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Infectious Diseases of Mice and Rats (1991)

Chapter: 6. Respiratory System

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Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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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

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

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

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

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.

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

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).

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×
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:

  1. 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).
  2. 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,
Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×
  • 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

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

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).

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

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

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

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).

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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In 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.)

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

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).

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

M. pulmonis can be preserved indefinitely by lyophilization or freezing at -70°C (Razin and Freundt, 1984).

Hosts

Rats and mice are considered the natural hosts. Isolated on occasion from wild rats, cotton rats, rabbits, Syrian hamsters and guinea pigs (Lindsey et al., 1982).

Epizootiology

M. pulmonis infection and disease are common in conventionally reared rats and mice. Subclinical (often noncultivable) infection occurs in some cesarean derived, barrier maintained stocks (Cassell et al., 1981, 1983a,b; 1986).

Sites of predilection for the organism in the host are nasopharynx and middle ears (Davidson et al., 1981). M. pulmonis also has been reported from up to 40% of genital tracts in conventionally reared LEW rats (Cassell et al., 1979; Cassell, 1982).

Transmission is thought to be by the intrauterine route and by aerosol between cagemates, including from dam to offspring, and between adjacent cages (Hill, 1972; Lindsey et al., 1982).

M. pulmonis poorly resists environmental conditions outside the host, particularly drying (Vogelzang, 1975).

Clinical

Infection occurs most commonly without clinical signs (as is true of most indigenous pathogens of rats and mice). Signs are nonspecific, but may include: "snuffling" in rats, "chattering" in mice, rales, polypnea, weight loss, hunched posture, ruffled coat, inactivity, "head tilt" and in rats, accumulation of porphyrin pigment around the eyes and external nares (Lindsey et al., 1971, 1982).

Pathology

M. pulmonis is an extracellular parasite that preferentially colonizes the luminal surface of respiratory epithelium. Organisms (and lesions if present) tend to decrease from proximal to distal airways. Under ideal conditions for the host, the organism probably is a commensal (Cassell, 1982; Lindsey et al., 1986b).

Murine respiratory mycoplasmosis (MRM) is the disease of laboratory rats and mice which is caused by M. pulmonis but varies greatly in expression

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

because of environmental, host, and organismal factors that influence this host-parasite relationship (Lindsey et al., 1985, 1986b). Intracage ammonia concentrations of 19 µg/liter of air or greater (Broderson et al., 1976a; Saito et al., 1982; Schoeb et al., 1982), concurrent Sendai virus infection (Howard et al., 1978; Saito et al., 1981; Schoeb et al., 1985), or concurrent sialodacryoadenitis virus infection (Schoeb and Lindsey, 1987) promote MRM by increasing growth of M. pulmonis in the respiratory tract. Other factors that influence expression of MRM include: concurrent infection with the CAR bacillus (see page 48); administration of hexamethylphosphoramide (Overcash et al., 1976; K. T. Lee and Trochimowicz, 1982a,b) or cyclophosphamide (Singer et al., 1972); vitamin A or E deficiency (Tvedten et al., 1973); inhalation of tobacco smoke (Wynder et al., 1968); genetic susceptibility of the host [e.g., LEW rats are more susceptible than F344 rats (Davis and Cassell, 1982; Davis et al., 1982); and C3H/HeN mice are more susceptible than C57BL/6N mice (Davis et al., 1985)]; and virulence of the M. pulmonis strain (Lindsey et al., 1971; Whittlestone et al., 1972; Howard and Taylor, 1979; Davidson et al., 1988a).

Characteristic microscopic lesions of MRM at any level in the respiratory tract include: neutrophils in the airways, hyperplasia of mucosal epithelium, and a lymphoid response in the submucosa. Lesions may be acute or chronic, and include: rhinitis, otitis media, laryngitis, tracheitis, bronchitis, bronchiectasis, pulmonary abscesses, and alveolitis (Lindsey et al., 1978b, 1982). Pleuritis and emphysema are rare. The dramatic hyperplasia of bronchus associated lymphoid tissue (BALT) characteristic of MRM in the rat has been related to the finding that M. pulmonis is a potent non-specific mitogen for rat lymphocytes (Naot et al., 1979a,b). Syncytial epithelial giant cells may occur in nasal and bronchial mucosa in mice (Lindsey and Cassell, 1973).

Athymic mice are no more susceptible to pneumonia and death due to M. pulmonis than immunocompetent mice. However, they often develop arthritis after intranasal inoculation of the organism (Cassell, 1982).

Natural infections of M. pulmonis also occur in the genital tract of female rats (Graham, 1963; Juhr and Obi, 1970; Casillo and Blackmore, 1972; Ganaway et al., 1973; Cassell et al., 1979; Davidson et al., 1981). LEW rats are highly susceptible to severe genital (as well as respiratory) disease due to M. pulmonis, and this is characterized by purulent endometritis or pyometra, salpingitis and perioophoritis (Cassell et al., 1979; Cassell, 1982). Similar genital lesions attributable to M. pulmonis are rare in rats of other strains.

In mice, humoral antibody is protective and can be passively transferred. In the rat, little metabolic inhibiting, complement fixing, or hemaglutinating antibody is produced and BALT may be a nonspecific response. Cellular immunity appears to be more important in rats than in mice (Cassell, 1982).

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Diagnosis

Cultural isolation of M. pulmonis from rats or mice with clinical MRM usually is readily accomplished by culturing lavage or swab samples from the respiratory tract using medium which has been protested and shown to support growth (Cassell et al., 1983a). The nasopharynx is considered the best single site for culture (Davidson et al., 1981), but culturing multiple sites increases the isolation rate (Davidson et al., 1981). Diagnostic workups should include a battery of procedures (bacterial, viral, and histopathologic) designed to identify the responsible agent(s) and exclude other possible causes or contributors. Efforts should be made to identify all promoters (e.g., Sendai virus infection, intracage ammonia, etc.) that contribute to expression of MRM within the affected rodent population. Cultural isolation also is effective in diagnosing M. pulmonis infection of the genital tract in rats (Cassell, 1982, 1983a).

The ELISA is the method of choice for rodent health surveillance as it is far more sensitive and cost effective than culture. However, since the mycoplasma ELISAs currently in use are only genus specific (Horowitz and Cassell, 1978; Cassell et al., 1981, 1983a,b), ELISA positive sera should be tested by immunoblot (Davis et al., 1987; Minion et al., 1984) in order to differentiate between M. pulmonis and other mycoplasma infections. The detection of subclinical infection often is a major problem as ELISA seropositivity may occur only sporadically, the percentage of ELISA positive animals may be very small, and animals once seropositive may become negative again (Cassell et al., 1986; Cox et al., 1988). Animals with subclinical infections usually have so few mycoplasmas that they cannot be detected by routine culture (Davidson et al., 1981; Cox et al., 1988). These problems may be counteracted to some extent by testing only adults (weanlings with subclinical infection usually are ELISA negative), increasing the sample size, and testing repeatedly. Once a subclinically infected stock is shown to be ELISA positive, immunoblot and culture methods should be helpful in making decisions about suitability of the stock for research purposes (Cassell et al., 1983a; Lindsey, et al., 1986b; Davis et al., 1987).

Control

Cesarean derivation and barrier maintenance programs appear to have reduced the prevalence of M. pulmonis disease but may not have been as successful in reducing the prevalence of M. pulmonis infection (i.e., subclinical infection) in contemporary rodent stocks (Cassell et al., 1981, 1983a,b; Lindsey et al., 1986b; Cox et al., 1988). Thus, major emphasis must be given to selection of mycoplasma free breeding stocks. This probably can be achieved in some instances by housing small groups of young adult breeders (known to be free of non-mycoplasmal pathogens) in plastic film

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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isolators and repeatedly testing them by the ELISA over many months (e.g., monthly until 12 months of age). Stocks found to be consistently negative could then be used for establishment of breeder production populations under barrier programs.

Definitive information on the elimination of M. pulmonis from clinically or subclinically infected stocks is lacking but the following suggestions might prove useful. For cesarean derivations, use only dams that are several months old and have been repeatedly found to be ELISA negative. Candidate stocks for rederivation may be maintained for a few months on tetracycline or other antibiotics for the purpose of suppressing the mycoplasma flora as much as possible prior to cesarean operation. A separate isolator should be used for the pups of each donor female and a single foster female (from a stock known to be free of mycoplasmas and other pathogens). The placental membranes from each donor female should be cultured for mycoplasmas using a variety of media (Tram et al., 1970; Cassell et al., 1983a).

Control of environmental factors that favor MRM may be helpful in preventing clinical disease or ameliorating outbreaks (e.g., more frequent cage sanitation and reduction of cage population density to reduce intracage ammonia concentrations, and prevention of Sendai and sialodacryoadenitis virus infections). While administration of antimicrobials such as tetracyclines may help to control clinical signs, such agents are not curative and may introduce variables if animals on experiment are treated.

Interference with Research

MRM can cause morbidity and mortality, particularly in long term studies (Lindsey et al., 1971, 1982).

M. pulmonis respiratory infection can alter results of many experimental responses of the respiratory tract, including: carcinogenesis (Schrieber et al., 1972), ciliary function (Irvani and van As, 1972; Westerberg et al., 1972), cell kinetics (Wells, 1970), and immunity (Cole et al., 1975; Naot et al., 1979a,b; Davis et al., 1982).

M. pulmonis infection of the genital tract can alter histology (Cassell et al., 1979) and reproductive efficiency (Leader et al., 1970; Goeth and Appel, 1974; Fraser and Taylor-Robinson, 1977; Lal et al., 1980; Cassell, 1982).

M. pulmonis infection in LEW rats has been found to delay onset and reduce the severity of adjuvant arthritis, reduce the incidence of experimental collagen-induced arthritis, and reduce antibody response to collagen (Taurog et al., 1984).

M. pulmonis infection has been found to increase natural killer cell activity in mice (Lai et al., 1987), and suppress humoral antibody response to sheep red blood cells in rats (Aguila et al., 1988).

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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M. pulmonis can contaminate transplantable tumors and cause arthritis in recipient mice (Barden and Tully, 1969).

Subclinical M. pulmonis infection can be exacerbated by known experimental procedures (e.g., deficiencies of vitamin A or E, administration of hexamethylphosphoramide, etc.), and probably by many others yet to be identified (Lindsey et al., 1986b).

M. pulmonis has been a frequent contaminant of rodent cell cultures (Barile, 1973).

Mycoplasmas have been shown to produce "lymphokine-like" substances that are mitogenic for B and T lymphocytes in vitro (Proust et al., 1985).

Cilia-Associated Respiratory Bacillus
Significance

Uncertain.

Perspective

1980: van Zwieten et al. (1980a) in The Netherlands reported this organism in a colony of aged laboratory rats with severe murine respiratory mycoplasmosis (MRM). Morphologically similar organisms had been observed previously in the U.S. in Mystromys albicaudatus with chronic pulmonary lesions (A. E. McKee, Naval Medical Research Institute, Naval Medical Center, Bethesda, Md., personal communication, 1977), and as an apparent incidental finding in laboratory rats in Sweden (Afzelius, 1979).

1981: Mackenzie et al. (1981) reported that they found the organism in wild rats with an MRM-like disease, and in rabbits and a mouse in the United States.

1985: Ganaway et al. (1985) named the organism the "cilia-associated respiratory (CAR) bacillus". They also reported success in propagating the bacillus in embryonated hen's eggs and in experimentally infecting gnotobiotic rats with the organism to produce severe respiratory disease.

1986: Matsushita (1986) and Matsushita et al. (1987) reported natural infections of CAR bacillus in rats in Japan.

Agent

A Gram-negative, argyrophilic, filamentous, rod-shaped bacillus, measuring approximately 0.2 µm x 6.0 µm. Presently it is not classified, but it possibly belongs to the group called gliding bacteria (Ganaway et al., 1985). It has been cultivated in embryonated hen's eggs but has not been

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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grown in cell free media (Ganaway et al., 1985; van Zwieten et al., 1980a). The organism withstands freezing and thawing, and has been stored at -70°C and 23°C for short periods (Ganaway et al., 1985).

Hosts

Laboratory and wild rats (Rattus norvegicus), African white-tailed rats (Mystromys albicaudatus), laboratory rabbits, and laboratory mice (Mackenzie et al., 1981; Waggie et al., 1987; Griffith et al., 1988). However, the assumption that morphologically similar bacteria observed in different hosts are all the same organism may or may not be true.

Epizootiology

Unknown.

Clinical

The clinical manisfestations are those of severe MRM. Signs are nonspecific, but may include: hunched posture, ruffled coat, inactivity, "head tilt," and accumulation of porphyrin pigment around the eyes and external nares in rats (Lindsey et al., 1978b; van Zwieten et al., 1980a,b; Ganaway et al., 1985). No description of clinical disease in mice has been published.

Pathology

The pathology of natural CAR bacillus infection has been described only for rats and mice. The predominant lesions are those of advanced MRM (see "Pathology" of Mycoplasma pulmonis infection, pp. 44) with added distinctive features, as follows. Severe bronchiectasis and bronchiolectasis, pulmonary abscesses, and atelectasis of entire lung lobes are common and may be seen in rats only one month old. Severe bronchiectasis and bronchiolectasis are associated with accumulation of purulent or mucopurulent exudate in airways. An abundance of mucus often is present in peribronchiolar alveoli. Multifocal necrosis of bronchiolar and bronchial epithelia with an acute inflammatory response occurs and often progresses to severe granulomatous inflammation in walls of airways and abscess formation in airway lumens. Disordered repair may result in distorted, scarred bronchioles and bronchiolitis obliterans. The ciliated border of respiratory epithelium in affected airways often appears quite dense in hematoxylin and eosin stained sections because of the large numbers of CAR bacilli present between the cilia. The CAR bacillus also may be found on epithelial surfaces associated with lesions of MRM in nasal passages, larynx, trachea, and middle ears (van Zwieten et al., 1980a,b; Mackenzie et

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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al., 1981; Griffith et al., 1988: J. R. Lindsey, Department of Comparative Medicine, University of Alabama at Birmingham, personal communication).

In instances where the CAR bacillus has been found in rats with natural disease the predominant lesions have been those of MRM and mycoplasmas compatible with M. pulmonis have been present (van Zwieten et al., 1980a,b; Mackenzie et al. 1981; J. R. Lindsey, Department of Comparative Medicine, University of Alabama at Birmingham). An epizootic of Sendai virus infection preceded one outbreak (van Zwieten et al., 1980a,b). It may be that in rats M. pulmonis is the primary pathogen and the CAR bacillus is a promotor of MRM. Whether the CAR bacillus can cause natural disease in rats in the absence of M. pulmonis is unknown.

CAR bacillus-associated respiratory lesions similar to those in rats were recently reported in C57BL/6J-ob/ob mice (Griffith et al., 1988). These mice also had Sendai virus and pneumonia virus of mice infections, and ob/ ob mice are known to have impaired cell mediated immunity (Sheena and Meade, 1978).

Diagnosis

At present, diagnosis is dependent upon recognition of the argyrophilic CAR bacillus on respiratory epithelium in lesions of the respiratory tract. The Warthin-Starry silver stain gives by far the best results, and should be used as a standard procedure in pathologic evaluation of rat lungs with the characteristic lesions described above. Also, the organism can be demonstrated by transmission electron microscopy (van Zwieten et al., 1980a,b).

Enzyme-linked immunosorbent assays for CAR bacillus infection are under development, but their specificity and sensitivity have not been reported (Ganaway et al., 1985; Lukas et al., 1987). An indirect immunofluorescence test for CAR bacillus infection has been developed in Japan (Matsushita et al., 1987).

Control

Uncertain. The infection probably can be eliminated by cesarean derivation but definitive studies have not been done.

Interference with Research

Uncertain. The organism may be an important contributor to the morbidity and mortality due to MRM in rats and mice.

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Streptococcus pneumoniae
Significance

Low.

Perspective

1950: Recognition of S. pneumoniae as a natural pathogen of laboratory rats was a relatively recent development as the first reported outbreak was by Mirick et al. (1950). Only an occasional report has appeared since that time, bringing the present total to only about seven papers (Mirick et al., 1950; Ford, 1965; Baer, 1967; Baer and Preiser, 1969; Weisbroth and Freimer, 1969; Tucek, 1971; Fallon et al., 1988).

1969: Weisbroth and Freimer (1969) found the organism to be present in 19 of 22 breeding colonies of conventionally reared rats in seven states in the United States, but none of five pathogen free (cesarean derived, barrier maintained) colonies in four states.

1988: Fallon et al. (1988) reported S. pneumoniae infections not associated with disease in rats and mice free of other pathogens at a large commercial breeding facility. This was the first report of natural S. pneumoniae infection in mice.

Agent

Streptococcus pneumoniae is a bacterium with the following synonyms: Diplococcus pneumoniae, Pneumococcus pneumoniae. An encapsulated, Gram positive, lancet-shaped diplococcus. Although found mainly in pairs, the organism may occur in short chains or singly. As cultures age they become Gram negative (Deibel and Seeley, 1974).

It grows on blood agar producing α-hemolysis. Growth may be facilitated by 10% CO2. Some isolates require microaerophilic environments. On solid medium the organism forms round, glistening, unpigmented colonies that reach 0.5-1.5 mm in diameter after 24-36 hours. It differs from other streptococci of the viridans group in being bile soluble, sensitive to optochin (ethylhydrocupreine), and virulent for mice (Deibel and Seeley, 1974).

More than 80 different capsular types are known. Typing is by the Quellung reaction (swelling of carbohydrate capsules of individual organisms in the presence of type-specific antiserum). The most common capsular types reported from rats have been 2, 3, and 19; types 8, 16 and 35 have been encountered less frequently (Weisbroth, 1979; Fallon et al., 1988).

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Hosts

Humans are the main natural host. Between 40% and 70% of normal human adults carry one or more serologic types of pneumococci in their throats. Severe epizootics have been reported occasionally in rats, guinea pigs, and monkeys (Deibel and Seeley, 1974; Quie et al., 1981).

Epizootiology

The agent is rarely seen in rats except in some conventionally reared stocks. Within colonies, isolates are usually monotypic, i.e., one capsular type (Weisbroth and Freimer, 1969).

Host sites of greatest predilection for the infection are nasal passages and middle ears. The carrier state is common in infected colonies.

Transmission is mainly by aerosol. The organism can remain viable for days on fomites, but fomites are of doubtful importance in transmission.

Clinical

Signs are non-specific. Dyspnea, weight loss, hunched posture, snuffling respiratory sounds, and abdominal breathing have been reported. Clinical onset can appear to be sudden. Young rats are affected most often (Weisbroth and Freimer, 1969).

Pathology

Pneumococcal capsules consist of large polysaccharide polymers that form hydrophilic gels on the surface of the microorganisms. Only encapsulated strains are pathogenic.

Very little is known about mechanisms by which the organism spreads from nasopharynx to lungs in rats or man. Infection becomes established in a bronchopulmonary segment and spreads centrifugally. As shown by Wood (1941) in his classic studies using rats, affected alveoli at the front of the spreading infection form an edema zone; an influx of polymorphonuclear leukocytes gives rise to consolidation; the organisms are removed by phagocytes leading to resolution; and the persistence of macrophages characterizes the macrophage reaction, the final healing stage of the local lesion. The infection readily spreads from the lung to the pleural space and pericardium, and into the blood stream.

Host defense is dependent in large part on efficient phagocytosis, with type-specific antibody (Wood, 1941; Wood and Smith, 1950) and complement opsonization (Coonrod and Yoneda, 1982) playing key roles. Evidence also has been advanced to suggest a nonphagocytic mechanism for pulmonary clearance of S. pneumoniae (Coonrod et al., 1983), perhaps mediated by

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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surfactant (O'Neill et al., 1984). C-reactive protein may have a protective role against pneumococcal septicemia (Horowitz et al., 1987). Athymic (nu/nu) mice do not have increased susceptibility (Winkelstein and Swift, 1975). Organisms often persist in the nasopharynx long after recovery from disease.

Susceptibility to S. pneumoniae is influenced by splenectomy (Biggar et al., 1972; Bogart et al., 1972; Leung et al., 1972; Cooney et al., 1979a,b; Cohn and Schiffman, 1987; Harding et al., 1987), pulmonary edema (Johanson et al., 1974), and iron deficiency (Shu-heh et al., 1976).

A circadian periodicity of susceptibility to experimental infection with S. pneumoniae has been observed in mice (Feigin et al., 1969; Wongwiwat et al., 1972; Shackelford and Feigin, 1973). Hyperthermia has been reported to protect mice against the experimental infection (Liddle et al., 1987).

The predominant lesions in affected rats are suppurative rhinitis and otitis media. With extension of infection into distal airways, there is acute tracheitis and fibrinous lobar pneumonia. Extension to organs adjacent to the lungs is common, resulting in fibrinous pleuritis or empyema, fibrinous pericarditis, and/or acute mediastinitis. In some cases, these pleural and pericardial lesions occur in the absence of identifiable pulmonary lesions and thus, may result from bacteremia rather than direct spread of infection from the lungs (Mirick et al., 1950; Baer, 1967; Baer and Preiser, 1969; Weisbroth and Freimer, 1969; Tucek, 1971).

Severe bacteremia is an important part of advanced disease due to S. pneumoniae in the rat. One or more of the following may occur: suppurative arthritis, meningitis, hepatitis, splenitis, peritonitis, and orchitis. There may be multiple infarcts in the spleen or infarction of entire testicles. Enormous numbers of organisms can be demonstrated in such lesions by Gram stain. In some cases, the primary cause of death is one of these abdominal lesions (Weisbroth and Freimer, 1969; J. R. Lindsey, Department of Comparative Medicine, University of Alabama at Birmingham).

Diagnosis

Cultural isolation of S. pneumoniae from the respiratory tract is diagnostic of infection but, because of the common carrier state, is not necessarily diagnostic of disease. Isolation of the organism from blood, body cavity, or a diseased organ is much stronger evidence to implicate the organism as a cause of disease.

Necropsy diagnosis must correlate presence of the organism at a given site with characteristic lesions, while excluding other possible causes and contributors to the disease process. This is of critical importance. Many of the reported outbreaks attributed to S. pneumoniae in the literature documented lesions more characteristic of murine respiratory mycoplasmosis

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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than pneumococcal infection, but failed to exclude the possibility of M. pulmonis infection. Similarly, possible concurrent viral infections were not excluded.

Imprints of diseased organs and exudates due to S. pneumoniae often contain myriads of the organisms that are readily demonstrated by Gram stain. These methods can provide strong evidence for a preliminary necropsy diagnosis.

Several serologic tests, including enzyme-linked immunosorbent assays and a radioimmunoassay, have been developed for human patients (Schiffman et al., 1980) but have not been used in testing rats.

Control

Cesarean derivation and barrier maintenance have been extremely effective. The high rate of carriers in man suggests the need for personnel working with rats to wear masks, but this has not been borne out by practical experience. Vaccines are available for people and conceivably could prove useful for animals in special situations.

Interference with Research

Septicemia due to S. pneumoniae in the rat has been shown to alter hepatic metabolism (Powanda et al., 1972; Canonico et al., 1975; DeRubertis and Woeber, 1972; Thompson and Wannemacher, 1980), serum biochemistries (Mitruka, 1971), blood pH and electrolytes (Elwell et al., 1975), and thyroid function (Shambaugh and Beisel, 1966).

Infection with S. pneumoniae can jeopardize studies in rats involving the respiratory tract.

Corynebacterium kutscheri
Significance

Uncertain: subclinical infections may be common in conventionally reared mice and rats.

Perspective

1894: The organism was first isolated from mice in Germany by Kutscher (1894) who called the disease it causes, "pseudotuberculosis."

1964: Pierce-Chase et al. (1964) and Fauve et al. (1964) studied mice from 21 conventionally reared colonies and demonstrated that 9 of these colonies had "latent" C. kutscheri infection. Latency was explained on the

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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basis of two forms of the organism, avirulent and virulent. They proposed that "latent" infections were due to the avirulent form, and that administration of cortisone provoked reversion to the virulent form which was responsible for disease expression.

1978: Hirst and Olds (1978a,b) refuted the claims of Pierce-Chase et al. (1964) and Fauve et al. (1964), and presented evidence that the avirulent organism was a group N streptococcus, not C. kutscheri.

1984: Ackerman et al. (1984) reported development of an enzyme-linked immunosorbent assay (ELISA) for C. kutscheri, and compared its efficacy with that of the tube agglutination test for detection of experimental infections in rats.

1986: Saltzgaber-Muller and Stone (1986) developed and tested a number of C. kutscheri DNA probes for detection of early infections due to this organism in rats.

Agent

Corynebacterium kutscheri is a Gram positive, metachromatic, diphtheroid bacillus. Synonyms are Corynebacterium murium, Bacillus pseudotuberculosis murium, Corynethrix pseudotuberculosis murium, and Bacterium kutscheri. Colonies on blood agar are 1 to 2 mm in diameter after 24 hours; they appear circular, entire, dome-shaped, yellow or gray, smooth, and nonhemolytic.

The organism reduces potassium tellurite and produces acid from glucose, fructose, maltose, mannose, salicin, and sucrose. Does not produce acid from dulcitol, lactose, or mannitol. Usually hydrolyzes urea, does not produce indole or grow on MacConkey agar, and is catalase positive (Weisbroth, 1979; Coyle et al., 1985).

Hosts

Mice, rats, and rarely, guinea pigs.

Epizootiology

Natural infections of C. kutscheri are usually subclinical, occur in conventionally reared mice and rats, and result in disease expression only after severe immunosuppression of hosts by experimental regimens, dietary deficiencies, or concurrent infections of other agents (Weisbroth, 1979). A few epizootics have occurred in which the provoking factors could not be identified (Giddens et al., 1968). The infection is rare in cesarean-derived, barrier-maintained stocks.

The host-parasite relationships in naturally infected colonies (with

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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inapparent, subclinical infection) are poorly understood. Infected animals presumably become chronic carriers but the natural habitat(s) of the organism remain uncertain. The oropharynx, submaxillary lymph nodes, and large intestine may be the main sites of predilection (Brownstein et al., 1985; Barthold and Brownstein, 1988; Suzuki et al., 1988), with transmission being mainly by the fecal-oral route. Other sites of infection include the respiratory tract, middle ears and preputial glands (Weisbroth, 1979). Whether naturally infected colonies have dormant or latent infections remains unresolved (Fox et al., 1987; Suzuki et al., 1988).

In rats experimentally infected by the oronasal route, the organism has been isolated from the oral cavity and submaxillary lymph nodes for at least eight weeks post infection (Brownstein et al., 1985). Whether this simulates the natural subclinical infection is unknown.

Clinical

Signs are not present in animals with the inapparent infection. In active infection of rats, signs are most often those of respiratory disease: dyspnea, rales, weight loss, humped posture, and anorexia. In mice, findings are usually those of a severe septicemia, particularly dead and moribund animals. In either species, arthritis or abscesses (in most any organ) may occur (Weisbroth, 1979).

Pathology

Inbred C57BL/6 mice are much more resistant to infection than are outbred S (Swiss) mice. Resistance reportedly is due to greater efficiency of C57BL/6 mononuclear phagocytes and is controlled by a single autosomal dominant gene (Hirst and Wallace, 1976; Hirst and Campbell, 1977).

Active disease characteristically begins as a septicemia which results in lodgement of septic emboli in many organs, most notably:

  1. In mice, kidney and liver, less frequently in lungs, skin and joints (Weisbroth and Scher, 1968a).
  2. In rats, the lungs. Subsequent expansion from the initial embolic foci in the lungs explains the characteristic pneumonia (i.e., parenchymal infection which spreads by contiguity, not a broncho-pneumonia) due to this agent in rats (J. R. Lindsey, Department of Comparative Medicine, University of Alabama at Birmingham).

In mice, during the septicemic phase large bacterial emboli are trapped in capillary beds, particularly in kidney and liver. Embolic glomerulitis is characteristic. If the animal survives the acute episode, each focus of infection may enlarge forming an abscess (Weisbroth and Scher, 1968a).

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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In rats, bacterial emboli lodge in capillaries of the lungs, break out and enlarge, often coalescing with adjacent foci. Along each advancing front alveoli contain edema fluid and a few polymorphonuclear leukocytes (PMNs), along with a high concentration of the organism. As this front expands, the alveoli behind become packed with PMNs and eventually may form large necropurulent centers. The expanding infection often involves the pleura resulting in fibrinous to fibrous pleuritis. The lungs usually have gross lesions of varying size (0.25 mm to 1 cm), indicating lesions of different ages related to repetitive showers of emboli, perhaps arising in part from active lesions in the lungs. Occasionally, abscesses may occur in the liver, kidneys, subcutis, peritoneal cavity, and other sites (Ford and Joiner, 1968; Giddens et al., 1968; J. R. Lindsey, Department of Comparative Medicine, University of Alabama at Birmingham).

The term "pseudotuberculosis" usually is inappropriate for the infection in mice and rats as septicemia and acute inflammation tend to predominate without formation of tubercle-like lesions. However, this term may be applicable to occasional focal lesions (e.g., abscesses) which reach chronicity (Giddens et al., 1968).

Diagnosis

Detection of the infection in subclinically infected colonies is a major problem. Culture methods give inconsistent results and are not reliable for routine purposes (Fujiwara, 1971; Suzuki et al., 1988). The agglutination reaction, indirect fluorescent antibody technique, and agar-gel immunodiffusion methods are unsatisfactory for the detection of subclinical infections (Weisbroth and Sher, 1968b). The C. kutscheri ELISA holds great promise for detection of antibodies in subclinical infections (Ackerman et al., 1984; Fox et al., 1987). Similarly, a C. kutscheri DNA probe method for the diagnosis of C. kutscheri infection has been reported (Saltzgaber-Muller and Stone, 1986), but whether it has the specificity and sensitivity required to detect subclinical infections has not been determined.

In Japan, cortisone provocation followed in 6 days by the tube agglutination test for an anamestic rise in titer has been used as a routine diagnostic test (Takagaki et al., 1967; Fujiwara, 1971). In persistently infected mice, a single dose of 10 mg of cortisone acetate given intraperitoneally is sufficient to provoke active disease (Fauve et al., 1964). In rats, 10 mg cortisone acetate daily for 28 days, given by subcutaneous injection, has been successful in activating the subclinical, persistent infection (LeMaistre and Tompsett, 1952).

The diagnosis of active disease is made by culture of the organism, demonstration of characteristic lesions, and exclusion of other infectious agents and disease processes (Giddens et al., 1968; Fox et al., 1987). Efforts

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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should be made to identify those factors responsible for provocation of natural epizootics.

Primary isolation of the organism from mucosal sites such as nasopharynx can be very difficult because colonies of C. kutscheri on blood agar have the same appearance as colonies of Staphylococcus epidermidis (M. Davidson, Department of Comparative Medicine, University of Alabama at Birmingham).

Presumptive diagnosis of active disease due to C. kutscheri can be made at necropsy by identifying the characteristic lesions and demonstrating typical colonies of the organism in Gram or Giemsa stained tissue imprints. In tissue sections the organism often can be demonstrated in greater numbers by the methenamine silver method than by Gram stain methods (J. R. Lindsey, Department of Comparative Medicine, University of Alabama at Birmingham).

Control

Cesarean derivation and barrier maintenance have proved very successful. There is one report of possible vertical transmission (Juhr and Horn, 1975).

Interference with Research

Experimental procedures which immunocompromise mice or rats can be complicated by C. kutscheri unless stocks free of this agent are used. Latent infection has been provoked to active disease:

  1. In mice, by cortisone as a single dose of 10 mg cortisone acetate given intraperitoneally (Antopol, 1950; Antopol et al., 1951, 1953; Fauve et al., 1964), x-irradiation (Shechmeister and Adler, 1953), concurrent ectromelia (Lawrence, 1957), or concurrent salmonellosis (Topley and Wilson, 1920; Wolff, 1950).
  2. In rats, by cortisone (LeMaistre and Tompsett, 1952) or deficiency of pantothenic acid (Seronde, 1954; Zucker, 1954, 1956, 1957; Seronde et al., 1955; Seronde et al., 1956; Zucker and Zucker, 1956).

In one study, experimental infections of rats with Sendai virus, sialodacryoadenitis virus or Kilham rat virus were unsuccessful in causing disease expression due to prior C. kutscheri infection (Barthold and Brownstein, 1988).

Rat Coronavirus

The virus originally reported as "Rat Coronavirus" by Parker et al. (Arch. Ges. Virusforsch. 31:239-302, 1970c) is now considered a strain of sialodacryoadenitis virus (see page 97, this volume).

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Pneumonia Virus of Mice
Significance

Low.

Perspective

Two laboratory groups (Horsfall and Hahn, 1939, 1940; Mills and Dochez, 1944, 1945) independently discovered this virus during attempts to isolate influenza virus and other agents from human patients with respiratory infections. Nasopharyngeal washings or homogenates of diseased lung were serially passaged in mice resulting in high rates of "pulmonary consolidation" and mortality in the mice. As of this date, confirmation is lacking that pneumonia virus of mice (PVM) is a significant pathogen for immunocompetent rodents under natural conditions.

Agent

PVM is an RNA virus, family Paramyxoviridae, genus Pneumovirus. It is antigenically distinct from other members of the paramyxoviridae. All known strains of PVM have antigenic homology (Parker and Richter, 1982).

The virus particles are pleomorphic, occurring either as filaments 100 nm in diameter and up to 3,000 nm in length or as spheres 80-200 nm in diameter. The virus is labile under environmental conditions such as room temperature and heating to 56°C for 30 minutes. It agglutinates erythrocytes of mice, rats, and hamsters at room temperature or 5°C (Parker and Richter, 1982).

Hosts

Mice, rats, and hamsters. Possibly, guinea pigs and rabbits based on serologic evidence without virus isolations (Jacoby et al., 1979; Parker and Richter, 1982).

Epizootiology

PVM is a very common infection of laboratory rodents worldwide, the general prevalence rates being >50% of colonies of mice, rats, and hamsters. Prevalence rates within colonies vary greatly but tend to be higher (»50%) in rats and hamsters than in mice (»20%). Thus, the virus has relatively low infectivity for mice and tends to cause focal enzootics of infection within mouse colonies. Hemagglutination inhibition antibody titers also tend to be lower in mice (Parker and Richter, 1982).

Active infection in mice (and presumably rats and hamsters) is short

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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lived, lasting only up to about 9 days (Horsfall and Ginsberg, 1951; Carthew and Sparrow, 1980a; Smith et al., 1984). Persistent infections do not occur in euthymic mice. Transmission is exclusively horizontal via the respiratory tract, mainly by direct contact and aerosol. Fomites are probably not important in transmission (Tennant et al., 1966; Parker and Richter, 1982).

Athymic (nu/nu) mice infected with PVM develop chronic pneumonia with terminal emaciation and death (Richter et al., 1988; Weir et al., 1988).

Clinical

Natural infections are subclinical in euthymic rodents. Athymic (nu/nu) mice infected with PVM have chronic illness with emaciation and deaths (Richter et al., 1988.

Pathology

Very little useful information is available on pathogenesis and morphologic expression of natural PVM infection. There are no reports of lesions demonstrated to be due to PVM alone in naturally infected mice or rats.

The pathologic description of Horsfall and Hahn (1940) is generally cited as the prototype for PVM infection in the mouse. However, the severe lung disease which they observed was produced only after serial passage of lung tissue in mice. Furthermore, their description of dense peribronchial and perivascular cuffs of mononuclear cells, purulent bronchitis, hyperplasia of bronchial epithelium, and a mononuclear response in alveoli, plus the isolation of mycoplasmas from consolidated lungs provide the essential criteria for a diagnosis of murine respiratory mycoplasmosis due to Mycoplasma pulmonis (see pp. 232, Horsfall and Hahn, 1940)! In sharp contrast, later investigators (Tennant et al., 1965, 1966) using cultures of the virus as inocula found it necessary to use ether anesthesia to increase host susceptibility in their standard experimental model using mice.

It may be that natural infections in mice are due to relatively small intranasal doses of virus with viral replication (and mild lesions) occurring principally in the nasal passages. Experimental infections of PVM in mice have given different results that appear to relate to the dose of virus given. In one study (Smith et al., 1984), 25 day old Crl:CF1® mice were given 2.5 x 102 median tissue culture infectious doses (TCID50) of PVM intranasally and they developed only mild rhinitis. In another study (Carthew and Sparrow. 1980a), NMRI mice (age not specified) given either 104 or 105 TCID50 of PVM intranasally developed severe interstitial pneumonia. The latter authors stated that similar lung lesions due to PVM had been seen in naturally infected mice, but they gave no details.

Chronic pneumonia, emaciation, and deaths have been reported in athymic (nu/nu) mice naturally infected with PVM (Richter et al., 1988; Weir et al.,

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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1988). Athymic (nu/nu) mice given 105 TCID50 of PVM intranasally have been reported to have interstitial pneumonia that persisted at least until 20 days post infection (Carthew and Sparrow, 1980b). Susceptibility to PVM may be increased by ether anesthesia (Tennant et al., 1966), urethane administration (Mirick et al., 1952), and pyridoxine deficiency (Leftwich and Mirick, 1949; Mirick and Leftwich, 1949). Susceptibility to the virus has been decreased by the intranasal administration of polysaccharides from a variety of bacterial and nonbacterial sources (Horsfall and McCarty. 1947; Ginsberg and Horsfall, 1951).

Diagnosis

For routine monitoring purposes, the enzyme-linked immunosorbent assay is the most sensitive (Payment and Descoteaux, 1978; Descoteaux et al., 1980; Descoteaux and Payment, 1981; London et al., 1983), but the hemagglutination inhibition test is highly reliable and has served as the standard for PVM for many years (Parker and Richter, 1982). With either of these tests, serum antibody is first detected around day 9 post infection (Parker and Richter, 1982; Smith et al., 1984). The complement fixation (CF) test is useful in detection of recent infections as CF antibody appears about 9 days post infection, begins to decline after 2 weeks post infection, and disappears altogether by 3 months post infection (Tennant et al., 1966). The antibody response to PVM infection may be delayed and reduced in mice previously infected with mouse hepatitis virus (Carrano et al., 1984).

The mouse antibody production (MAP) test may be used for testing biologic specimens for presence of the virus (Rowe et al., 1962). PVM may be isolated using primary hamster kidney, BHK-21, Vero, and hamster embryo cells (Parker and Richter, 1982).

Control

Cesarean derivation and barrier maintenance have given excellent results. Since the normal pattern of infection within mouse populations is focal enzootics and active infection is present in the individual mouse for only about 9 days, it may be possible to remove a few breeding pairs to individual gnotobiotic isolators and select those that are free of the infection by serologic testing. Alternatively, pairs which are serologically positive but produce only seronegative young while being maintained under gnotobiotic conditions can provide large numbers of breeders.

Interference with Research

PVM conceivably could alter the experimental results of some studies involving the respiratory tract in euthymic mice but, no examples have been

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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reported. Athymic (nu/nu) mice with natural infections of PVM develop chronic pneumonia and emaciation with deaths (Richter et al., 1988; Weir et al., 1988).

Mycobacterium avium-intracellulare
Significance

Very low.

Perspective

Natural infection and disease due to this agent have been observed only in breeder C57BL/6N mice housed in a single room at a commercial breeding facility (Waggie et al., 1983a). Koch's postulates were fulfilled by oral and subcutaneous inoculation of the organism into pathogen free C57BL/6N mice (Waggie et al., 1983b).

Agent

A bacterium, family Mycobacteriaceae, Mycobacterium avium-intracellulare. Mycobacterium avium and Mycobacterium intracellulare are virtually indistinguishable organisms that belong to the "M. avium complex" within the "Runyon Group III" of the "nontuberculous" (or, "atypical") mycobacteria (i.e., mycobacteria other than Mycobacterium tuberculosis and Mycobacterium bovis). Thus, the species designation is avium-intracellulare. However, avium is frequently used as the species name because identification beyond the complex level usually is not practical (Sommers and Good, 1985).

Mycobacteria are aerobic, acid fast, nonsporeforming, nonmotile bacilli. Growth in artificial medium is usually slow. For methods of cultivation and identification of M. avium-intracellulare, see Sommers and Good (1985).

The nontuberculous (atypical) mycobacteria generally are considered normal inhabitants of soil and water (Chapman, 1982).

Hosts

Birds, man, swine, and rarely, mice (reported only in the C57BL/6N strain).

Epizootiology

The source of infection for the C57BL/6N mice was thought to be

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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contaminated drinking water. The affected C57BL/6N population received chlorinated (9 ppm) water while an unaffected population of C57BL/6N mice elsewhere in the same facility had been given acidified (pH 2.5) water. M. avium-intracellulare is known to be resistant to sodium hypochlorite Waggie et al., 1983a).

M. avium-intracellulare has been isolated from public water supplies (Goslee and Wolinsky, 1976; Saito and Tsukamura, 1976; DuMoulin and Stottmeier, 1978).

Clinical

Clinical signs have not been reported to occur in naturally infected mice (Waggie et al., 1983a).

Pathology

Approximately 8% of 5 month old C57BL/6N females had gross lung lesions consisting of raised, tan, subpleural foci 1 to 5 mm in diameter. Sixty-three percent had microscopic lung lesions. These were foci in which large foamy macrophages and multinucleate giant cells filled the alveoli, particularly in the vicinity of terminal bronchioles but sometimes extending out to the pleura. Rarely, a few small nests of polymorphonuclear neutrophils were seen among the macrophages. Alveolar septae in affected foci often were widened and sometimes the interstitium was infiltrated by lymphocytes. Microgranulomas also occurred in livers, spleens, and mesenteric lymph nodes of a few mice. Acid fast bacilli were demonstrated in 37% of lung lesions using the Ziehl-Neelson method (Waggie et al., 1983a).

Sixty-seven percent of the breeder C57BL/6N females from the affected population also had either unilateral or bilateral chronic suppurative otitis media in addition to lesions in other organs. Acid fast bacilli were demonstrated in these ear lesions using the Fite Faraco method (J. R. Lindsey, Department of Comparative Medicine, University of Alabama at Birmingham).

C3H/HeN and (C57BL/6 x C3H) F1 mice, and F344/N rats housed in the same room with affected C57BL/6N mice did not have lesions attributable to the mycobacterial organism, nor was the organism isolated from these animals by culture. This suggests that they were less susceptible to the disease and possibly, to the infection (Waggie et al., 1983a).

Other investigators (Gangadharam et al., 1981) also have found C57BL/6 to be one of the more susceptible mouse strains. Also, an experimental model has been established using beige (C57BL/6J-bg/bg) mice (Gangadharam et al., 1983; Bertram et al., 1986). Athymic (nu/nu) mice are no more susceptible than immunocompetent mice (Ueda et al., 1976).

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Diagnosis

Diagnosis of the disease in C57BL/6N mice is made by demonstration of typical lesions and cultural isolation of the organism. In tissue sections stained by acid fast methods the organisms are often beaded in appearance and measure 0.5 µm x 2.0 µm (Waggie et al., 1983a).

Cultural isolations of M. avium-intracellulare from C57BL/6N mice were made as follows. The left lobe of the lung was removed aseptically and ground in 5 ml of sterile saline using a tissue grinder. A loopful of the suspension was transferred to 10 mm deep blood agar plates and these were incubated in a humidified atmosphere with 10% carbon dioxide. Small (<1 mm) translucent colonies appeared after one week of incubation (Waggie et al., 1983a).

For purposes of differential histologic diagnosis, it is instructive to compare the lung lesions due to M. avium-intracellulare in C57BL/6N mice with the idiopathic lung lesion known as "alveolar histiocytosis" that has been reported to occur in both germfree as well as conventional rats (Beaver et al., 1963; Yang et al., 1966; Flodh et al., 1974). The latter condition is characterized by focal subpleural accumulations of foamy macrophages in alveoli immediately beneath the pleura, especially over the dorsal surfaces of the right caudal lobe and the posterior half of the left lobe. Grossly, these lesions appear as slightly raised, 1 to 2 mm, pale foci with discrete margins. Their characteristic location subpleurally distinguishes them from the mycobacterial lesions in C57BL/6N mice which primarily are located around the terminal airways.

Control

Uncertain, probably by acidification or ultrafiltration of water supply.

Interference with Research

Infection of susceptible mice such as C57BL/6N could complicate studies of the respiratory tract. Immunosuppression probably could result in active disease in less susceptible mice and possibly, in rats.

Pneumocystis carinii
Significance

Low, except in immunodeficient and immunosuppressed animals.

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Perspective

1966: Although small numbers of Pneumocystis carinii had been described in the lungs of rats as early as 1912, it was not until the development and experimental use of cortisone in rats that this infection began to take on significance (Frenkel et al., 1966). Following the administration of cortisone, Frenkel et al. (1966) demonstrated latent P. carinii in six of eight rat stocks from different commercial breeders in the U.S.

1977: Ueda et al. (1977a) reported natural disease due to P. carinii in athymic (nu/nu) mice. Walzer et al. (1977) transmitted rat- and human-derived organisms to athymic (nu/nu) mice.

1979: Walzer et al. (1979) demonstrated widespread natural infections in mice by giving cortisone, low (8%) protein diet, and tetracycline. Using a similar immunosuppressive regimen, Walzer et al. (1980) demonstrated a high prevalence of natural infection in rats.

1989: Walzer et al. (1989) reported natural outbreaks of P. carinii pneumonia in colonies of athymic (nu/nu) and severe combined immunodeficient (scid/scid) mice at four institutions.

Agent

Unclassified, but generally regarded as a protozoan or fungus, with the consensus favoring classification as a protozoan (Long et al., 1986). The recent ultrastructural demonstration of membrane surface rosettes during endocytosis and exocytosis is more supportive of classification as a protozoan (Yoneda et al., 1982). P. carinii of human, mouse, and rat origin have been found to have shared as well as host species-specific antigenic determinants (Walzer and Linke, 1987; Walzer et al., 1989).

The life cycle consists of four morphologically distinct stages, and occurs entirely within alveoli of the lung (Barton and Campbell, 1967; Campbell, 1972; Vossen et al., 1978; Yoneda et al., 1982).

  1. Trophozoite—1.5 to 2.0 µm diameter. Uninucleate, pleomorphic (round, oval, or crescent shaped), limited by a double-layered membrane or pellicle. May have filopodia extending from the surface. The filopodia often intermesh with the filopodia of other organisms forming an intra-alveolar cluster or aggregate. Thought to be an obligate parasite of the type 1 pneumocyte (Long et al., 1986).
  2. Precyst—2 to 4 µm diameter. Uninucleate, oval, thick cell wall, smooth surface without pseudopodial tubular extensions.
  3. Cyst—5 to 7 µm diameter. Thick (three layered) cyst wall. Filopodial extensions may be present (but are not as prominent as those in trophozoites). The cytoplasm contains up to 8 nuclei, each representing a sporozoite.
Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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  1. Sporozoite—1.0 to 1.7 µm diameter. Undergoes development in the cyst and transforms into extracystic trophozoites that escape from the cyst and multiply (? asexually), or can develop into precysts or cysts.

Limited success has been achieved in growing the organism in cell cultures, including the A549 and WI-38 VA 13 cell lines (Pifer et al., 1977; Bartlett et al., 1979; Cushion and Walzer 1984a,b; Cushion et al., 1985).

Hosts

Natural hosts include man, mice, rats, rabbits, ferrets, and numerous other mammals.

Epizootiology

P. carinii is an ubiquitous opportunistic pathogen that inhabits pulmonary alveoli. Active pulmonary infection and disease are practically always manifestations of deficiency or compromise in host resistance.

The organism is extremely prevalent as a persistent, subclinical infection in mice and rats (Walzer et al., 1979, 1980, 1983; Walzer and Rutledge, 1981, 1982; Pifer, 1983).

Transmission is thought to be mainly by inhalation of infective cysts expelled during exhalation or coughing. Vertical transmission has been suggested (Pifer, 1983, 1984; Pifer et al., 1984), but has not been proved.

Clinical

Immunocompetent rats and mice have subclinical infection (unless immunosuppressed). Immunodeficient and immunocompromised animals with P. carinii infection have chronic wasting and respiratory insufficiency that may persist for months. Clinical signs can include rough hair coat, dyspnea, cyanosis, severe weight loss and death (Ueda et al., 1977a; Tamura et al., 1978a; Walzer et al., 1979, 1980, 1983, 1989; Weir et al., 1986).

Pathology

Active infections may occur or be produced as follows:

  1. Athymic (nu/nu) mice—natural (Ueda et al., 1977a; Tamura et al., 1978a: Weir et al., 1986; Walzer et al., 1989) or experimental (Walzer et al., 1979) infections.
  2. Severe combined immunodeficient (scid/scid) mice—natural infections (Walzer et al., 1989).
  3. C3H/HeJ mice—when C3H/HeJ (lipopolysaccharide unresponsive)
Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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  1. mice and C3HeB/FeJ (lipopolysaccharide responsive) mice with persistent infection are given 1 mg cortisone acetate subcutaneously (s.q.) twice weekly, with or without low (8%) protein diet, the C3H/HeJ mice develop more severe P. carinii infections and lung lesions than the immunologically normal C3HeB/FeJ mice (Walzer et al., 1983).
  2. Adult immunocompetent mice—To produce lesions in persistently infected animals, give: 1 mg cortisone acetate s.q. twice weekly (or dexamethasone, 1 mg/1,000 ml in the drinking water), low (8%) protein diet, and tetracycline (1 mg/ml tap water) in drinking water for 8 weeks (Walzer et al., 1979).
  3. Adult immunocompetent rats—To produce lesions in persistently infected animals, give: 25 mg cortisone acetate (for rats weighing 200 gm or less, 40 mg for rats weighing over 200 gm) s.q. twice weekly, low (8%) protein diet, and tetracycline (0.5 to 1.0 mg/ml) in drinking water for 8 to 10 weeks. The organism usually can be found in the lungs after 6 to 8 weeks of immunosuppression (Walzer et al., 1980; Bartlett et al., 1987b). The development of large numbers of trophozoites in alveoli and their attachment to the type I pneumocytes lining the alveoli appear to be central events in pathogenesis. The attachment mechanism is unknown, but probably is not by filopodia as has been suggested (Barton and Campbell, 1969). Attachment of large numbers of trophozoites to type I pneumocytes apparently alters alveolar capillary membrane permeability resulting in fluid accumulation along the basement membrane beneath type I pneumocytes leading to death of these pneumocytes (Lanken et al., 1980; Yoneda and Walzer, 1980, 1981, 1983).

Host defense mechanisms are poorly understood. The increased susceptibility of nude mice (Walzer et al., 1977), the protective effect of spleen T cells from previously infected immunocompetent mice (Furuta et al., 1985), evidence that specific antibody is required to opsonize the organism for phagocytosis by alveolar macrophages (Masur and Jones, 1978), and the protective effect of specific monoclonal antibody against P. carinii pneumonitis in animal models (Gigliotti and Hughes, 1988), indicate roles for both cellular and humoral immunity. Polymorphonuclear leukocytes also may have an important role (Pesanti, 1982).

Animals with active disease usually become progressively emaciated. The lungs are enlarged, rubbery in consistency, plum colored, and heavier than normal. On histopathology, the alveolar septae are variably thickened and there is a meager inflammatory response of lymphoid cells. Many alveoli are distended by homogeneous, foamy, eosinophilic material characteristic of P. carinii pneumonia. Ultrastructural studies have demonstrated that this material consists of serum protein, myelin figures consistent with pulmonary surfactant, degenerate host cells and organisms, and viable organisms (Yoneda and Walzer, 1980; Walzer et al., 1989).

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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In imprints or histologic sections of lung, definitive identification of P. carinii depends on demonstration of cysts (characteristically containing 8 sporozoites) and trophozoites. The cysts measure 5 to 7 µm in diameter and have a thick wall that stains with methenamine silver, cresyl violet, periodic acid-Schiff, or toluidine blue-0 (Shiota, 1986). Giemsa stain traditionally has been preferred for demonstration of trophozoites and sporozoites in imprints (Frenkel et al., 1966). Indirect fluorescent antibody (IFA) methods also have been used (Milder et al., 1980; Kovacs et al., 1986). An immunoperoxidase method using a monoclonal antibody has been reported (C.-H. Lee et al., 1986). Acridine orange has been proposed for rapid screening of imprints; the trophozoites stain yellow to orange while the cyst walls do not stain (Thompson and Smith, 1982).

Diagnosis

For rats or mice with active infection and disease, the most reliable method is necropsy and demonstration of typical organisms in diseased lungs by the use of special staining methods, e.g., methenamine silver. Athymic (nu/nu) and severe combined immunodeficient (scid/scid) mice are unable to mount a significant antibody response to P. carinii and thus, serologic tests are of no value (Walzer et al., 1989).

Diagnosis of latent infection in mouse and rat stocks is a major problem. Two different approaches have been used:

  1. An immunosuppressive regimen followed by attempts to demonstrate organisms in diseased lung tissue (Barton and Campbell, 1969; Ogino, 1978; Walzer et al., 1979, 1980; Bartlett et al., 1987a,b). Use of this method to test a given immunocompetent rodent population for latent P. carinii infection requires either (i) testing representative animals within the confines of the domiciliary space occupied by that population, or (ii) if removed to another location, animals must be transported and maintained in a containment device such as a gnotobiotic isolator to avoid contamination during transportation and the period of immunosuppression.
  2. Serologic testing. This approach is possible but may be of limited usefulness because of the high prevalence of serum antibodies due to persistent infection in contemporary rat and mouse stocks. By IFA technique, young rats from two commercial sources were found to be negative and retired breeders were usually positive for serum antibodies to P. carinii. Serum antibodies were detected in five of six strains of immunocompetent mice up to 3 months of age. In both rats and mice the predominant serum antibody class was IgG. Nude mice rarely produced serum antibodies to P. carinii (Walzer and Rutledge, 1981, 1982).
Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Several serologic tests including IFA (Walzer et al., 1987) and enzymelinked immunosorbent assay (Maddison et al., 1982) have been developed for detection of P. carinii infection but have not been shown to be useful for health surveillance or diagnostic purposes in laboratory rodents.

Control

Subclinical infection is probably common in conventionally reared and "pathogen free" colonies (Bartlett et al., 1987a). Gnotobiotic methods probably are useful in excluding the infection but, even this approach may not be completely effective because of possible vertical transmission (Pifer, 1983, 1984; Pifer et al., 1984). Cesarean derivation followed by maintenance under gnotobiotic methods has been found to exclude the infection in rats Wagner, 1985).

Prolonged treatment with trimethoprim-sulfamethoxazole or dapsone, may be useful in controlling disease but does not eradicate the infection (Hughes, 1979, 1988).

Interference with Research

Animals for use in studies involving long term, severe immunosuppression require careful selection and maintenance to avoid complication by P. carinii (Bartlett et al., 1987a). Gnotobiotic conditions are preferred for this purpose. Athymic (nu/nu) mice and mice with severe combined immunodeficiency (scid/scid) may develop active infection under natural conditions (Ueda et al., 1977a; Walzer et al., 1989).

Chlamydia trachomatis
Significance

Low.

Perspective

This latent pathogen of the mouse was discovered at Yale University in 1939 by Dr. Clara Nigg during attempts to isolate influenza virus by intranasally inoculating throat washings from human patients into mice (Nigg, 1942; Nigg and Eaton, 1944). It was subsequently reported as a cause of pneumonia after serial passages of tissues in mice in Australia (DeBurgh et al., 1945), Germany (Gonnert, 1941, 1942), and Chicago (Gordon et al., 1938; Karr, 1943).

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Agent

A bacterium, order Chlamydiales, family Chlamydiaceae, C. trachomatis biovar mouse. The mouse biovar does not share type-specific antigens with the two human strains of C. trachomatis (biovars trachoma and lymphogranuloma venereum), and has only 30-60% DNA homology with them. The type strain is ATCC VR-123 (Nigg II). Synonyms are Nigg agent and mouse pneumonitis agent (Moulder, 1984).

Members of the genus Chlamvdia are nonmotile, Gram-negative, coccoid organisms that measure 0.2-1.5 µ in diameter and are obligate intracellular parasites. They multiply within membrane-bound vacuoles in the cytoplasm of host cells. The life cycle consists of elementary bodies (0.2-0.4 µ in diameter) and reticulate bodies (0.6-1.5 µ in diameter). They can be propagated in cell cultures, particularly in McCoy and HeLa 220 cells, and in the yolk sac of chick embryos (Moulder, 1984). C. trachomatis has compact or oval glycogen-positive inclusions. Growth in the chick embryo yolk sac is inhibited by sulfadiazine (Moulder, 1984).

Host

Mice.

Epizootiology

Natural infections are persistent and subclinical. All reported instances of disease have been the result of passaging infected mouse tissues, particularly lung, in mice (Gonnert, 1941, 1942; Nigg, 1942; Karr, 1943; Nigg and Eaton, 1944; DeBurgh et al., 1945).

Natural transmission is thought to be by inhalation and. possibly, cannibalism (Genest, 1959). There is no information available on the prevalence of this agent in contemporary mouse stocks.

Clinical

Natural infections are persistent and subclinical. The clinical signs seen in mice with active disease during serial passage of mouse tissues have been nonspecific. They have included chattering, dyspnea, cyanosis, reluctance to move, humped posture, and weight loss. The interval from inoculation to death is dose dependent, ranging from 1 to 20 days (Gonnert, 1941, 1942; Nigg, 1942; Karr, 1943; Nigg and Eaton, 1944; DeBurgh et al., 1945).

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Pathology

The major lesion is multifocal to diffuse interstitial pneumonia. Following intranasal inoculation of the organism, alveolar macrophages are the predominant responding cell until about 30 hours post infection, when the first C. trachomatis developmental cycle in macrophages and bronchial epithelium is completed. Death of these infected cells is associated with edema and infiltration of polymorphonuclear neutrophils (PMNs), beginning at about 48 hours after infection. Thereafter, the developmental cycle continues, spreading by contiguity into adjacent lung parenchyma. Macrophages and PMNs are the predominant cell types at the margins of advancing lesions, and lymphohistiocytic cells predominate in the older central zones. Peribronchial lymphoid cuffing is not a prominent feature. The lesions are not completely resolved by 6 weeks post infection. The organism can be demonstrated in alveolar macrophages and bronchial epithelium by using special strains, such as Machiavello, on either tissue sections or imprints of affected tissues (Gonnert, 1941; Weiss, 1949; Gogolak, 1953; Moulder, 1984).

Athymic (nu/nu) mice are significantly more susceptible to C. trachomatis than their immunocompetent heterozygous littermates. T-cell-dependent cellular immunity is important in host defense (Williams et al., 1984).

Woodland et al. (1983) have summarized the information on animal models that are currently in use for the study of C. trachomatis and C. psittaci infections.

Diagnosis

Definitive diagnosis requires the isolation and identification of the organism. Isolations are made by using McCoy and HeLa 229 cell cultures, inoculation of the yolk sacs of embryonating eggs, and inoculation of pathogen free mice (Moulder, 1984). The microtiter indirect immunofluorescence method is a practical, sensitive test that is used extensively for the detection of antigens and antibody in man (Wang, 1971). This test has been used experimentally for the detection of antibody to C. trachomatis in mice (Williams et al., 1981, 1982).

Control

Mouse stocks for the study of chlamydial infections should be rigorously monitored to ensure that the mice are free of these agents and maintained by a barrier program to exclude contamination. Cesarean derivation should be successful in eliminating the agent from an infected stock of mice.

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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Interference with Research

Probably the greatest risk is the inadvertent use of persistently infected mice for the study of experimental infections of C. trachomatis. Also, the infection can be activated in infected mice being used for passage of mouse tissues (Gonnert, 1941, 1942; Nigg, 1942; Karr, 1943; Nigg and Eaton, 1944; DeBurgh et al., 1945).

Chlamydia psittaci

There are two reports in which the serial passage of mouse tissues in conventionally reared mice led to the isolation of C. psittaci.

Ata et al. (1971) serially passaged mouse lung tissue intranasally in three strains of mice that had been maintained in their laboratory for 25-38 years. "Small areas of consolidation and hyperemia" were noted in the lungs of mice receiving the second and third passages of lung, and chlamydial elementary bodies were identified in imprints of these lungs stained by the Gimenez method. The Chlamydia was isolated from diseased mouse lungs in the yolk sacs of embryonated eggs. The Chlamydia was sulfadiazine resistant and negative for glycogen-containing inclusions, thus identified as C. psittaci.

Gerloff and Watson (1970) serially passaged liver and spleen suspensions by intraperitoneal inoculation of mice from a stock that had been maintained in their laboratory for 32 years. In mice of the 46th and subsequent passages there was splenomegaly, hepatomegaly, serofibrinous peritonitis, and up to 2 ml of ascitic fluid in the abdomen. A sulfadiazine-resistant Chlamydia isolate compatible with C. psittaci was established in yolk sacs of chick embryos. The intranasal inoculation of the yolk sac fluid into mice resulted in pneumonia with 50% mortality.

The significance of these reports is unclear. However, they raise the possibility that C. psittaci can occur as a persistent infection in some conventionally reared mouse stocks and that serial passage of tissues in such mice can result in activation of the infection. The prevalence of this agent in contemporary mouse stocks is unknown. It is also not known whether the strains of C. psittaci isolated from mice were variants peculiar to the mouse.

Klebsiella pneumoniae
Significance

Low.

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×
Perspective

Organisms of this species are considered part of the normal gastrointestinal flora in humans and animals. Evidence that they may be associated with natural disease in mice and rats is limited to two reports for mice (Flamm, 1957; Schneemilch, 1976) and two for rats (Hartwich and Shouman, 1965; Jackson et al., 1980).

Agent

Klebsiella pneumoniae is a bacterium, family Enterobacteriaceae, K. pneumoniae, subspecies ozaenae. Gram-negative, nonmotile, capsulated bacillus, 0.3-1.5 µ x 0.6-6.0 µ, occurs in pairs or short chains. Lacks special growth requirements. Does not utilize malonate. Voges-Proskauer test negative. Produces large mucoid colonies. Four or five capsular types have been identified for K. pneumoniae ozaenae (Orskov, 1984).

The taxonomy of the genus Klebsiella has been revised recently. K. pneumoniae now has three subspecies: pneumoniae, ozaenae, and rhinoscleromatis (replacing the three former species: K. pneumoniae, K. ozaenae, and K. rhinoscleromatis). Capsular types 3, 4, 5, and 6 have been regarded as belonging to K. pneumoniae ozaenae (Edwards and Fife, 1952; Orskov, 1957, 1984). Capsular type 5 has been associated with one outbreak of the disease in rats (Jackson et al., 1980), and capsular type 6 has been associated with an outbreak in mice (Schneemilch, 1976). The organisms from two similar outbreaks, one in rats and one in mice, were not typed (Flamm, 1957; Hartwich and Shouman, 1965). Thus, it is possible that K. pneumoniae ozaenae was the organism responsible for all four reported outbreaks.

In the past, K. pneumoniae included isolates from natural settings such as soil, water, grain, and forest products (Duncan and Razzell, 1972; Newman and Kowalski, 1973). Many of those organisms now belong to two proposed new species: K. terrigena and K. planticola. Indole-positive Klebsiella organisms are now classified as K. oxytoca (Orskov, 1984).

Hosts

Mice, rats, humans, and others.

Epizootiology

The organism is presumably a normal inhabitant of the gastrointestinal tract in man and animals, including mice and rats. It is an opportunistic pathogen. Transmission is by feces, air, and water (Ostrom, 1958).

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×
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.

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×
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.

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×
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.

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×
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-

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

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-

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

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).

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×
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.

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×
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;

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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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

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×

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).

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
×
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).

Suggested Citation:"6. Respiratory System." National Research Council. 1991. Infectious Diseases of Mice and Rats. Washington, DC: The National Academies Press. doi: 10.17226/1429.
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This new edition—a must for all researchers who use these lab animals—provides practical suggestions for breeding, keeping, and identifying pathogen-free laboratory rodents. It contains three informative sections. The first, Principles of Rodent Disease Prevention, summarizes methods for eliminating infectious agents. It offers information on pathogen terminology; pathogen status of rodents; and breeding, transporting, isolating, testing, and diagnosing rodents. The second section, Individual Disease Agents and Their Effects on Research, describes the diagnosis and control of each infectious agent, and the last section, Diagnostic Indexes: Clinical Signs, Pathology, and Research Complications, contains informative tables covering all the diseases listed in the volume, arranged to help in the diagnosis of infected animals.

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