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--> 6 Respiratory System Overview Diseases of the respiratory tract are among the most common health problems encountered in mice and rats. Numerous reports have dealt with inapparent respiratory infections as well as the respiratory diseases due to infectious agents in these animals. Nevertheless, the subject is large and tends to be confusing to persons not intimately involved in the study of these infections. At present there are 14 specific agents that have been recognized as respiratory pathogens in laboratory mice and rats (at some point in history and under some set of circumstances). They are extremely varied in pathogenicity and importance as research complications (which are not always directly related). Subclinical infection is far more common than overt disease for all of the agents. Synergistic interactions in which combined infections have more than an additive effect in producing disease are common (probably far more common than currently recognized). Dual or multiple infections usually are responsible when severe respiratory disease occurs (thus, diagnostic efforts must test for multiple agents and must obtain positive evidence for incriminating some and negative evidence for excluding others). Table 9 gives a perspective to the relative importance of respiratory infections as causes of clinical and morphologic disease. Agents are listed in order of descending importance (this is a rough approximation) for mice and rats. Those agents listed in group I are by far the major causes of overt respiratory disease in the species indicated. Mycoplasma pulmonis is deemed
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--> TABLE 9 Agents Grouped According to Importance as Causes of Natural Respiratory Disease Groupª Mouseb Ratb I Sendai virus Mycoplasma pulmonis Mycoplasma pulmonis Sendai virus CAR bacillus Streptococcus pneumoniae Corynebacterium kutscheri II Pneumonia virus of mice Rat coronavirus Pneumocystis carinii Sialodacryoadenitis virus Mycohaterium avium-intracellulare Pneumonia virus of mice Chlamydia trachomatis Pneumocystis carinii Klebsiella pneumoniae Klebsiella pneumoniae Streptococcus pyogenes Mycoplasma collis Mycoplasma neurolyticum Mycoplasma collis K virus III Corynebacterium kutscheri Pasteurella pneumotropica Chlamydia psittaci Bordetella bronchiseptica Pasteurella pneumotropica Adenovirus Bordetella bronchiseptica Adenovirus a Group Key: I = Agents that are unquestionably important respiratory tract pathogens. II = Agents of questionable importance or pathogenicity as respiratory tract pathogens, except in special circumstances. III = Agents that are not primary respiratory tract pathogens in 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 respiratory pathogens for that rodent species. the most important in the rat and Sendai virus the most important in the mouse. In actual practice, however, severe natural respiratory disease in the rat usually is due to M. pulmonis in combination with Sendai virus and/ or the cilia-associated respiratory (CAR) bacillus. In the mouse, combined infections of Sendai virus and M. pulmonis are responsible for the most severe outbreaks of natural respiratory disease, although Sendai virus infection alone also can cause severe disease when first introduced into a naive population of genetically susceptible mice. Streptococcus pneumoniae and Corynebacterium kutscheri are potent respiratory pathogens in the rat but seldom in the absence of some combination involving M. pulmonis, Sendai virus, and/or CAR bacillus. The agents listed in group II of Table 9 are relatively unimportant as natural respiratory pathogens in comparison to those of group I. Some of
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--> them (Mycobacterium avium-intracellulare, Chlamydia trachomatis, Klebsiella pneumoniae ozaenae, and Streptococcus pyogenes) have been reported to cause natural disease in only a few instances. Under natural conditions current evidence indicates that pneumonia virus of mice causes minimal upper respiratory tract disease and very mild transient lung disease (the rodent equivalent of man's common cold?). Sialodacryoadenitis virus (see "Digestive System" later in this volume) is listed here as some strains are also mild respiratory pathogens. Disease expression due to Pneumocystis carinii requires immunodeficiency or immunosuppression. Active disease due to Chlamydia psittaci and K virus are laboratory-induced occurrences. Mycoplasma neurolyticum and Mycoplasma collis are probably commensals. The agents included in group III of Table 9 are not primarily respiratory tract pathogens in the species indicated (Corynebacterium kutscheri in the mouse, Pasteurella pneumotropica, and adenovirus) or are not conclusively demonstrated to be natural pathogens of mice or rats (Bordetella bronchiseptica). Sendai Virus Significance Very high. Perspective 1950s: The early history of Sendai virus (SV) is confusing. The original isolations of the virus were made in the 1950s from mice that had been inoculated for diagnostic purposes using specimens from: (a) human infants with "newborn pneumonitis" in Japan, (b) swine with an influenza-like disease in Japan, or (c) humans with influenza in Russia. In subsequent years, evidence accumulated to show that an indigenous virus of the mice had been isolated (rodents are the exclusive natural hosts of SV). The seropositives among the human patients probably were due to a closely related, serologically cross reactive virus, parainfluenza 1, hemadsorption type 2, for which man is the natural host (Parker and Richter, 1982). 1968: Degre and Glasgow (1968) published the first in a series (Degre and Solberg, 1971) of papers from their laboratory demonstrating that SV infection increases susceptibility of mice to bacterial infection of the respiratory tract. Subsequently, major contributions in that area were made by Jakab and his colleagues (Jakab, 1981). 1975: Fukumi and Takeuchi (1975) reported development of a formalin killed SV vaccine.
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--> 1976: Ward et al. (1976) reported that athymic (nu/nu) mice had increased susceptibility to the virus, resulting in chronic infection with progressive emaciation. Increased susceptibility to SV infection was later reported to occur in athymic (rnu/rnu) rats (Carthew and Sparrow, 1980c). 1978: Parker et al. (1978) demonstrated an extremely wide range of susceptibility to SV among 24 strains of mice, and also reported that two-thirds or more of the mouse, rat, and hamster colonies in the United States were infected. Their work attracted great interest in SV infection and probably helped to stimulate the flurry of papers since 1978 that have documented the great importance of this agent as a complication of research. 1978: Howard et al. (1978) presented evidence that SV infection exacerbates Mycoplasma pulmonis infection in mice. Their findings in mice were confirmed by Saito et al. (1981). Schoeb et al. (1985) reported similar findings for rats. Agent An RNA virus, family Paramyxoviridae, genus Paramyxovirus, species parainfluenza 1 (Sendai). All known strains of SV are antigenically homologous. Some of the more common laboratory strains are: 52 (ATCC VR-105), Fushimi, Akitsugu, MN, and Z (Parker and Richter, 1982). The virus particles are spherical, 150-250 nm in diameter, and have a helical nucleocapsid and a continuous single stranded RNA genome. The virus contains HN glycoprotein with hemagglutinating and neuraminidase activities that are responsible for adsorption to host cells, and F glycoprotein with cell fusion and hemolytic activities that mediate virus entry into host cells. Entry of wild type Sendai virus into host cells requires conversion of the F glycoprotein to the biologically active form by host proteases. The HN glycoprotein also has been shown to be an inducer of type I interferon. The HN and F glycoproteins also are T cell-dependent B cell mitogens. The virus agglutinates erythrocytes of many species (Parker and Richter, 1982; Ito and Hosaka, 1983; Kizaka et al., 1983; Tashiro and Homma, 1983, 1985; Brownstein, 1986). SV is commonly grown in embryonated hen's eggs, and BHK-21 and primary monkey kidney cell cultures. It is inactivated by UV light, temperatures above 37°C, and lipid solvents (Parker and Richter, 1982). Hosts Laboratory mice, rats, and hamsters. Possibly, guinea pigs (based on serological evidence only, not confirmed by virus isolation).
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--> Epizootiology SV is EXTREMELY CONTAGIOUS, one of the most contagious infections of laboratory rodents. First time infections usually are epizootic within rooms, but can become epizootic throughout entire facilities or institutions (Zurcher et al., 1977)! The virus is highly prevalent (»70% of colonies) in laboratory mice and rats worldwide (Parker and Richter, 1982). Natural infection occurs via the respiratory tract. Contact and airborne transmission are born highly efficient (Parker and Reynolds 1968; van der Veen et al., 1970, 1972; Iida, 1972). Airborne transmission can occur over a distance of 5-6 feet (van der Veen et al., 1970, 1972). Viral replication is thought to be limited to the respiratory tract and occurs for only about 1 week post infection under usual circumstances. Viremia probably is a seldom occurrence. Transfer of embryos from infected mice to noninfected recipient mothers has been used successfully in eliminating the virus (Carthew et al., 1983; Parker and Richter, 1982). Clinical Natural infections of SV alone (i.e., not complicated by other agents) in rats are usually inapparent or cause only small reductions in litter size and growth rate of pups (Makino et al., 1972). Experimental infections of pregnant females have been reported to cause prolonged gestation, fetal resorptions, retarded embryonic development, and mortality of neonates (Coid and Wardman, 1971, 1972). Natural SV infections alone in mice usually follow one of two clinical patterns: Enzootic (subclinical) infection. This is the common pattern occurring in breeding populations. Adults have active immunity due to prior infection, and do not carry the virus. Newborn mice are passively protected by maternal antibody until around 4 to 8 weeks of age when they become infected. Recovery is prompt without morbidity or mortality. Infection is maintained by continuous supply of young susceptible mice (Iida et al., 1973; Fujiwara et al., 1976; Goto and Shimizu, 1978; Parker and Richter, 1982). Epizootic (clinically apparent) infection. This is the pattern that occurs when a population is first infected by the virus. Infection spreads through the entire population within a short time. Signs are variable but may include chattering, mild respiratory distress, and prolonged gestation in adults, deaths (even whole litters) in neonates and sucklings, and poor growth in weanling and young adult mice. Breeding colonies return to normal productivity in 2 months, and thereafter maintain the enzootic pattern of infection (Fukumi et al., 1962; Parker and Reynolds, 1968; Bhatt and Jonas,
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--> 1974; Fujiwara et al., 1976; Zurcher et al., 1977; Itoh et al., 1978; Parker and Richter, 1982). Epizootics of disease involving SV virus infection in mice and rats which exceed the above general patterns in clinical severity should arouse suspicion of complication by other agent(s), particularly concurrent M. pulmonis and/ or CAR bacillus infection (Lindsey et al., 1985a; Schoeb et al., 1985). Pathology Strains of mice vary markedly in susceptibility to SV. The more susceptible stocks include 129/ReJ, 129/J, S-nu/nu (Swiss nude), DBA/1J, and DBA/2J. Stocks of intermediate susceptibility are A/HeJ, A/J, SWR/J, C57BL/1OSn and BALB/c. The most resistant stocks include SJL/J, RF/J, C57BL/6J and S. Strain 129/J is 25,000 times as susceptible to lethal infection as SJL/J (Parker et al., 1978). The mode of inheritance and mechanisms of host resistance are poorly understood (Brownstein, 1983, 1986, 1987a,b; Brownstein and Winkler, 1986, 1987). SV infections have been studied most in mice of the resistant stocks. In resistant stocks of mice and in rats, pathogenesis is approximately as follows. After intranasal infection, descending infection follows with virus replication occurring in respiratory epithelium of the nasal passages, trachea, bronchi, bronchioles, and in type I and II pneumocytes and macrophages of the alveoli. Virus titer peaks in tracheobronchial epithelium at 5 to 6 days, then decreases to undetectable levels throughout the respiratory tract around day 14 post infection. Serum antibody appears at 6 to 8 days and remains detectable for approximately 1 year depending on sensitivity of the test used. Secretory antibody may appear as early as day 3, but is usually difficult to demonstrate before days 6 to 10 post infection (Sawicki, 1962; Parker and Reynolds, 1968; Robinson et al., 1968; van der Veen et al., 1970; Appell et al., 1971; Blandford and Heath, 1972; Charlton and Blandford, 1977; Parker et al., 1978; Brownstein et al., 1981; Castleman, 1983, 1984; Castleman et al., 1987; Garlinghouse et al., 1987 ). Morphogenesis of lesions in SV infection of mice and rats proceeds by the following general pattern. Following intranasal infection, there is descending infection with transient hypertrophy, necrosis, and repair of airway epithelium occurring in rapid succession. Necrosis of respiratory epithelium is mild and focal in nasal passages beginning at 2-3 days, becomes progressively more severe distally with peak severity in the distal trachea and major bronchi around day 5. Regeneration of airway epithelium becomes evident by day 9, with epithelial hyperplasia, squamous metaplasia, and occasional syncytial giant cell formation. Focal interstitial pneumonia occurs with alveolar septal thickening by edema, mononuclear cell infiltration, alveolar epithelial hypertrophy and hyperplasia, and atelectasis. Resolution is in progress well before 21 days, although residual inflammatory lesions
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--> may persist up to 1 year or longer. These most often consist of lymphocytes around airways and vessels, always in loose concentration rather than dense cuffs. Some reports mention vasculitis. Despite these impressive histologic changes, few gross lesions are seen in uncomplicated SV infections. The lungs may appear focally reddened or atelectatic and serous fluid may be visible in the pleural and pericardial cavities (Sawicki, 1961, 1962; Fukumi et al., 1962; Robinson et al., 1968; Appell et al., 1971; Degre and Midtvedt, 1971; Blandford and Heath, 1972; Richter, 1973; Ward, 1974; Brownstein et al., 1981; Parker and Richter, 1982; Castleman, 1983, 1984; Hall et al., 1985; Schoeb et al., 1985; Castleman et al., 1987; Giddens et al., 1987). The most severe lesions due to SV are seen in fully susceptible mice infected while very young (as sucklings or weanlings) and in mice of the more susceptible stocks (such as DBA/2J and 129/ReJ). The terminal bronchioles are particularly susceptible to severe injury. During the period of severe necrotizing bronchitis and bronchiolitis, there may be intense inflammatory injury to terminal bronchioles. This may result in scarring with severe distortion of the smaller airways and formation of polypoid outgrowths into the bronchiole lumens. Also, there may be pronounced hyperplasia of airway epithelium resulting in peribronchiolar "adenomatous hyperplasia" (also called "adenomatoid change" and "alveolar bronchiolization") that may persist throughout life of the animal. In aged mice the air spaces in these lesions may be filled with mucus, large macrophages, and cellular debris. There may be large eosinophilic crystals in the air spaces, and in the cytoplasm of the macrophages and cells forming the "adenomatoid" structures (Yang and Campbell, 1964; Richter, 1970, 1973; Parker and Richter, 1982; Zurcher et al., 1977). The terminal bronchioles of rats also may be scarred and distorted but do not show the hyperplastic peribronchiolar changes seen in mice (Castleman, 1983, 1984). Athymic (nu/nu) mice have increased susceptibility to SV. They develop chronic pneumonia similar to that in immunocompetent mice but have abundant intranuclear and intracytoplasmic inclusions in laryngeal, tracheal, bronchial, and bronchiolar epithelium, as well as in type I and II pneumocytes and alveolar macrophages. The virus persists for 10 weeks or longer (Ward et al., 1976; Ueda et al., 1977b; Iwasaki, 1978; Iwai et al., 1979). Nude (rnu/rnu) rats also have increased susceptibility to SV and develop a similar chronic lung disease (Carthew and Sparrow, 1980c). The immune responses to SV that confer protection have not been completely defined. However, it appears that both T and B cells have important roles (Kizaka et al., 1983; Ertl and Finberg, 1984a,b). Passive immunization of mice using monoclonal antibodies against specific subgroup antigens of the viral F and HN glycoproteins has given protection against experimental SV challenge (Orvell and Grandien, 1982; Mazanec et al., 1987). In mice, L3T4+ (and Lyt-1+) and Lyt-2+ subsets of T cells may be important in clearing the virus from infected lungs (Iwai et al., 1988).
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--> Some reports of SV infection in the literature include lesions that are not attributable to SV alone (Burek et al., 1977). Lesions such as suppurative bronchitis, pulmonary abscesses, and dense peribronchial and perivascular lymphoid cuffs are suggestive of M. pulmonis infection, possibly superimposed on SV infection. SV is a strong promoter of murine respiratory mycoplasmosis due to M. pulmonis in mice (Howard et al., 1978; Saito et al., 1981) and rats (Schoeb et al., 1985). Diagnosis The enzyme-linked immunosorbent assay (ELISA) is the test of choice for routine serologic monitoring. It is 100 times more sensitive than the complement fixation (CF) test and 300 times more sensitive than the hemagglutination inhibition (HI) test. Because of the high contagiousness of the virus, typically about 90% of animals in infected populations will be positive by the ELISA (Parker et al., 1978, 1979; Ertl et al., 1979; Parker, 1980). The ELISA successfully detects anti-SV antibody in infected athymic (nu/nu) mice (Iwai et al., 1984), compared to the CF test that is sometimes positive at low titer (Ward et al., 1976; Iwai et al., 1977), and the HI and neutralization tests that usually do not detect antibody to SV in infected nude mice (Iwai et al., 1977). A quantitative immunofluorescence test for detection of serum antibody to SV has been reported (Lucas et al., 1987). In instances where natural SV infection is associated with clinical disease or gross lung lesions, other intercurrent infection(s), e.g., M. pulmonis, often have a contributory role. Definitive diagnosis of such disease states requires detection of each of the agents involved, demonstration of the characteristic lesions due to each agent, and exclusion of other agents and disease processes. An avidin-biotin-peroxidase complex method has been used successfully for demonstrating SV antigen in histologic sections (Hall and Ward, 1984). Isolation of SV may be achieved using BHK-21 or primary monkey kidney cell cultures, or 8 to 10 day embryonated hen's eggs inoculated into the amniotic or allantoic sac (Parker and Richter, 1982). The mouse antibody production (MAP) test may be used in testing transplantable tumors and other biologic materials for contamination by SV (Rowe et al., 1962). Control Exclusion of SV is EXTREMELY DIFFICULT in most institutions that receive rodents from outside sources. Ordinarily, exclusion requires very strict adherence to systematic measures for preventing entrance of the infection into an entire facility or institution. SV free subpopulations of rodents must be identified by regular health surveillance of a supplier, transported to the user facility in containers which prevent contamination
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--> en route, quarantined by barrier system at the receiving institution until tested and shown to be free of infection, and subsequently maintained by strict barrier protocol. In addition, all biological materials such as transplantable tumors coming into the institution must be pre-tested and shown to be free of the virus before experimental use (Collins and Parker, 1972; Parker and Richter, 1982). Once infection has been diagnosed in a facility, prompt elimination of infected subpopulation(s) is essential to prevent spread of the infection to other rodents on the premises. A less effective alternative is to place the infected animals under strict quarantine, remove all young and pregnant females, suspend all breeding, and prevent addition of other susceptible animals for a period of 6-8 weeks until the infection has run its course and the virus has been eliminated naturally. Because of this alternative, cesarean derivation of infected stocks usually is not justified. Vaccination may prove useful in some situations (Parker, 1980; Eaton et al., 1982). A number of killed vaccines (Fukumi and Takeuchi, 1975; Nedrud et al., 1987; Tsukui et al., 1982), a temperature sensitive mutant strain vaccine (Kimura et al., 1979), and a trypsin-resistant mutant strain vaccine (Tashiro and Homma, 1985; Tashiro et al., 1988) have been tested experimentally. A formalin-killed SV vaccine is available commercially in the United States (Microbiological Associates, Bethesda, Md.). Interference with Research Experimental infection of mice with SV decreases pulmonary bacterial clearance (Degre and Glasgow, 1968; Degre and Solberg, 1971), probably through a variety of mechanisms including altered phagocytic function. Altered functions in pulmonary macrophages that have been identified include: decreased Fc receptor and non-Fc receptor mediated attachment, decreased Fc receptor and non-Fc receptor mediated ingestion, inhibited phagosome-lysosome fusion, decreased intracellular killing, decreased degradation of ingested bacteria, and decreased lysosomal enzyme content (Jakab, 1981; Jakab and Warr, 1981). Concurrent SV and M. pulmonis infections are synergistic in mice (Howard et al., 1978; Saito et al., 1981) and rats (Schoeb et al., 1985), causing disease of far greater severity than either alone. SV infected mice have been reported to have deficiencies in T and B cell function that persist throughout life (Kay, 1978, 1979; Kay et al., 1979). (Unfortunately, these results have not been confirmed by other investigators). SV infection transiently increased splenic IgM and IgG plaque forming cell responses to sheep red blood cells in mice (Brownstein and Weir, 1987). SV infection inhibited in vitro mitogenesis of lymphocytes (Wainberg and Israel, 1980; Roberts, 1982).
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--> In rats, infection altered the mitogenic responses of T cells, reduced severity of adjuvant arthritis, and decreased antibody response to sheep erythrocytes (Garlinghouse and Van Hoosier, 1978). Mice naturally infected with SV have been found to have increased natural killer cell mediated cytotoxicity (Clark et al., 1979), and increased cytotoxic lymphocyte responses after stimulation with SV-coated syngeneic cells (Finberg et al., 1980). SV infection altered isograft rejection in mice (Streilein, et al., 1981). SV infection altered host responses to transplantable tumors (Wheelock, 1966, 1967; Collins and Parker, 1972; Matsuya et al., 1978; Takeyama et al., 1979). Previous or concurrent infection in mice may increase or decrease neoplastic response to respiratory carcinogens (Nettesheim et al., 1974, 1981; Parker, 1980; Peck et al., 1983; Hall et al., 1985). SV infection delayed wound healing in mice (Kenyon, 1983). Athymic (nulnu) mice (Ueda et al., 1977b; Iwasaki, 1978; Iwai et al., 1979) and nude (rnulrnu) rats (Carthew and Sparrow, 1980c) had increased susceptibility and developed chronic lung disease when infected with SV. Cyclophosphamide increased clinical and pathological severity of SV infection in mice (Robinson et al., 1969; Blandford, 1975; Anderson et al., 1980). SV infection can cause deaths and retarded growth of young mice (Parker and Reynolds, 1968; Bhatt and Jonas, 1974) and rats (Makino et al., 1972). Mycoplasma pulmonis Significance Very high, particularly in long term studies. Perspective 1937: Nelson (1937a,b,c) described the proximal airway disease called "infectious catarrh" in mice and attributed it to "coccobacilliform bodies" (later identified as M. pulmonis). 1937: Klieneberger and Steabben (1937) described pulmonary "bronchiectasis" in rats, and subsequently (Klieneberger and Steabben, 1940) recognized the association of their "L3" organism (later identified as M. pulmonis) with this lesion. 1957: Nelson (1957) advanced the term "chronic respiratory disease" and proposed that it was due to two agents: M. pulmonis which caused "infectious catarrh" (proximal airway disease), and "enzootic bronchiectasis virus" alleged to cause the bronchopulmonary (distal airway) disease. (This putative virus still has not been identified.)
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--> 1966: Lutsky and Organick (1966) fulfilled Koch's postulates (for both proximal and distal airway disease) by the inoculation of M. pulmonis into pathogen free mice; their work was confirmed by Lindsey and Cassell (1973). 1969: Kohn and Kirk (1969) fulfilled Koch's postulates (for proximal and distal airway disease) by inoculation of M. pulmonis into pathogen free rats. Their work was confirmed by Lindsey et al. (1971), Whittlestone et al. (1972), and Jersey et al. (1973). 1976: Broderson et al. (1976a) demonstrated that intracage ammonia promotes respiratory disease due to M. pulmonis in rats. The work was confirmed in rats by Schoeb et al. (1982), and in rats and mice by Saito et al. (1982). 1978: Howard et al. (1978) showed that Sendai virus infection promotes respiratory disease due to M. pulmonis in mice; this was confirmed in mice by Saito et al. (1981) and in rats by Schoeb et al. (1985). 1978: Horowitz and Cassell (1978) developed an enzyme-linked immunosorbent assay (ELISA) for detection of rodent mycoplasma infections. The test was extensively field tested by Cassell et al. (1981, 1983b). 1984: Minion et al. (1984) introduced the immunoblot method for discriminating between infections due to M. pulmonis and Mycoplasma arthritidis using mycoplasma ELISA positive sera. 1987: Schoeb and Lindsey (1987) demonstrated that sialodacryoadenitis virus infection exacerbates respiratory disease due to M. pulmonis in rats. Agent This is a bacterium, class Mollicutes, order Mycoplasmatales, family Mycoplasmataceae (sterol-requiring mycoplasmas). Gram negative, lacks a cell wall, pleomorphic but usually spherical to pear-shaped, 0.3 to 0.8 um in diameter. Grows on conventional horse serum-yeast extract mycoplasma medium, usually under facultatively anaerobic conditions at pH 7.8, 37°C, and 95% relative humidity. Ferments glucose. Rarely produces "fried egg" appearance when grown on solid medium (Razin and Freundt, 1984). For details of methodology for cultural isolation, see Cassell et al. (1983a). Speciation of mycoplasmas is based on biochemical and serological tests (Razin and Freundt, 1984). Rapid presumptive identification of M. pulmonis can be made by the hemadsorption test (Manchee and Taylor-Robinson, 1968), but some strains do not hemadsorb (Tamura et al., 1981). Type strain is ATCC 19612 [NCTC 10139; Ash (PG34)]. Other well known strains include: Peter C, Negroni, WRAIR, JB and Ogata T. All strains are currently considered members of a single serotype. Different strains vary greatly in virulence (Davidson et al., 1988a), but virulence factors of M. pulmonis have not been defined (Razin and Freundt, 1984; Davidson et al., 1988b).
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--> Clinical Nonspecific signs of dyspnea, sneezing, cervical lymphadenopathy, inappetence, hunched posture, and rough hair coat have been observed in diseased mice (Flamm, 1957; Schneemilch, 1976). In the reported outbreaks in rats, there were a few deaths, and some rats had abscesses in the cervical and inguinal lymph nodes with fistulous tracts to the adjacent skin surface (Hartwich and Shouman, 1965; Jackson et al., 1980). Pathology Mice with natural disease have cervical lymphadenitis; cervical, pharyngeal. renal, and hepatic abscesses; empyema; and granulomatous pneumonia. Experimental inoculation of organisms into the buccal mucosa induced a syndrome identical to that of the naturally occurring disease (Flamm, 1957; Schneemilch, 1976). Rats with natural disease had submaxillary, parotid, or inguinal lymph node abscesses, often with fistulous tracts draining to the skin; abscesses in mesenteric nodes; and renal abscesses. Respiratory lesions either were not observed or were considered a minor part of the disease (Hartwich and Shouman, 1965; Jackson et al., 1980). Rats are commonly used for studies of experimental pneumonia induced by the inoculation of capsular type 1 K. pneumoniae (Berendt et al., 1977; Coonrod, 1981; Domenico et al., 1982). The extent to which such studies are relevant to the natural disease in rats is uncertain. Diagnosis The few reports of natural disease associated with this agent are insufficient to allow firm conclusions about its role as a primary pathogen in mice and rats. Like other opportunistic pathogens, host factors probably are extremely important determinants of disease caused by this organism. Diagnostic efforts must differentiate between possible roles of K. pneumoniae: primary pathogen, secondary pathogen, or coincidental infection. Case studies should include isolation, identification, and serotyping of the organism; serologic and culture procedures to exclude other infectious agents; necropsy with histopathologic examination of all major organs and gross lesions; and efforts to identify possible contributing host factors. Also, efforts should be made to fulfill Koch's postulates through experimental infections of pathogen-free mice or rats by using an isolate of K. pneumoniae from a natural lesion.
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--> Control Uncertain. K. pneumoniae ozaenae is presumably a part of the normal gastrointestinal flora of mice and rats. Interference with Research K. pneumoniae is an opportunistic pathogen that may complicate studies in which host defenses are compromised. Streptococcus pyogenes Significance Very low. Perspective Nelson (1954) described a natural outbreak of cervical lymphadenitis caused by a group A streptococcus in laboratory mice; the organism was later identified as S. pyogenes serotype 50 (Hook et al., 1960). A second occurrence of this disease was reported by Hook et al. (1960), who also obtained evidence that two epizootics of the disease had occurred previously in mice of two additional laboratories in the vicinity of New York City, one in 1935 and the other in 1959. Agent A bacterium, family Streptococcaceae, S. pyogenes, group A, serotype 50. S. pyogenes is composed of b-hemolytic, microaerophilic, bacitracinsusceptible, Gram-positive cocci that usually form chains. The species is subdivided into groups on the basis of Lancefield's group antigens and into serotypes based on cell wall M and T antigens. Serotyping is sometimes helpful in tracking common source outbreaks (Lancefield, 1972; Facklam and Carey, 1985). Hosts Humans are considered the natural host of b-hemolytic group A S. pyogenes (Lancefield, 1972). There are only two reports (Nelson, 1954; Hook et al., 1960) of natural infection in laboratory mice.
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--> Epizootiology Man is the natural reservoir of b-hemolytic group A streptococci. Transmission is mainly via close contact or contaminated food and usually involves an asymptomatic carrier colonized in the nasopharynx, skin, vagina, or rectum (Facklam and Carey, 1985). Hook et al. (1960) found that up to 52% of mice from a commercial breeding facility harbored the organism in their throats. One-third of infected mice developed cervical lymphadenitis, and approximately 50% of those observed for 3 months died of the streptococcal infection. The source of infection for the mice studied by Nelson (1954) and Hook et al. (1960) was not determined. The fact that the mice of all epizootics reported by Nelson (1954) and Hook et al. (1960) were in the vicinity of New York City suggests that there could have been a common source of infection. Hook et al. (1960) were unsuccessful in culturing group A streptococci from 40 throat cultures obtained from 15 people who worked with their mice. At least four stocks of mice, S (Swiss), Princeton (the noninbred forerunner of strain PL), C57 (strain designation incompletely given), and A (an unspecified stock designated A by Jacob Furth), have been involved in four spontaneous epizootics (Hook et al., 1960). Clinical Some mice carried the organism in their throats for more than 90 days without developing clinical signs of infection. Affected mice showed ruffled hair coats and inactivity for a few days before death. In the more advanced cases the cervical lymph nodes were enlarged and often had purulent exudate draining through fistulous tracts to the skin (Nelson, 1954; Hook et al., 1960). Pathology Only gross descriptions of the pathology in this disease have been published. The lesions reported included suppurative cervical lymphadenitis (with or without drainage to the skin), otitis media, rhinitis, and pneumonia. Myriads of organisms were demonstrated in exudates from cervical nodes. Septicemia caused by S. pyogenes was considered an important cause of death because the organism was often cultured from heart blood of animals that died (Nelson, 1954; Hook et al., 1960). Wildfeuer et al. (1978) carried out experimental studies in which mice were infected intranasally with the organism. Suppurative cervical lymphadenitis was produced regularly, and the infection in mice was proposed as an experimental model of human streptococcal pharyngitis.
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--> Diagnosis The diagnosis is based on demonstration of characteristic lesions, isolation and identification of the organism, and exclusion of other possible causes. Control Uncertain. Cesarean derivation and barrier maintenance should be effective in eliminating the organism from an infected stock of mice. Interference with Research Mortality due to this organism reached 50% during one epizootic in mice (Hook et al., 1960). Hook et al. (1960) observed increased numbers of deaths in naturally infected mice injected intracerebrally with either sterile saline or sublethal doses of bacterial endotoxin. Mycoplasma neurolyticum Significance Uncertain, probably very low. Perspective M. neurolyticum has been isolated occasionally from mice and rats since 1938 (Findlay et al., 1938; Sabin, 1938a,b), but there is only one instance in which it has been thought to be a natural pathogen. Nelson (1950a,b) described a colony of mice in which he associated the occurrence of conjunctivitis with presence of this organism. However, that alleged association has not been confirmed in the intervening 35 years since Nelson's reports; and experimental inoculations of M. neurolyticum into mice by the conjunctival, intranasal, or intravenous route have consistently failed to cause conjunctivitis (Cassell and Hill, 1979). Thus, this organism is not considered a natural pathogen. Agent Mycoplasma neurolyticum is a bacterium, class Mollicutes, order Mycoplasmatales, family Mycoplasmataceae (sterol-requiring mycoplasmas). It is Gram negative, lacks a cell wall, pleomorphic but usually spherical to pear-shaped, and measures 0.3-0.8 µm in diameter. It may produce filaments up to 160 µm long. M. neurolyticum grows on conventional horse serum-
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--> yeast extract mycoplasma medium, usually under facultatively anaerobic conditions at pH 7.8, 37°C. and 95% relative humidity. Unlike other rodent mycoplasmas, growth is inhibited by penicillin G (Hottle and Wright, 1966). It ferments glucose. M. neurolyticum rarely produces "fried egg" appearance when grown on solid medium. For details of methodology for cultural isolation, see Cassell et al. (1983a). Identification of the species of Mycoplasma is based on biochemical and serologic tests (Razin and Freundt, 1984). The type strain is ATCC 19988 (NCTC 10166). It produces a true exotoxin that is neurotoxic and causes "rolling disease" when injected intravenously into mice or young rats. Neurotoxicity also occurs when washed living organisms are given intravenously, intraperitoneally, or intracerebrally. The exotoxin is a protein with a molecular weight of greater than 200,000. It is thermolabile and is inactivated at 50°C in 10-30 minutes or 45°C in 15-90 minutes (Razin and Freundt, 1984). Hosts Laboratory and wild mice and laboratory rats (Cassell and Hill, 1979). Epizootiology M. neurolyticum has been isolated from the conjunctiva, nasal passages, Harderian glands, and brains of laboratory mice (Findlay et al., 1938; Sabin, 1938a,b, 1939; Sabin and Johnson, 1940; Nelson 1950a,b; Tully and Rask-Nielsen, 1967; Hill, 1974a; Cassell and Hill, 1979) and from the conjunctiva of wild mice and laboratory rats (Hill, 1974a; Cassell and Hill, 1979). Thus, the mucous membranes of the conjunctiva and upper respiratory tract are presumably the main sites of predilection for the organism. Data on the natural history of the infection are lacking. The prevalence of M. neurolyticum infection in contemporary rodent stocks is unknown. However, it appears to be very low because the organism is rarely isolated by those laboratories that routinely culture nasal passages with suitable media for monitoring the health of large numbers of mice and rats (M. K. Davidson, Department of Comparative Medicine, University of Alabama at Birmingham, personal communication). Clinical Infections due to M. neurolyticum are subclinical. Pathology No gross or microscopic lesions are associated with natural M. neuro-
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--> lyticum infection or experimental inoculation of the organism into the conjunctiva or nasal passages (Cassell and Hill, 1979). In natural infections the organism is apparently a commensal. The intravenous, intraperitoneal, or intracerebral inoculation of M. neurolyticum-infected tissues, broth cultures containing viable organisms, or cell-free filtrates of M. neurolyticum cultures into mice or young rats causes severe cerebral edema (spongiform degeneration) manifested clinically by rolling from side to side (Findlay et al., 1938; Tully and Ruchman, 1964; Aleu and Thomas, 1966; Thomas, 1967) and is caused by the exotoxin of M. neurolyticum (Tully, 1964; Thomas and Bitensky, 1966; Thomas, 1967; Tully and Rask-Nielsen, 1967). Ultrastructurally, there is extreme distension of astrocytes by fluid with mechanical displacement and compression of myelinated axons, accumulation of extracellular fluid in the white matter, and degeneration of myelin sheaths of axons (Aleu and Thomas, 1966). The neurotoxin is thought to bind to ganglioside receptors on astrocyte podocytes resulting in disruption of normal regulation of fluid transport (Thomas et al., 1966). [This so-called rolling disease experimentally induced by M. neurolyticum is not to be confused with the naturally occurring circling or rolling disease in mice that has been associated with inner ear disease caused by Pseudomonas aeruginosa (Gorrill, 1956; Ediger et al., 1971; Kohn and Mackenzie, 1980).] Diagnosis Cultural isolation of the organism is the only proven method for diagnosing M. neurolyticum infection. Lavage or swab samples from nasal passages and conjunctivas should be cultured in mycoplasma media without penicillin, which is inhibitory to some strains of the organism (Hottle and Wright, 1966; Cassell et al., 1983a). Control No data are available. Presumably, the organism can be eliminated from infected stocks by cesarean derivation and barrier maintenance techniques. Interference with Research The intracerebral passage in mice of Toxoplasma gondii (Sabin, 1938a) and lymphocytic choriomeningitis and yellow fever viruses (Findlay et al., 1938) was complicated by contamination of the passaged tissues with M. neurolyticum resulting in the occurrence of rolling disease. M. neurolyticum may be a common contaminant of transmissible mouse leukemia cell lines (Tully and Rask-Nielsen, 1967).
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--> Mycoplasma collis Significance Unknown. Perspective This is a recently described species of mycoplasma that appears to be a nonpathogenic inhabitant of the conjunctiva and nasopharynx in mice and rats. Agent Mycoplasma collis is a bacterium, class Mollicutes, order Mycoplasmatales, family Mycoplasmataceae (sterol-requiring mycoplasmas). Strains of mycoplasmas provisionally designated Gough from mice (Hill, 1974a) and 58b from rats (Young and Hill, 1974) were later found to be serologically identical and have been assigned to a new species, M. collis (Hill, 1983). It grows on standard medium for murine mycoplasmas and utilizes glucose (Cassell et al., 1983a: Hill, 1983). Hosts Mice and rats. Epizootiology The organism has been isolated from one mouse colony and four rat colonies in the United Kingdom. The isolates were from the conjunctiva in mice (Hill, 1974a) and from the conjunctiva, Harderian gland, and nasopharynx in rats (Young and Hill, 1974). The prevalence is unknown (Hill, 1983). Clinical Rats with the natural infection had conjunctivitis (Young and Hill, 1974), but attempts to reproduce the disease experimentally by inoculating pathogen-free rats with cultures of strain 58B failed (Hill, 1974b). Clinical signs have not been observed in infected mice (Hill, 1974a). Thus, M. collis infection alone is subclinical.
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--> Pathology The organism is considered a nonpathogen. Diagnosis Diagnosis is by cultural isolation (Hill, 1974a; Young and Hill, 1974; Cassell et al., 1983a). Use of the mycoplasma enzyme-linked immunosorbent assay (Cassell and Brown, 1983) for diagnosing this infection has not been investigated. Control No data are available. Presumably, cesarean derivation and barrier maintenance would be effective. Interference with Research Unknown. K Virus Significance Very low. Perspective This agent was originally isolated by Kilham (1952) from asymptomatic C3H mice carrying the Bittner agent. Although the virus initially attracted much attention for causing pneumonitis when passaged to infant mice (Fisher and Kilham, 1953; Kilham and Murphy, 1953), it is now mainly of interest as an experimental model of acute and persistent papovavirus infections in mice. Agent The agent is a small DNA virus, family Papovaviridae. genus Polyoma virus. K virus is taxonomically related to polyoma virus, but the two agents are immunologically distinct (Dalton et al., 1963; Mattern et al., 1963; Bond et al., 1978). Virions are spherical and measure 35-45 nm in diameter. Synonyms for K virus are K papovavirus (Jordan and Doughty, 1969;
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--> Takemoto and Fabisch, 1970), Kilham virus (Kraus et al., 1968), and mouse pneumonitis virus (Parsons, 1963). K virus is resistant to environmental conditions. In tissue suspensions at room temperature it has been found to remain stable for 11 weeks. It withstands ether, acid pH, repeated freezing and thawing, heating to 70°C for 3 hours, and exposure to 0.5% formalin. It agglutinates sheep erythrocytes (Kilham, 1952, 1961c; Holt, 1959). Limited success has been achieved in the culture of K virus in vitro using primary mouse embryo cells (Greenlee et al., 1982). Host Mice (Mus musculus), exclusively. Wild mice are considered the natural hosts. Epizootiology K virus is considered to have a worldwide distribution. It occurs as an enzootic, subclinical, persistent infection primarily in feral Mus musculus, but it has been found in conventionally reared populations of laboratory mice as well. Prevalence of infection within populations is usually low (about 10%). It is rare in contemporary cesarean-derived, barrier-maintained stocks in the United States (Rowe et al., 1962, 1963; Tennant et al., 1966; Parker and Richter, 1982). The natural history of infection within mouse populations is poorly understood. The virus is shed in milk, urine, and feces, and natural transmission is thought to be by ingestion. Contaminated food and bedding may be important as the virus is stable for long periods outside the host. The virus persists in the host for at least 8 months and perhaps, for life (Greenlee, 1979, 1981; Greenlee and Dodd, 1984; Parker and Richter, 1982). Clinical Natural infections are subclinical. Clinical disease results from experimental inoculation of the virus into infant mice less than 8 days of age (Kilham, 1952; Kilham and Murphy, 1953). Pathology In the experimental model of acute K virus infection, 1- to 3-day-old mice are given the virus. The intracerebral route is preferred, but almost any other route (intraperitoneal, intranasal, subcutaneous, or oral) is also satisfactory. After an incubation period of 6-15 days, there is a sudden onset of "chugging" (pumping) respiration followed by death within a few
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--> hours. The gross lesions include pulmonary congestion, hemorrhage, atelectasis, and edema, with hydrothorax (Fisher and Kilham, 1953; Kilham and Murphy, 1953; Holt, 1959; Margolis et al., 1976). The most striking histologic lesions in experimentally infected infant mice occur in the lungs. Characteristically, there is diffuse interstitial pneumonia with numerous prominent amphophilic to basophilic intranuclear inclusions in vascular endothelium throughout the lungs. Because of these findings, it was thought for many years that viral replication and cytopathic effects occurred exclusively in the pulmonary endothelium (Gleiser and Heck, 1972; Margolis et al., 1976). Subsequently, it has been shown that the major sites of K virus replication are the pulmonary endothelium and hepatic sinusoidal lining cells, with less involvement of cells in spleen, lymph nodes, and brain (Greenlee, 1979, 1981). Susceptibility of infant mice has been related to the inability to mount an antibody response (Mokhtarian and Shah, 1980; Greenlee, 1981). Athymic (nu/nu) mice produce low levels of virus-specific IgM and are no more susceptible to infection than immunocompetent mice (Mokhtarian and Shah, 1983). Persistent K virus infections have been reactivated 8 months post infection by the administration of 8 weekly injections of cyclophosphamide at a dose of 150 mg/kg (Greenlee and Dodd, 1984). K virus has been reported to transform cells in vitro (Takemoto and Fabisch, 1970; Greenlee and Law, 1984), but unlike polyoma virus, it is not known to be tumorigenic in vivo. Diagnosis The hemagglutination inhibition test and the complement fixation test are the most commonly used serologic tests. The intracerebral inoculation of the organism into infant mice and/or the mouse antibody production test may be used for testing biologic materials for K virus contamination (Parker and Richter, 1982). Even under the best of conditions, the detection of K virus in a population of mice can be difficult, as appropriately emphasized by the following quote from Parker and Richter (1982): "The predominant characteristics of K virus infection are latency, chronicity, low incidence, low antibody titers in recovered mice, and infection in older mice. Thus, testing large numbers of mice at frequent intervals and testing mice of all ages, especially those 7 months and older, may be required to certify a population free of infection." Control Cesarean derivation and barrier maintenance have been very successful in eliminating the infection (Parker and Richter, 1982).
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--> Interference with Research The virus can be a contaminant of transplantable murine tumors causing early deaths in suckling recipient mice (Fisher and Kilham, 1953; Rowe et al., 1962). K virus infection enhances the severity of hepatic necrosis caused by mouse hepatitis virus (Tisdale, 1963). K virus has been reported to transform cells in vitro (Takemoto and Fabisch, 1970; Greenlee and Law, 1984).
Representative terms from entire chapter: