely affected in rats. Ulcerative cecitis with severe pyogranulomatous inflammation in the lamina propria and purulent to chronic inflammation leading to atrophy and cyst formation in the paracecal lymph nodes are characteristic of salmonellosis in rats. Multifocal purulent, pyogranulomatous, or granulomatous inflammation occurs in the mesenteric lymph nodes, liver, and spleen. So-called cell fragment thrombi commonly occur in those organs in which necrotic foci break into venous channels. Peritonitis is commonly seen due to extension of the infection through the capsule of the liver, lymph nodes, or spleen. Cholangitis and cholecystitis are seen infrequently. Pyogranulomatous inflammation occasionally occurs in other organs such as the lungs (Buchbinder et al., 1935; Ratcliffe, 1949; Bakken and Vogelsang, 1950; Habermann and Williams, 1958; Bohme et al., 1959; Miller and Bohnhoff,

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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1962; Abrams et al., 1963; Maenza et al., 1970; Carter et al., 1975; Weisbroth, 1979; Casebolt and Schoeb, 1988).

The mechanisms of immunity to S. enteritidis have been studied extensively in mice (as the best animal models of typhoid fever in humans). According to Eisenstein and Sultzer (1983), many important conclusions can be made from this vast literature. There is a wide spectrum (10,000-fold range) of genetically determined susceptibility among different mouse strains to S. enteritidis infection. The genetic diversity (at the Ity, Lps, xid, and probably other loci) among mouse strains accounts for many of the discrepancies in results on protective immunity. In resistant stocks, such as Crl:ICR (CD®-1), humoral immunity alone is protective; but in strains that are highly susceptible, such as C3H/HeJ, antibody alone gives poor protection and cellular immunity is required. Living organisms are required for cellular immunity. Humoral immunity appears to be dependent on O antigens and can be produced by the inoculation of killed organisms. Monoclonal antibodies to O antigens have been found to be protective for C3H/HeN but not C3H/HeJ mice (Colwell et al., 1984).

Diagnosis

The diagnosis of S. enteritidis infection is dependent upon cultural isolation and identification of the organism. Typing of isolates is done by serology. Organs with suspicious lesions should be cultured, but it is usually best to culture liver, spleen, intestine, feces, and blood. Histopathology should be done to demonstrate the characteristic microscopic lesions of salmonellosis and to help rule out those diseases that can have similar gross lesions (e.g., mousepox, Tyzzer's disease, and streptobacillosis in mice) or exacerbate disease caused by S. enteritidis.

The detection of carrier animals is a special problem because few (less than 5%) animals in a population may be infected, and cultural isolation is necessary because there is no satisfactory serologic method for testing suspected carriers. The preferred procedure is to culture recently voided fecal pellets from individual mice or cages of mice in selenite F enrichment broth, followed by the inoculation of selective agar plates (brilliant green, salmonella shigella, or bismuth sulfite), and to test selected colonies on triple sugar iron agar (Margard and Litchfield, 1963; Margard et al., 1963). Sampling is a major problem in the detection of carriers. Assuming a carrier rate of 5% and random sampling of a population, 58 mice must be cultured to achieve 95% confidence of detecting one carrier mouse (Ganaway, 1982).

Control

Rodent populations infected with S. enteritidis are a zoonotic risk to personnel, a source of infection for other animals in the facility, and unsuitable

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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for research purposes. There are only two effective methods of eliminating the infection. The usual method is to destroy the entire population and replace them with animals from a pathogen-free source. In the case of extremely valuable stocks, cesarean derivations can be attempted. This requires the maintenance of all breeders in an isolation system (e.g., a Trexler-type plastic film isolator) to prevent the spread of the infection, housing of individual cesarean-derived litters and their foster dams in separate isolators, and repeated fecal cultures (see above) to assure that any litter to be used for restocking purposes is free of S. enteritidis.

Prevention of S. enteritidis infection is accomplished through barrier maintenance of rodent stocks. Particular attention must be given to the avoidance of Salmonella-contaminated food and water, and the exclusion of infected animals and wild rodents from the facility (Ganaway, 1982). Regular monitoring of all rodent populations must be done to assure that all rodents are free of S. enteritidis. Studies involving experimental infection of animals with S. enteritidis must be done by using appropriate containment systems (Habermann and Williams, 1958; Steffen and Wagner, 1983).

Interference with Research

Rodents infected with S. enteritidis can serve as a source of infection for other laboratory animals and humans. In addition, the infection can alter a number of biologic responses.

  1. S. enteritidis-infected mice can have nonspecific resistance to challenge with other intracellular parasites such as Listeria monocytogenes (Blanden et al., 1966; Zinkernagel, 1976).
  2. Prior immunization of mice with viable S. enteritidis results in the suppression of growth of transplantable tumors (Ashley et al., 1976).
  3. Concurrent infections of S. enteritidis and Plasmodium berghei in mice result in higher mortality than infection by either agent alone (Kaye et al., 1965).
  4. Concurrent infections of S. enteritidis and Schistosoma japonicum in mice result in higher mortality than infection by either agent alone (Tuazon et al., 1986).
  5. S. enteritidis infection results in reduced blood glucose and hepatic enzyme levels (Moore et al., 1977).
  6. Mice orally infected with S. enteritidis have reduced intestinal enzyme activities (Madge, 1973).
  7. Susceptibility to S. enteritidis infection is increased by:
    • lack of a gastrointestinal microflora (Bohnhoff and Miller, 1962; Margard and Peters, 1964; Ruitenberg et al., 1971; Collins and Carter, 1978);
Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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  • pretreatment with oral antibiotics (Bohnhoff et al., 1954; Bohnhoff and Miller, 1962; Watson, 1970; Collins and Carter, 1978);
  • food and/or water deprivation (Miller and Bohnhoff, 1962; Maenza et al., 1970; Collins, 1972; Tannock and Smith, 1972).
  • pretreatment with sodium bicarbonate by gavage (Collins, 1972);
  • •  

    slowing of gastrointestinal motility by morphine sulfate (Miller and Bohnhoff, 1962); or

    •  

    iron deficiency in rats (Newberne et al., 1968; Baggs and Miller, 1974) and iron overload in mice (Jones et al., 1977).

  • cecetomy of mice (Voravuthikunchai and Lee, 1987).
Citrobacter freundii (Biotype 4280)
Significance

Low.

Perspective

1965: Brennan et al. (1965c) associated Citrobacter freundii with an outbreak of diarrhea in laboratory mice.

1974: Ediger et al. (1974) reported an epizootic characterized by colitis, colonic mucosal hyperplasia, and rectal prolapse. C. freundii was isolated and given orally to pathogen-free mice, resulting in reproduction of the clinical and histopathologic features of the natural disease.

1976: Barthold et al. (1976) named the disease transmissible murine colonic hyperplasia and identified a biochemical variant (4280) of C. freundii as the etiologic agent. Bieniek and Tober-Meyer (1976) characterized the disease and also incriminated a biotype of C. freundii as the causative agent.

1977: Barthold and Jonas (1977) demonstrated that infection with C. freundii (Biotype 4280) enhances the responsiveness of mice to the colon carcinogen 1,2-dimethylhydrazine.

Agent

The agent is a bacterium, family Enterobacteriaceae, C. freundii (Biotype 4280). They are straight, Gram-negative, facultatively anaerobic rods, measuring 1.0 µm wide x 2.0 to 6.0 µm long, which occur singly and in pairs, and grow on ordinary medium. They may be a normal inhabitant of the gastrointestinal tract in humans and other species. C. freundii (Biotype 4280) is usually considered an opportunistic pathogen (Sakazaki, 1984).

Only one biotype (4280), defined on the basis of in vitro biochemical

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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reactions, is recognized as being pathogenic in mice. This biotype is positive for ornithine decarboxylase, ferments rhamnose, produces hydrogen sulfide, and reduces nitrite. This is in contrast to the nonpathogenic biotype which is negative for these reactions (Barthold et al., 1976).

Hosts

Mice. The organism does not colonize the intestines of rats or hamsters (Barthold et al., 1977).

Epizootiology

The natural history of this infection is poorly understood. Transmission has been shown to occur between cage contacts (Brennan et al., 1965c), presumably via fecal contamination and ingestion. It has been stated that the organism persisted in one colony for at least 3 years (Ediger et al., 1974). It is rarely found in cesarean-derived, barrier-maintained mouse populations.

Clinical

Signs are nonspecific. They include ruffled fur; listlessness; weight loss; stunting; pasty feces about the anus, base of the tail, and the perineum; and rectal prolapse (Brennan et al., 1965c; Ediger et al., 1974; Bieniek and Tober-Meyer, 1976; Barthold et al., 1977).

Suckling mice are more susceptible than adults. Mortality may reach 60% and rectal prolapse 15%. Mortality is significantly higher in C3H/HeJ than in DBA/2J, NIHS (Swiss), or C57BL/6J mice (Barthold et al., 1977).

Pathology

C. freundii (Biotype 4280) produces a transient infection in mice lasting only about 4 weeks. Even if the infection is eliminated as early as 2 days post infection by administration of neomycin sulfate and tetracycline hydrochloride, mucosal hyperplasia still occurs. Presence of the infection in the intestine for 10 days results in maximum hyperplasia (Barthold, 1980).

Following experimental infection, the organism attaches to the surface of the colonic mucosa within 4 to 10 days, hyperplasia is most severe at 16 days, and the mucosa reverts to normal by 45 days. Colonic hyperplasia possibly serves as a defense mechanism for replacement of infected epithelial cells (Johnson and Barthold, 1979).

The descending colon is most commonly affected, but the entire colon and cecum may be involved. Grossly, the affected bowel is thickened and

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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rigid in appearance. Microscopically, there is increased crypt height (to 3 times normal), increased mitotic activity, decreased numbers of goblet cells, and increased basophilia of the epithelium. Crypt abscesses are common, and mucosal erosions and ulcers can occur. Occurrence of necrotizing and inflammatory lesions tend to parallel mortality. Variable numbers of neutrophils or mononuclear leukocytes may be present in the lamina propria, but there is often a paucity of inflammatory cells. During regression of mucosal hyperplasia, goblet cell hyperplasia with mucinous distension of crypts and streaming of mucin into the gut lumen can occur (Brynjolfsson and Lombard, 1969; Bieniek and Tober-Meyer, 1976; Barthold et al., 1978).

Diagnosis

Diagnosis is made by demonstration of characteristic lesions in the large intestine and isolation of C. freundii (Biotype 4280). However, the organism may not be recovered from all cases because lesions persist beyond the period of infection. Both the pathogenic and the nonpathogenic biotypes may be isolated from a given animal (Barthold et al., 1976).

Control

Definitive data are lacking. Elimination of the organism from an infected colony probably requires depopulation and restocking with cesarean-derived mice. Neomycin and tetracycline in drinking water reduce losses during outbreaks but probably do not completely eliminate the infection.

Interference with Research

Cytokinetics of the mucosal epithelium in the large intestine is profoundly altered in infected mice. Susceptibility to the carcinogen 1,2-dimethylhydrazine is increased, and the latent period for neoplasia induction is reduced (Barthold and Jonas, 1977).

Pseudomonas aeruginosa
Significance

Low, except in immunosuppressed hosts.

Perspective

Pseudomonas aeruginosa, and to a lesser extent other species of Pseudomonas and coliform bacteria, are normal inhabitants of the

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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nasopharynx and lower digestive tract; however, they are occasionally associated with disease, primarily as opportunistic pathogens in immunosuppressed hosts (Weisbroth, 1979). P. aeruginosa has been particularly troublesome in mice following whole body irradiation (Hammond et al., 1954; Vincent et al., 1955; Wensinck et al., 1957; Flynn, 1963a,c; Hammond, 1963; Hightower et al., 1966) or administration of cyclophosphamide (Pierson et al., 1976; Hazlett et al., 1977; Rosen and Berk, 1977; Urano and Maejima, 1978; Harada et al., 1979). In addition, there are a few reported instances in which clinical inner ear disease in mice has been attributed to natural P. aeruginosa infection (Ediger et al., 1971; Kohn and MacKenzie, 1980).

Agent

P. aeruginosa is a bacterium, class Schizomycetes, order Eubacteriales, family Pseudomonadaceae. The organisms are Gram-negative, straight or slightly curved rods measuring 0.5-1.0 µm in diameter x 1.5-5.0 µm in length. They are motile and catalase and oxidase positive. Most strains produce a bluish green phenazine pigment (pyocyanin), as well as fluorescein, a greenish yellow pteridine that fluoresces. The organisms grow readily on media used in the routine isolation of bacteria (Palleroni, 1984; Gilardi, 1985).

Different strains of P. aeruginosa vary widely in virulence for mice (Pennington and Williams, 1979), and flagella appear to have a role in virulence (Montie et al., 1982). P. aeruginosa elaborates three potent exotoxins that are more pathogenic than endotoxin. Exotoxin A is thought to be the major toxin involved in virulence. In addition, the organism produces a variety of other toxins and enzymes that can contribute to its pathogenesis (Palleroni, 1984; Gilardi, 1985).

Hosts

Laboratory and wild mice and rats, humans, and numerous other species (Palleroni, 1984).

Epizootiology

The organism is ubiquitous, occurring widely in soil, water, sewage, and air. It frequently is present in small numbers in the normal intestinal flora of people, and also occurs normally on the human skin (Gilardi, 1985). It is widely distributed in conventional stocks of rodents. In one 10-year survey in Japan it was found in 20-50% of the rodent colonies (Nakagawa et al., 1984). In the same survey P. aeruginosa was isolated from nasal passages, oropharynx, large intestine, and skin in many healthy rodent colonies.

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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P. aeruginosa can be transmitted by fomites (such as food, bedding, and water), human carriers, and contact with infected rodents (either wild rodents or other laboratory animals). There is frequent shedding of the organism in infected, clinically normal rodents, but the normal flora of the nasopharynx and the gastrointestinal tract are effective in controlling the population in vivo. Disease occurs when this normal flora is altered or host defenses are impaired (Vincent et al., 1955; Hoag et al., 1965; Hightower et al., 1966).

Clinical

Under usual circumstances the organism is part of the normal flora in the digestive tract and clinical signs are not present. After immunosuppression fulminating septicemia can occur, resulting in death with few clinical signs (Flynn, 1963c). There are a few reports of "circling" or "rolling" in mice associated with otitis media and interna due to P. aeruginosa (Ediger et al., 1971; Olson and Ediger, 1972; Kohn and MacKenzie, 1980).

Pathology

In immunosuppressed animals the organism invades from the normal sites of localization into deeper tissues, resulting in bacteremia and high mortality (Hammond et al., 1954; Vincent et al., 1955; Hightower et al., 1966). There is disagreement on whether the major site of entry is via the intestine and portal circulation (Urano and Maejima, 1978) or through the nasal mucosa (Brownstein, 1978). Gross and histopathologic lesions are nonspecific (Flynn, 1963b).

There are two reported instances of inner ear disease in mice attributed to P. aeruginosa and characterized clinically by either circling (Ediger et al., 1971) or rolling (Kohn and MacKenzie, 1980). In both instances, affected animals had suppurative otitis media with extension into the inner ears and to the adjacent meninges or brain. P. aeruginosa was consistently isolated from the middle ears and associated lesions (Olson and Ediger, 1972; Kohn and MacKenzie, 1980). A similar disease has been produced experimentally by inoculating P. aeruginosa intravenously into mice (Gorrill, 1956; Ediger et al., 1971).

B-lymphocyte-deficient mice are more susceptible to P. aeruginosa infection than B-lymphocyte-immunocompetent mice and have been used as a model for studying the protective efficacy of monoclonal antibodies against the organism (Zweerink et al., 1988). Other models of increased susceptibility of mice to the organism have included whole body irradiation (Flynn, 1963a,b,c; Hammond, 1963; Hightower et al., 1966), administration of cyclosphosphamide (Buhles and Shifrine, 1977; Urano and Maejima, 1978), local burns (Stieritz and Holder, 1975; Cryz et al., 1984), and administration of ferric ions (Forsberg and Bullen, 1972).

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Diagnosis

Diagnosis is dependent on isolation and identification of the organism and exclusion of other possible causes of disease. In animals previously immunosuppressed, septicemia due to P. aeruginosa (or other opportunists) should be demonstrated by culture (Flynn, 1963b).

Control

Control of P. aeruginosa infection is not necessary for most studies using mice and rats, but it can be of great importance to the success of studies in which these animals are immunosuppressed by whole body irradiation or the administration of chemotherapeutic agents such as cyclophosphamide (Flynn, 1963b; Urano and Maejima, 1978).

P. aeruginosa can be completely eliminated from mice and rats by cesarean derivation followed by maintenance under gnotobiotic conditions (Trentin et al., 1966). However, for most studies employing immunosuppressive regimens, the complications caused by this organism can be avoided through more practical measures.

When mice and rats are maintained by standard husbandry practices, the nasopharynx and lower digestive tract frequently become colonized by P. aeruginosa. Maintenance of this colonized state depends on the repeated ingestion of large numbers of P. aeruginosa in the drinking water. Water provided by either water bottles or automatic watering systems can serve as an excellent medium for growth of the organism. The colonized state can be virtually eliminated by preventing the repeated ingestion of organisms through rigorous sanitation measures coupled with acidification and/or hyperchlorination of the water. However, hyperchlorination/acidification of water will not eliminate an established infection. Cages, water bottles, sipper tubes, and bedding should be autoclaved. The water can be treated with sodium hypochlorite to provide 10-12 µg/ml (ppm) chlorine or acidified to pH 2.5 to 2.8 with hydrochloric acid to prevent growth of P. aeruginosa. Water bottles must be changed and automatic water lines flushed frequently to assure these effective levels of chlorination or acidification (Wensinck et al., 1957; Beck, 1963; Flynn, 1963c; McPherson, 1963; Woodward, 1963; Hoag et al., 1965; Hightower et al., 1966; McDougall et al., 1967; Weisbroth, 1979). Hyperchlorination of drinking water to prevent infection with P. aeruginosa has been reported to depress macrophage function in mice (Fidler, 1977). Acidification and chlorination of drinking water does not adversely affect reproduction in mice (Les, 1968).

A number of vaccines against P. aeruginosa have been shown to have some protective effect in mice and rats (Lusis and Soltys, 1971; Pavlovskis et al., 1981; Joo et al., 1982; Leiberman et al., 1986) but appear to be of doubtful value for use in rodents.

Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Interference with Research

Indigenous infections of P. aeruginosa in laboratory mice and rats are generally of little importance except when immunosuppressive regimens are carried out:

  1. Mice and rats naturally or experimentally infected with P. aeruginosa typically show the early death syndrome (i.e., on average they die several days earlier than comparable animals not infected with this organism) when exposed to lethal doses of whole body irradiation (Hammond et al., 1954, 1955; Vincent et al., 1955; Wensinck et al., 1957; Flynn, 1963a,b,c; Hammond, 1963; Hightower et al., 1966). Similar early deaths in mice exposed to lethal irradiation have occasionally been attributed to other enteric bacteria including Enterobacter cloacae, Escherichia coli, Klebsiella pneumonia, and Proteus vulgaris (Matsumoto, 1980, 1982). In general, axenic rodents can tolerate more radiation than animals with a normal flora, i.e., they survive higher median lethal doses and have increased survival times (McLaughlin et al., 1964).
  2. Mice and rats infected naturally or experimentally with P. aeruginosa can have high mortality due to P. aeruginosa bacteremia following administration of cyclophosphamide (Pierson et al., 1976; Hazlett et al., 1977; Rosen and Berk, 1977; Urano and Maejima, 1978; Harada et al., 1979), cortisone (Millican, 1963), or other chemical immunosuppressants (Schook et al., 1977).
  3. Mice with streptozotocin-induced diabetes mellitus have been reported to have increased susceptibility to P. aeruginosa infection (Kitahara et al., 1981).
  4. Mice previously infected with murine cytomegalovirus have been reported to be more susceptible to experimental P. aeruginosa infection (Hamilton and Overall, 1978).
  5. Mice given P. aeruginosa intraperitoneally have depressed contact sensitivity to oxazolone (Campa et al., 1975, 1976, 1977).
  6. Mice given P. aeruginosa by gavage have reduced survival when exposed to cold stress of -20°C for 2.5 hours (Halkett et al., 1968).
  7. Indwelling jugular catheters in rats may become infected with P. aeruginosa and be causally associated with septic pulmonary emboli (Wyand and Jonas, 1967).
  • Common Endoparasites

    Traditionally, publications concerned with endoparasites of laboratory mice and rats have tended to be encyclopedic in coverage and monolithic in treatment of reports from older versus recent literature. That approach is no

  • Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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    longer acceptable inasmuch as earlier reports often reflected the diverse parasite flora of wild mice and rats, which persisted for many years after these species were adapted for laboratory use. The majority of parasites that were commonplace in rodent colonies only a few decades ago have now been either eliminated or greatly reduced in prevalence through the application of cesarean derivation and barrier maintenance methodologies. Occasional exceptions may be encountered in conventional colonies that have never been rederived by cesarean section and in cesarean-derived stocks that have been contaminated by wild rodents. Thus, those parasites of the digestive tract that are regularly encountered in contemporary stocks of laboratory mice and rats in the United States are listed in Table 11. Among parasites of this group only two, Spironucleus muris and Giardia muris, are considered important pathogens and have been reported to interfere significantly with research. The other pathogen in the group, Hymenolepis nana, is only considered a mild pathogen. The remainder (Syphacia obvelata, Syphacia muris, Aspicularis tetraptera, Entamoeba muris, and Tritrichomonas muris) are nonpathogenic under usual circumstances. These eight parasites are reviewed below.

    Spironucleus muris
    Significance

    Spironucleus muris is a common intestinal parasite in laboratory rodents, including those maintained by barrier methods, and there is increasing evidence that it alters immune responses (see below).

    Agent

    S. muris (formerly Hexamita muris) is a flagellated protozoan, subphylum Mastigophora, class Zoomastigophorea, order Diplomonadida, family Hexamitidae, subfamily Hexamitinae. The trophozoite is elongated, torpedo-shaped, bilaterally symmetrical, and measures 3-4 x 10-15 µm. It has six actively beating anterior flagella and two slow-moving flagella at the posterior tip. The cysts measure 4 x 7 µm (Kunstyr, 1977; Brugerolle et al., 1980).

    Life Cycle

    The life cycle is direct. The trophozoites reproduce by longitudinal fission and form highly resistant cysts. The minimal infectious dose for a mouse is one cyst (Kunstyr, 1977; Stachan and Kunstyr, 1983).

    Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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    Hosts

    Mice, rats, and hamsters (Wagner et al., 1974; Kunstyr et al., 1977a; Kunstyr and Friedhoff, 1980).

    Epizootiology

    Young mice, 2-6 weeks old, are most susceptible. The trophozoites usually inhabit the crypts of Lieberkuhn in the small intestine, but in young animals the lumen can also contain large numbers. In older mice and rats very few trophozoites may be present, and they can be found only in the glands of the gastric pylorus (J. R. Lindsey, Department of Comparative Medicine, University of Alabama at Birmingham, unpublished).

    Transmission is by ingestion of the cysts. The cysts are shed in the feces, in greatest numbers by young or immunocompromised hosts, e.g., athymic (nu/nu) or lethally irradiated mice (Kunstyr et al., 1977b). The cysts are inactivated by some disinfectants and high temperature (45°C for 30 minutes) but are highly resistant to most other environmental conditions. They retain infectivity after -20°C for 6 months, pH 2.2 for 1 day, room temperature for 14 days, and 0.1% glutaraldehyde for 1 hour (Kunstyr and Ammerpohl, 1978).

    Clinical

    The infection is usually subclinical in immunocompetent hosts. Severe chronic enteritis with weight loss has been associated with S. muris infection in athymic (nu/nu) and lethally irradiated mice (Meshorer, 1969: Kunstyr et al., 1977b).

    Young mice infected with S. muris have been reported to have diarrhea, dehydration, rough hair coats, weight loss, listlessness, hunched posture, abdominal distension, and sporadic mortality (Sebesteny, 1969; Lussier and Loew, 1970; Boorman et al., 1973a; Csiza and Abelseth, 1973; Wagner et al., 1974; von Mattheisen et al., 1976; Flatt et al., 1978; Van Kruiningen et al., 1978; Eisenbrandt and Russell, 1979). However, none of these studies excluded other possible causes such as mouse hepatitis virus infection. It is doubtful whether S. muris alone causes clinical disease in otherwise healthy hosts (Kunstyr and Friedhoff, 1980).

    Pathology

    Immunocompetent mice are susceptible to experimental infection until about 8 or 10 weeks of age, and rats are susceptible until 12 weeks of age. Athymic (nu/nu) mice are fully susceptible at any age. After ingestion of cysts by the host, trophozoite (and cyst) numbers in the intestines of

    Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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    TABLE 11 Common Endoparasites of Mice and Rats

    Site and Parasite(Common name, if any)

    Type of Parasite

    Stage Most Useful in Diagnosis(Size)

    Ecological Niche in Host

    Histologic Features

    Other Diagnostic Methods

    Small Intestine

     

     

     

     

     

    Spironucleus muris

    Flagellated protozoan

    Trophozoite(2-3 µm wide wide x 7-9µm long)

    Intervillous spaces and crypts of small intestine, glands of gastric pylorus

    Charcteristic granular appearance of trophozoites in crypts of small intestine and glands of pylorus; stain with periodic acid- Schiff and silver methods

    Demonstrate characteristic trophozoites in saline mounts of small intestine contents or cysts (4.0 x 7.4 µm) in wet mounts of feces: phase-contrast preferred

    Giardia muris

    Flagellated protozoan

    Trophozoite(5-10 µm wide x 7-13 µm long)

    Along and between villi of small intesine

    Size, shape, and location of trophozoites; stain with periodic acid-Schiff and silver methods

    Same as for S. muris

    Hymenolepis nana (dwarf tapeworm)

    Tapeworm

    Adults (25-40 µm long x 0.75 µm wide)

    Small intestine attached to mucosa [Note: larval stage(cercocystis) can be seen in mucosa of small intestine]

    Adults have scolices with armed rostellum, segments, no body cavity or digestive tract, calcareous corpuscles present; cercocystis in intestinal villi

    Demonstrate eggs(37-41 x 50-53 µm) by fecal flotation

    Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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    Site and Parasite(Common name, if any)

    Type of Parasite

    Stage Most Useful in Diagnosis(Size)

    Ecological Niche in Host

    Histologic Features

    Other Diagnostic Methods

    Large intestine

     

     

     

     

     

    Syphacia obvelata (mouse pinworm)

    Nematode

    Adults (female, 3.4-5.8 µm long; male, 1.1-1.5 µm long), egg

    Cecum and colon

    Nematodes have a digestive tract and a single layer of muscle cells; genus and species identification based on morphology of intact adults

    Demonstrate eggs (36 x 134 µm, flat on one side) by cellophane tape preparation of periannal area

    Syphacia muris rat pinworm)

    Nematode

    Adults (female, 2.8-4.0 mm long; male, 1.2-2.3 mm long), egg

    Cecum and colon

    Same as S. obvelata

    Same as for S. obvelata; eggs are 29 x 75 µm, football shaped to slightly flat on one side

    Aspicularis tetraptera (mouse pinworm)

    Nematode

    Adults (female, 2.6-4.7 µm long; male, 2.0-2.6 µm long), egg

    Large intestine, except cecum

    Same as S. obvelata

    Demonstrate eggs (41 x 90 µm, ellipsoidal shape) by fecal flotation

    Entamoeba muris

    Amoeba

    Trophozoite (8-30 µm diam.)

    Fecal/mucosal interface, cecum, and colon

    Trophozoites have distinct magenta nucleus and vacuolated violet cytoplasm; cell membrane indistinct (hematoxylin and eosin stain)

    Demonstrate characteristic trophozoites in saline mounts of cecal or colon contents, cysts (9-20 µm diameter) in feces

    Tritrichomonas muris

    Flagellated protozoan

    Trophozoite (10-15 µm x 16-26 µm)

    In fecal stream of cecum and colon

    Trophozoites have indistinct nucleus and no cytoplasmic vacuolation; outer membrane is distinct and may appear wrinkled (hematoxylin and eosin stain)

    Demonstrate trophozoites with characteristic wobbly movement in saline mount of cecal or colon contents, fresh feces

    Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
    ×

    immunocompetent rodents peak at 1-2 weeks and decline to low numbers by 4-5 weeks (BALB/c mice), 7-9 weeks (CBA, SJL, and C3H/He mice), or 13 weeks (A and B.B1O mice). By comparison, the numbers in athymic (nu/ nu) mice persist indefinitely at high levels (Kunstyr et al., 1977a,b; Kunstyr and Friedhoff, 1980; Brett and Cox, 1982a).

    In severe infections, such as those in athymic (nu/nu) mice, the small intestine may appear reddened and contain watery fluid and gas. Wet mounts of the contents of the small intestine are useful for demonstrating the motile trophozoites; the cysts can be demonstrated in the contents of the cecum and colon. In hematoxylin and eosin-stained sections of the small intestine, the best indicator of S. muris infection is distension of the crypts of Lieberkuhn by masses of granular-appearing trophozoites. They are seen less frequently in the intervillous spaces and gut lumen. The trophozoites can cause shortening of microvilli on the crypt epithelium and increased turnover of enterocytes. There usually is little or no inflammatory response in immunocompetent animals, but heavily parasitized animals that are immunodeficient can have moderate to severe enteritis (Kunstyr et al., 1977a,b; Brugerolle et al., 1980; Kunstyr and Friedhoff, 1980).

    Diagnosis

    Definitive diagnosis of disease caused by S. muris requires exclusion of other possible causes of digestive tract disease, e.g., enterotrophic strains of mouse hepatitis virus. In many laboratories the diagnosis is made by using wet mounts to demonstrate the characteristic trophozoites in contents of the small intestine or cysts in contents of the large intestine or feces (Kunstyr, 1977). For routine health surveillance that includes histopathology, the examination of multiple histologic sections of small intestine and gastric pylorus is probably superior to other methods because very few parasites may be present and they may be localized in distribution. The trophozoites can be stained by silver or periodic acid-Schiff methods (Flatt et al., 1978).

    Control

    Cesarean derivation and barrier maintenance are the recommended procedures. Treatment of spironucleosis in mice with 0.04-0.1% dimetridazole in drinking water for 14 days can ameliorate clinical signs but does not completely eliminate the infection (Herweg and Kunstyr, 1979).

    Interference with Research

    There is considerable evidence that S. muris infection interferes with research, including the following:

    Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
    ×
    1. S. muris can increase the severity and mortality of wasting syndrome (presumably due to mouse hepatitis virus) in athymic (nu/nu) mice (Boorman et al., 1973a,b).
    2. Susceptibility to S. muris infection and disease is greatly enhanced by whole body irradiation (Meshorer, 1969; Meyers, 1973; Kunstyr et al., 1977b; Gruber and Osborne, 1979).
    3. S. muris infection increases mortality in cadmium-treated mice (Exon et al., 1975).
    4. S. muris infection alters macrophage function (Keast and Chesterman, 1972; Ruitenberg and Kruyt, 1975).
    5. S. muris infection reduces spleen plaque-forming cell responses to sheep erythrocytes (Brett, 1983) and lymphocyte responsiveness to mitogens such as phytohemagglutinin, concanavalin A, and pokeweed mitogen (Kunstyr and Friedhoff, 1980).
    6. S. muris infection alters immune responsiveness to tetanus toxoid and type 3 pneumococcal polysaccharide in mice (Ruitenberg and Kruyt, 1975) but not in rats (Mullink et al., 1980).
    7. Concurrent infections of Babesia microti, Plasmodium yoelii, or Plasmodium berghei with S. muris decreases the numbers of S. muris trophozoites and cysts (Brett and Cox, 1982b).
    Giardia muris
    Significance

    Giardia muris causes a common, subclinical infection of laboratory rodents that has been shown in recent years to alter a number of immune responses in mice (Belosevic et al., 1985).

    Agent

    G. muris is a flagellated protozoan, subphylum Mastigophora, class Zoomastigophorea, order Diplomonadida, family Hexamitidae, subfamily Giardinae. The trophozoite is 7-13 µm long x 5-10 µm wide, pear-shaped, and bilaterally symmetrical. The anterior end is broadly rounded, while the posterior end is drawn out. There are two dark-staining median bodies that are small and round and located just posterior to the nucleus. The trophozoites are easily recognized in wet preparations of intestinal contents because of their characteristic cupped shape and rolling and tumbling motion. The cysts are ellipsoidal, 15 x 17 µm, have thick walls and 4 nuclei, and are found in the large intestine and feces (Hsu, 1979, 1982; Brugerolle et al., 1980).

    Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
    ×
    Life Cycle

    The life cycle is direct. The trophozoites reproduce by longitudinal fission and form cysts that are passed in the feces. Transmission is by ingestion of cysts. The minimal infectious dose for a mouse is approximately 10 cysts (Hsu, 1979, 1982; Stachan and Kunstyr, 1983).

    Hosts

    Mice, rats, hamsters, humans, and many other species (Kunstyr and Friedhoff, 1980).

    Epizootiology

    The trophozoites colonize the proximal one-fourth of the small intestine in which they are found mainly adhering to columnar cells near the bases of villi and free in the adjacent mucus layer. The number of trophozoites in the small intestine correlates directly with the number of cysts in the large intestine and feces (Owen et al., 1979; Brett and Cox, 1982a; Belosevic and Faubert, 1983; Belosevic et al., 1984).

    The cysts are resistant to most environmental conditions but are inactivated by treatment with a 2.5% phenol solution and by temperatures above 50°C (Hsu, 1979).

    Clinical

    G. muris infections in mice and rats are usually subclinical but can cause reduced weight gain, rough hair coats, and enlarged abdomens (Sebesteny, 1969; Roberts-Thomson et al., 1976b; Owen et al., 1979). Infection has been associated with morbidity and mortality in athymic (nu/nu) and thymectomized mice (Boorman et al., 1973a), and in mice immunocompromised by x-irradiation or protein-deficient diets (Owen et al., 1979). However, these studies did not exclude concurrent infection(s) by other enteric pathogens.

    Pathology

    Pathogenesis of G. muris infection has been studied most extensively in mice. The acute phase of the infection involves the proliferation of trophozoites in the small intestine and the peak period of cyst release during week 2 of infection. The elimination phase is the period during which the cysts released in the feces are reduced to undetectable levels. The DBA/2, B1O.A, C57BL/6, BALB/c, and SJL/J strains eliminate the infection in 5 weeks and are said to be resistant, whereas the C3H/He, A/J, and Crl:ICR

    Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
    ×

    (CD®-1) mice require 10 weeks and are considered susceptible (Brett and Cox, 1982a; Belosevic et al., 1984). Genetic analyses have shown that resistance during the acute phase of the infection may be controlled by several genes not linked to the H-2 locus (Belosevic et al., 1984), while resistance during the elimination phase is inherited as a dominant trait (Roberts-Thomson et al., 1980; Belosevic et al., 1984).

    Protective immunity is thought to be dependent upon both antibody-and cell-mediated mechanisms (Brett and Cox, 1982a; Stevens, 1982). Both IgA and IgG antibodies against G. muris have been demonstrated in milk of immune mice (Andrews et al., 1980), and such milk conveys passive protection (Stevens and Frank, 1978; Roberts-Thomson and Mitchell, 1979). Athymic (nu/nu) mice also are highly susceptible and have prolonged infections (Roberts-Thomson and Mitchell, 1978; Stevens et al., 1978).

    The morphologic changes in the small intestine associated with uncomplicated G. muris infection are usually minimal. The villus to crypt ratio may be reduced and variable numbers of lymphocytes may be present (Roberts-Thomson et al., 1976b; Stevens and Roberts-Thomson, 1978; Brett and Cox, 1982a).

    Diagnosis

    Definitive diagnosis of overt disease thought to be attributable to G. muris requires exclusion of other possible primary or contributing pathogens. Infection due to G. muris can be diagnosed histologically by identifying the characteristic "monkey-faced" trophozoites in sections of small intestine. The trophozoites also can be demonstrated in wet mounts of intestinal contents, and the cysts can be demonstrated in wet mounts of feces (Hsu, 1979, 1982).

    Control

    The most practical approach is procurement of rodents from breeding populations shown by health surveillance testing to be free of G. muris, followed by barrier maintenance in the user facility. Cesarean derivation is required to eliminate the parasite from infected stocks. Metronidazole can be used for treatment of infected animals (Belosevic et al., 1985) but does not completely eradicate the infection.

    Interference with Research

    G. muris infection can alter research results, and the course of experimental G. muris infections can be influenced by a variety of experimental procedures as follows:

    Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
    ×
    1. G. muris infection causes a transient reduction in the immunoresponsiveness of mice to sheep erythrocytes during weeks 2 and 3 of infection (Brett, 1983; Belosevic et al., 1985).
    2. G. muris infection alters intestinal fluid accumulation and mucosal immune responses caused by cholera toxin in mice (Ljungstrom et al., 1985).
    3. Concurrent G. muris and Trichinella spiralis infection in mice results in suppression of the G. muris infection (Roberts-Thomson et al., 1976a).
    4. Concurrent infections of Babesia microti, Plasmodium yoelii, or Plasmodium berghei with G. muris decreases the number of G. muris trophozoites and cysts (Brett and Cox, 1982b).
    5. Susceptibility to G. muris infection is increased by x-irradiation or protein-deficient diets (Owen et al., 1979).
    6. G. muris infection can increase the severity and mortality of wasting syndrome (presumably due to mouse hepatitis virus) in athymic (nu/nu) mice (Boorman et al., 1973a).
    Hymenolepis nana (Dwarf Tapeworm)
    Significance

    Hymenolepis nana is a small tapeworm found infrequently in contemporary rodents. It is only mildly pathogenic but has importance as a zoonotic agent for humans.

    Agent

    H. nana belongs to the order Cyclophyllidea, family Hymenolepididae. The adult is a slender, white worm, 25-40 mm long and less than 1 mm wide. It has a scolex with 4 suckers and an armed, retractable rostellum with a row of 20-27 hooks. Mature proglottids are trapezoidal in shape. The eggs are oval, thin shelled, and colorless and have prominent polar filaments. They contain an oncosphere with 3 pairs of hooklets enclosed in an inner envelope. They measure 30-56 x 44-62 µm and are unable to survive long outside the host. The spherical embryo measures 16-25 x 2430 µm and possesses 3 pairs of hooks (Flynn, 1973b; Wescott, 1982).

    Life Cycle

    The life cycle includes adult, egg (with embryo or oncosphere), and larval (cercocystis) stages. The life cycle can be direct. Eggs in the feces of a definitive host are directly infectious when ingested by another potential definitive host, or the eggs hatch within the intestine of the definitive host, resulting in autoinfection of the same host. In either case, the eggs hatch in the small intestine, the larvae penetrate and develop in the intestinal villi as

    Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
    ×

    cercocystis, and return to the lumen to become mature adults. By direct transmission the life cycle requires only 14-16 days. Alternatively, the cycle can be indirect. The egg is ingested by an arthropod intermediate host such as a flour beetle, the cercocystis develops in the intestine of the beetle, the intermediate host is eaten by the definitive host, and the adult develops in the lumen of the small intestine. The entire life cycle by indirect transmission requires 20-30 days (Flynn, 1973b; Wescott, 1982).

    Hosts

    Mice, rats, hamsters, other rodents, simian primates, and humans (Hsu, 1979; Kunstyr and Friedhoff, 1980; Wescott, 1982).

    Epizootiology

    Weanling and young adult rodents are most frequently infected. The duration of infection by the adult worms in the small intestine is usually only a few weeks (Flynn, 1973b; Wescott, 1982).

    Clinical

    Most infections are subclinical. However, severe infections have been reported to cause retarded growth and weight loss in mice and intestinal occlusion, impaction, and death in hamsters (Flynn, 1973b; Wescott, 1982).

    Pathology

    The presence of adult worms in the small intestine is usually associated with mild enteritis. The finding of typical cercocystis with an armed rostellum in the intestinal villi of a rodent is diagnostic of H. nana.

    Larval stages occasionally reach the mesenteric lymph nodes, liver, or lung where they incite a granulomatous inflammatory response (Flynn, 1973b; Hsu, 1979; Wescott, 1982). This is reportedly a common occurrence in mice of the RFM strain (C. Zurcher, Institute for Experimental Gerontology TNO, Rijswijk, The Netherlands, unpublished).

    Diagnosis

    The most common method is demonstration and identification of the adult tapeworms in the small intestine. Eggs may be demonstrated in the feces. Also, histologic sections occasionally are successful in demonstrating the cercocystis in intestinal villi and mesenteric nodes (Flynn, 1973b; Hsu, 1979; Wescott, 1982).

    Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
    ×
    Control

    The most practical method usually is to obtain rodents from stocks that have been demonstrated to be free of H. nana. Cesarean derivation and barrier maintenance are the most effective methods for eliminating the infection from infected rodent stocks (Flynn, 1973b; Wescott, 1982).

    Interference with Research

    Infected rodents are deemed unsuitable for research use because of the potential for zoonotic infection and the fact that H. nana is pathogenic. H. nana infection can interfere with studies involving the intestinal tract (Flynn, 1973b; Wescott, 1982).

    Syphacia obvelata (Mouse Pinworm) and Syphacia muris (Rat Pinworm)
    Significance

    Syphacia obvelata and Syphacia muris are among the more common endoparasites of contemporary rodents, including stocks that have been derived by cesarean section and maintained in barrier facilities (Flynn, 1973c).

    Agents

    These rodent pinworms belong to class Nematoda, order Ascarida, suborder Oxyurina, genus Syphacia. They are roundworms, with three broad lips; a prominent esophagus with a well-developed single bulb at the posterior end; and long, pointed tails. Male worms possess three mamelons; a long, prominent spicule; a gubernaculum; and a ventrally bent tail. On the female, the vulva is located on the anterior quarter of the body, behind the excretory pore. Eggs are asymmetrical, more or less flattened on one side, thin shelled, and transparent (Flynn, 1973c; Wescott, 1982).

    For most purposes there is no reason to differentiate infections due to S. obvelata and those due to S. muris; however, the two species can be distinguished by the following criteria (Wescott, 1982):

    1. Male worms. The adult male S. obvelata is 1.1-1.6 mm long and 125 µm wide. The adult male S. muris is 1.2-1.3 µm long and 100 µm wide. The tail length of S. muris is about two times its body width, whereas the tail length of S. obvelata is about equal to its body width.
    2. Female worms. The adult female S. obvelata is 3.4-5.8 µm long and 240-400 µm wide. The adult female S. muris is 2.8-4.0 mm long and 250
    Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
    ×
    • µm wide. The vulva of S. muris is further posterior in relation to the esophageal bulb than that of S. obvelata.
    • c.  

      Eggs. S. muris eggs (75 x 20 µm) are slightly more than half the length of S. obvelata eggs (134 x 36 µm), although nearly the same width. S. muris eggs are slightly asymmetrical, being slightly flatter on one side than the other, but somewhat football shaped. S. obvelata eggs are decidedly asymmetrical or somewhat banana shaped.

    Life Cycle

    The life cycle is direct and requires only 11-15 days for completion. Gravid females migrate from the large intestine to deposit their eggs in the perianal area. The eggs become infective in about 6 hours. Following ingestion by another host, the eggs hatch in the small intestine, and the larvae reach the cecum in 24 hours. The parasites spend 10-11 days in the cecum where they mature and mate. Gravid females again migrate to the perianal area, deposit their eggs, and die (Wescott, 1982).

    Hosts

    Laboratory mice, rats, hamsters, gerbils, and wild rodents. S. obvelata has been reported to occur in people, but it has no known public health significance (Flynn, 1973c; Wightman et al., 1978; Ross et al., 1980; Wescott, 1982).

    Epizootiology

    The adults occur primarily in the cecum and colon of infected hosts. Eggs are deposited in the perianal area of the host, from which they are efficiently disseminated into the cage and room environments. The eggs can survive for weeks under most animal room conditions. Transmission is by ingestion of embryonated eggs (Flynn, 1973c; Wescott, 1982).

    Clinical

    Infections due to Syphacia spp. alone are subclinical. Poor condition, rough hair coats, reduced growth rate, and rectal prolapse have been attributed to natural Syphacia infections in mice (Hoag, 1961; Harwell and Boyd, 1968; Jacobson and Reed, 1974). Unfortunately, such reports have failed to exclude other possible causes, e.g., Citrobacter freundii (Biotype 4280) infection, which has been shown to cause rectal prolapse in mice (Barthold et al., 1977). Clinical signs have not been observed in rodents experimentally infected with S. obvelata or S. muris (Flynn, 1973c; Wescott, 1982).

    Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
    ×
    Pathology

    The prevalence of pinworms in an infected rodent population is a function of age, sex, and host immune status. In enzootically infected colonies, weanling animals develop the greatest parasite loads, males are more heavily parasitized than females, and Syphacia numbers diminish with increasing age of the host (Wescott, 1982).

    Athymic (nu/nu) mice were reported by Jacobson and Reed (1974) to have increased susceptibility to pinworm infection. When naturally infected athymic and immunocompetent mice were housed together in the same room for 130 days, the athymic mice obtained increasingly heavy worm burdens, while the immunocompetent mice maintained consistently low worm counts.

    Pinworms of laboratory rodents are generally not considered pathogens. Heavy infections have been reported to cause rectal prolapse, constipation, intussusception, or fecal impaction but usually without use of diagnostic procedures appropriate to exclude other intercurrent diseases as primary or contributing causes (Flynn, 1973c; Wescott, 1982).

    Diagnosis

    Diagnosis is based on the demonstration of eggs on the perianal region (cellophane tape technique) or the finding of adult worms in the cecum and colon at necropsy (Flynn, 1973c; Wescott, 1982).

    Control

    The use of cesarean derivation and barrier maintenance methods are effective (Phillips, 1960), but subsequent reinfection of such stocks with Syphacia spp. has been a common occurrence (Flynn, 1973c).

    Hygienic methods, such as frequent cage and room sanitization, can aid in controlling the Syphacia burden in an infected rodent population. Cageto-cage transmission can be prevented by the use of filter-top cages (Wescott et al., 1976). Several anthelminthics are effective in eliminating a high percentage of the adult worms but are inefficient in clearing immature worms or eggs. Thus, treatment must be repetitive and is not generally recommended, except in special circumstances (Wagner, 1970; Flynn, 1973c; Hsu, 1979; Wescott, 1982).

    Interference with Research

    Pinworm infections in rats have been reported to reduce the occurrence of adjuvant-induced arthritis (Pearson and Taylor, 1975).

    Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
    ×
    Aspicularis tetraptera (mouse pinworm)
    Significance

    Aspicularis tetraptera ranks second (after Syphacia obvelata) in prevalence in contemporary rodents, but like the other rodent pinworms, it is considered nonpathogenic (Flynn, 1973c; Wescott, 1982).

    Agent

    These rodent pinworms belong to class Nematoda, order Ascarida, suborder Oxyurina, genus Aspicularis. The males are 2.0-2.6 mm long and 120-190 µm wide; the females are 2.6-4.7 mm long and 215-275 µm wide. Both sexes have broad cervical alae and conical tails. The vulva in the female is located near the center of the body. The males lack spicules and mamelons. The eggs are ellipsoidal and measure 41 x 90 µm (Flynn, 1973c; Hsu, 1979; Wescott, 1982).

    Life Cycle

    The life cycle is direct and requires 23-25 days. The adults reside in the colon. Females lay their eggs in the colon (not in the perianal area as is true of Syphacia spp.), and the eggs subsequently leave the host on fecal pellets. The eggs become infective after 6-7 days at room temperature. Transmission occurs when the infective eggs are ingested by another host. The eggs hatch in the colon, where the larvae develop to maturity, and the cycle begins again (Flynn, 1973c; Wescott, 1982).

    The life cycle of A. tetraptera differs from that of Syphacia obvelata and Syphacia muris in that it is 10-12 days longer, and the eggs of A. tetraptera require an additional 6 days for embryonation to the infective stage (Flynn, 1973c; Wescott, 1982).

    Hosts

    Mice, rats (rarely), and wild rodents (Flynn, 1973c; Wescott, 1982).

    Epizootiology

    Unlike S. obvelata and S. muris, which primarily inhabit the cecum (except for gravid females), A. tetraptera primarily inhabits the colon. A. tetraptera females lay eggs in the colon, not in the perianal area as is characteristic of S. obvelata and S. muris. Like the other pinworms, the eggs of A. tetraptera survive for weeks in animal room environments (Flynn, 1973c; Wescott, 1982).

    Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
    ×
    Clinical

    Same as for S. obvelata and S. muris (see above). Infections caused by A. tetraptera are subclinical.

    Pathology

    Same as for S. obvelata and S. muris (see above). A. tetraptera is not considered pathogenic.

    Diagnosis

    The more common methods are demonstration of the distinctive eggs by fecal flotation (the cellophane tape method is of no value) and demonstration and identification of the adult worms in the colon at necropsy (Flynn, 1973c; Wescott, 1982).

    Control

    Same as for S. obvelata and S. muris (see above).

    Interference with Research

    Same as for S. obvelata and S. muris (see above).

    Entamoeba muris
    Significance

    Entamoeba muris is a commensal amoeba found in the large intestine of rodents. It lacks significance both in terms of public health and as a complication of research.

    Agent

    This organism is a protozoan, phylum Sarcomastigophora, subphylum Sarcodina, superclass Rhizopoda, order Amoebida, family Entamoebidae, genus Entamoeba. The trophozoites measure 8-30 µm in diameter and have 8 nuclei (Levine, 1961).

    Life Cycle

    The life cycle is direct. The trophozoites form cysts that are passed in the feces. Transmission is by ingestion of cysts. Excystment occurs in the

    Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
    ×

    intestinal tract, and the trophozoites inhabit the cecum and colon (Levine, 1961).

    Hosts

    Mice, rats, hamsters, and other rodent species (Levine, 1961, 1974).

    Epizootiology

    The trophozoites reside in the cecum and colon, where they are most commonly found at the interface between the fecal stream and the intestinal epithelium. The cysts are quite resistant to environmental conditions (Levine, 1961).

    Clinical

    E. muris infections are always subclinical (Levine, 1961, 1974).

    Pathology

    The organism is nonpathogenic (Levine, 1961, 1974).

    Diagnosis

    The infection can be diagnosed by demonstrating the trophozoites in wet mounts of intestinal contents from the cecum or colon or the cysts in feces (Levine, 1961, 1974). In histologic sections of the cecum or large intestine prepared without disturbing the luminal contents, the trophozoites are readily identified at the margin of the fecal stream. In sections stained by hematoxylin and eosin, the trophozoites usually have a distinct magenta-stained nucleus and violet-stained cytoplasm that may or may not appear vacuolated. The outer cell membrane of the trophozoites is usually distinctly visible (J. R. Lindsey, Department of Comparative Medicine, University of Alabama at Birmingham, unpublished).

    Control

    Infection with this amoeba is considered inconsequential, and control measures are not usually justified. The infection can be eliminated by cesarean derivation and barrier maintenance.

    Interference with Research

    No examples have been reported.

    Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
    ×
    Tritrichomonas muris
    Significance

    Tritrichomonas muris is a commensal organism that occurs in the large intestine of rodents. It has no known significance as a complication of research. It is not known to infect people.

    Agent

    A flagellated protozoan, subphylum Mastigophora, class Zoomastigophorea, order Trichomonadida, genus Tritrichomonas. The trophozoite is pear-shaped and measures 10-14 x 16-26 µm. It has an anterior nucleus. More anteriorly there is a blepharoplast that gives rise to three anterior flagella and one posterior flagellum attached to the body by an undulating membrane. The trophozoite has a characteristic wobbly motility. Reproduction is by binary fission (Levine, 1974; Hsu, 1982). In the past, transmission has been thought to be by ingestion of the trophozoites, but there is a recent suggestion that T. muris forms cysts (Kunstyr and Friedhoff, 1980).

    Life Cycle

    In addition to the trophozoite stage, the organism may have a cyst stage (Kunstyr and Friedhoff, 1980). If so, the cyst stage is probably the one primarily involved in transmission.

    Hosts

    Mice, rats, hamsters, and rodents (Levine, 1974; Hsu, 1982).

    Epizootiology

    The trophozoites are found throughout the fecal mass in the cecum and colon (Levine, 1974; Hsu, 1982).

    Clinical

    T. muris infections are not known to cause clinical signs (Levine, 1974; Hsu, 1982).

    Pathology

    The organism is considered a commensal (Levine, 1974; Hsu, 1982).

    Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
    ×
    Diagnosis

    T. muris infection can be diagnosed by demonstrating the trophozoites in wet mounts of contents from the cecum or colon. Their wobbly or jerky movements are very characteristic (Levine, 1974; Hsu, 1982). In histologic sections of cecum or colon prepared without disturbing the luminal contents, the trophozoites are found dispersed throughout the fecal stream. In hematoxylin and eosin-stained sections, the nucleus stains poorly, the nuclear membrane is indistinct, and the cell wall often appears wrinkled or folded upon itself (J. R. Lindsey, Department of Comparative Medicine, University of Alabama at Birmingham, unpublished).

    Control

    The organism is a commensal, and control measures are not likely to be justified (Levine, 1974; Hsu, 1982).

    Interference with Research

    No examples have been reported.

    Other Endoparasites

    Numerous other endoparasites have been reported from wild mice and rats and are encountered occasionally in animals maintained by conventional methods. For information on those parasite species, more comprehensive works on this subject should be consulted (Levine and Ivens, 1965; Oldham, 1967; Griffiths, 1971; Owen, 1972; Flynn, 1973a; Levine, 1974; Hsu, 1979, 1982; Wescott, 1982).

    Suggested Citation:"7. Digestive System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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    This new edition—a must for all researchers who use these lab animals—provides practical suggestions for breeding, keeping, and identifying pathogen-free laboratory rodents. It contains three informative sections. The first, Principles of Rodent Disease Prevention, summarizes methods for eliminating infectious agents. It offers information on pathogen terminology; pathogen status of rodents; and breeding, transporting, isolating, testing, and diagnosing rodents. The second section, Individual Disease Agents and Their Effects on Research, describes the diagnosis and control of each infectious agent, and the last section, Diagnostic Indexes: Clinical Signs, Pathology, and Research Complications, contains informative tables covering all the diseases listed in the volume, arranged to help in the diagnosis of infected animals.

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