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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
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--> 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.
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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.
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--> 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,
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--> 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
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--> 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: Antibody production (Osborn and Medearis, 1967; Osborn et al., 1968; Howard and Najarian, 1974; Tinghitella and Booss, 1979; Schilt, 1987). Interferon induction (Osborn and Medearis, 1967; Kelsey et al., 1977). 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). Allogeneic skin graft rejection (Howard et al., 1974, 1977; Lang et al., 1976; Dowling et al., 1977). 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).
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--> 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
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--> 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.
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--> 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.
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--> 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
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--> 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).
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--> (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:
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--> 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). G. muris infection alters intestinal fluid accumulation and mucosal immune responses caused by cholera toxin in mice (Ljungstrom et al., 1985). Concurrent G. muris and Trichinella spiralis infection in mice results in suppression of the G. muris infection (Roberts-Thomson et al., 1976a). 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). Susceptibility to G. muris infection is increased by x-irradiation or protein-deficient diets (Owen et al., 1979). 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
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--> 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).
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--> 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): 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. 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
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--> 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).
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--> 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).
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--> 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).
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--> 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
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--> 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.
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--> 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).
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--> 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).
Representative terms from entire chapter: