Thursday, July 7, 2016
Infectious diseases are caused by microorganisms such as viruses, bacteria, fungi or parasites and can spread between individuals.
Mouse pox
Mouse pox is a highly infectious disease to which all strains of mice are susceptible, some more than others. Ectromelia is an excellent example of a virus carried and spread by apparently healthy mice. There may be no signs of illness, yet active virus may be readily recovered from viscera by blind passage. The virus obviously not only persists and multiplies despite a certain suppression by antibody, but also is shed through the feces. The virus was first described in England, but has since been reported from continental Europe as well as the United States ( Saunders, 1958a; Trentin, 1953). It is from 100 to 150 μ in diameter, is destroyed by exposure to 55°C for 30 minutes, but withstands 50 per cent glycerol and 0.5 per cent phenol for several months at refrigerator temperature. Clinical aspects of the disease have been described in detail by Fenner ( 1949) and Trentin ( 1953). The disease is generalized, the virus multiplying in cells of the skin, lymph nodes, liver, spleen, and other organs. In all these tissues intracellular eosinophilic inclusion bodies develop. The acute form, occurring in previously unexposed mice, is characterized by visceral lesions, usually hepatic necroses, the animals dying within days without external signs of illness. Sometimes inconspicuous external primary lesions are present such as swollen eyelids or pocked noses. The disease usually spreads rapidly throughout a susceptible colony, killing 50 to 95 per cent of the mice within weeks. The surviving mice develop circulating antibodies and often show a chronic form of the disease characterized by necrosis of the extremities, gangrene, crusting and scarring of skin, and regenerative lesions of internal organs. The foot lesions must be distinguished from those caused by Streptobacillus moniliformis. The most reliable diagnostic test for ectromelia infection is hemagglutination inhibition (HI). Either vaccinia or ectromelia virus is used as a red blood cell-agglutinating antigen. Serum from mice recovered from acute mouse pox will inhibit such agglutination of blood cells ( Burnet and Boake, 1946; Briody, 1959). The test can be quickly and conveniently carried out with as little as 0.05 ml of serum. The virus is demonstrated by intraperitoneal inoculation of visceral suspensions into susceptible mice. These animals ordinarily die 4 to 6 days after inoculation and show the visible lesions of multiple focal necroses of the livers.
Mouse colonies can be protected from ectromelia by strict isolation to preclude contamination by infected mice and other materials. Once the disease is recognized, however, infected colonies should be destroyed and all presumably infected animals incinerated. Another preventative measure, applicable to valuable inbred stocks, is vaccination, either by scarification or intranasal instillation with vaccinia virus or with formolized liver suspensions from infected mice ( Shope, 1954). Although both Levaditi and IHD-T strains of vaccinia virus have been successfully employed, IHD-T is preferred because it is more immunogenic, does not induce HI antibodies, and will not interfere with the HI test for diagnosis of infection. It is therefore possible to distinguish between antibodies formed as a result of infection and those formed as a result of vaccination. Vaccinated mice may transfer the infection to unvaccinated cage mates but not to mice in other cages of the same room. DBA mice seem to be more resistant to contact infection than other stocks ( Briody, 1959).
Hepatoencephalitis group of viruses
Some viruses are capable of causing hepatitis, encephalitis, or both in suckling mice. These are designated as MHV, JHM, EHF 120, and H747 and are related by degrees of cross-immunity (complement-fixation, neutralization tests). All produce qualitatively similar histological lesions affecting mesothelial and endothelial surfaces. These viruses, particularly the neurotropic ones, have similar susceptible host species ranges and occur in latent form in mouse stocks. All are heat-labile and ether-sensitive and range in size from 80 to 120 μ. The MHV or mouse hepatitis virus gives rise to intranuclear inclusion bodies, but to produce active disease requires the synergistic action of Eperythrozoön coccoides, a red blood cell parasite. The link between the two agents is complex. References may be found in Gledhill and Niven ( 1955) and Gledhill ( 1962).
Neurotropic viruses causing spontaneous encephalitis
There are three groups of viruses causing spontaneous encephalitis with distinctions based on the type of pathological lesion. Theiler's FV, FA, FO, and GD-VII affect the gray matter primarily, producing lesions comparable to those found in poliomyelitis ( Theiler and Gard, 1940; Thompson et al., 1951). The second group includes the JHM virus which causes demyelinization ( Olitsky and Lee, 1955; Pappenheimer, 1958a). The third group, which does not cause demyelinization, consists of SK, the Columbia strain of Jungeblut, LCM or lymphatic choriomeningitis ( Traub, 1939; Haas, 1954), EK virus, herpes simplex, and eastern equine encephalitis.
Theiler's mouse encephalomyelitis. Spontaneous infections of the central nervous system due to this virus are considerably more prevalent in younger mice. Unless the disease is rapidly fatal, gradual flaccid paralysis, usually of the hind limbs, develops. Microscopically, anterior-horn lesions quite similar to those of human poliomyelitis (ganglionic cell destruction) are observed. However, there is no serological relationship between Theiler's virus and that of poliomyelitis. Theiler's virus is recoverable from the intestinal tract or central nervous system. Artificial infections are possible by virtually all routes, although the level of infective dose differs considerably. For a more detailed review see Maurer ( 1958a).
Lymphocytic choriomeningitis. Although mice of laboratory colonies may harbor this virus, the disease is carried in latent form with few animals showing clinical signs or dying. Clinical illness occurs only in young animals which may show photophobia, conjuctivitis, and convulsive leg movements. Tremors, spastic convulsions, and paralysis are observed in Artificial infections. Microscopically, leukocytic infiltration of the meninges, pleural exudation, and splenomegaly are found. Virus is detected by intracerebral inoculation of mice with sterile suspensions of brain, blood, or spleen from suspect animals. In view of the wide host range (mice, monkeys, dogs, guinea pigs, and man) LCM represents a hazard to both experimental animals and man. Aside from direct contact transmission, as by the urine of infected mice, the virus may also be spread by ectoparasites and mosquitoes. For a review see Maurer ( 1958b).
Pneumotropic viruses
A number of viruses have been implicated as causes of respiratory disease in mice. Among these are Nigg's pneumonitis ( Nigg and Eaton, 1944) and PVM or pneumonia virus of mice ( Mirick et al., 1952; Volkert and Horsfall, 1947).
K virus
K virus infection is also a respiratory disease. In suckling mice the clinical signs are labored breathing and early death. Large basophilic inclusion bodies are observed in greatly hypertrophied alveolar lining cells. On occasion, focal disseminated fatty liver dystrophy is found. Since the first description of the disease and isolation of the virus, the K virus has been isolated in several parts of the world, notably Australia ( Fisher and Kilham, 1953; Holt, 1959; Derrick and Pope, 1960). The virus presumably spreads by way of the urine and saliva, yet serological surveys of both laboratory and wild mouse colonies indicate that antibodies occur in only a small proportion of mice from infected colonies.
Livers of moribund suckling mice provide a source for a suitable complement-fixing antigen so that the virus may be detected by the presence of specific antibody, although intracerebral inoculation of newborn mice with suspect tissues is a more sensitive test. It has been suggested that the K virus may be generically related to polyoma in view of certain common biological properties such as size, stability, etc. (Kilham, cited by Rowe et al., 1962.
Salivary gland virus
This virus is ubiquitous in wild mouse populations, but has been observed in only a few laboratory colonies. Apparently the virus does not spread with ease ( Rowe et al., 1962) since even in infected colonies its incidence is 3 per cent or less. Virus isolation techniques must be used for laboratory diagnosis. Serological techniques are of little value, and inclusion bodies are rarely observed despite continual excretion of virus in saliva ( Rowe et al., 1962; Brodsky and Rowe, 1958). Two procedures (utilizing mouth swabs as a convenient source of virus) are generally employed: (1) isolation of the virus in mouse embryo tissue culture, and (2) inoculation of newborn mice. The disease may be suspected in suckling mice by their malnourished appearance and at necropsy by the gross yellow discoloration of the edges of the liver.
Thymic virus
The thymic agent has been discovered in as high as one-half the mice of one laboratory colony and is highly prevalent in wild mice. It is pathogenic only for newborn mice and is recognizable by the production of gross thymic necroses, visible about 2 weeks after inoculation. Direct virus isolation is the most reliable diagnostic means, although infected mice produce neutralizing antibodies in low titer. The salivary glands represent the best source of virus since naturally or artificially infected mice excrete the virus in saliva for periods of more than one year. In contrast to the mouse salivary gland virus, the thymic agent does not propagate in tissue culture ( Rowe and Capps, 1961).
Adenovirus
This virus regularly infects recently weaned mice after prolonged exposure to urine of carrier animals. Infection induces a good complement-fixing and neutralizing-antibody response. Spontaneous clinical disease rarely occurs. However, virus inoculation into suckling mice induces a fatal disease consisting in inflammation and necrosis of the heart, adrenals, and brown fat. Acidophilic intranuclear inclusion bodies occur in these and other tissues ( Hartley and Rowe, 1960). The virus produces cytopathic effects in mouse kidney tissue cultures. Fluids from such infected cultures contain an excellent complement-fixing antigen which reacts strongly with adenoviral antisera prepared in guinea pigs (but not with human, monkey, or dog antisera). Apparently the virus does not cross the placental barrier, since it has not been encountered in caesarian-derived colonies ( Rowe et al., 1962).
Reoviruses
The reovirus group is composed of three serotypes ( Sabin, 1959). Antibodies to reoviruses are assayed by hemagglutination inhibition, neutralization, or complement-fixation tests. Reo 3 virus can be detected by tissue-culture cytopathogenicity, inoculation of suckling mice, or mouse antibody production tests. The sera of some mice contain hemagglutination inhibitor which is undoubtedly nonspecific since it is commonly encountered even in specific-pathogen-free and germ-free mouse colonies.
Only Reo 3 is indigenous to mice and has been isolated from tissue suspensions containing Molony virus. The oncolytic virus of Nelson, obtained from a transplanted ascites tumor ( Nelson and Tarnowski, 1960) has been identified as Reo 3. Reo 2 has been encountered as a focal infection in several wild mouse populations which may have acquired infection by contact with other species, such as man and cattle ( Rowe et al., 1962).
Oncogenic and tumor-associated viruses
Gross ( 1961) has discussed the status of virus-induced neoplasms, including mouse leukemias as produced by cell-free leukemic filtrates, the induction of leukemia by filtrates of solid mouse tumors, and the filterable agent causing mouse mammary tumors (Bittner's milk factor). Sinkovics ( 1962) has reviewed viral leukemias in mice and oncogenic viruses are discussed in Chapters 27 and 28. We refer here only to mouse polyoma infection and the lactic dehydrogenase-stimulating viruses ( Gross, 1953; Stewart, 1953; Riley et al., 1960).
A detailed epidemiology of mouse polyoma infection has been developed after intensive studies. The classic techniques of virology for detection of antibodies reveal that mouse polyoma virus is widely disseminated among laboratory colonies and wild mouse populations. Focal reservoirs of infection are maintained by routes of transmission involving virus excretion in urine, feces, and saliva. Apparently the virus is not transferred transplacentally since mice derived by caesarean section for specific-pathogen-free or germ-free colonies have been negative to antibody tests. Polyoma virus (e.g., infected tissue culture fluid) induces multiple tumors when inoculated into newborn mice. Such tumors are of multicentric and multiple histological origins, with mixed tumors of the salivary glands most frequent. The virus is capable of producing tumors in other species as well.
From the epidemiology of polyoma virus infection several points are notable: (10 Resistance of infant mice is perfectly correlated with presence of maternal antibody titers; no tumors develop in offspring of positive mothers; (2) spontaneous parotid tumors are rarely found in naturally infected mouse colonies; (3) high antibody titers are present in milk of infected mice; and (4) maternally derived antibodies are presumably present in fetuses, since it has been shown that mice born of resistant mothers but not having access to immune mouse milk are resistant to polyoma infection.
Certain transmissible agents have been found to be associated with many transplanted and induced spontaneous tumors of mice ( Riley et al., 1960). These are manifested by biochemical response. Susceptible animals respond with five- to tenfold increases in serum lactic dehydrogenase (LDH) and by induction of other glycolytic enzymes after inoculation with plasma or organ extracts from tumor-bearing hosts. Natural transmission of the lactic dehydrogenase-stimulating agents(s) (LDH) occurs when normal mice are placed in the same cage with agent-infected mice or tumor-bearing mice. In most infected animals moderate splenomegaly occurs. Serum LDH elevation has been induced by inoculation with preparations of cells from infected mice ( Riley et al., 1961). Although there is a close correlation between LDH levels and the growth of several transplanted tumors, elevation is not directly related to the neoplastic process. There is evidence to indicate that the LDH factor is a virus, specifically of mouse origin, and that changes in the LDH level of a tumor-bearing animal depend on whether or not the LDH factor has become associated with that tumor ( Notkins et al., 1962). An increase in lactate dehydrogenase levels can also be induced by injection of several of the mouse hepatitis viruses. LDH virus may be eliminated as a contaminant of transplantable tumors by passage of such material through tissue culture. However, it has been reported that the agent can be propagated and maintained by serial passage on primary mouse embryo tissue-culture ( Yaffee, 1962). It can be inactivated by radiation and is unstable in the presence of ether ( Notkins et al., 1962).
Infantile diarrhea
Diarrheal disease of unweaned mice is a complex syndrome caused by a number of agents ( Pappenheimer, 1958b; Runner and Palm, 1953; Kraft, 1962). As is true for diarrheal disease in human infants, there is considerable evidence that no single pathogen is the causative agent ( Thomlinson, 1964; Erlandson et al., 1964; Altman, 1964; Payne, 1960; Barua et al., 1962). Rather, under certain environmental conditions (crowding, poor sanitation, improper nutrition, variable temperatures, too low or too high humidity, inadequate ventilation, etc.) ubiquitous viral or bacterial organisms may be incriminated as producing diarrheal or septicemic signs in unweaned mice. We find that host factors, such as strain of mouse and parity play a determining role in morbidity and degree of clinical signs.
The clinical signs of the syndrome are: slight-to-severe diarrhea in which fecal material ranges from bright yellow to light brown in color. Nursing mothers will sometimes very efficiently clean up the anal region of the affected animal so that only a slight "pasting up" condition is noted. Animals affected by diarrhea (usually 4 to 10 days of age) usually recover, but many are discarded as runts at weaning time. The diarrhea is often followed by constipation contributing to later mortality, Mortality more often occurs in animals not showing signs of diarrhea. Such deaths occur early, 1 to 2 days after onset of the disease in a litter. Survivors in these litters show varying degrees of diarrhea.
At necropsy, the only common finding is the presence of light colored and watery fecal material in the intestinal tract, with occasional bubbles of gas. Histologically the tissues demonstrate a mild catarrhal inflammation. Some workers have reported inclusion bodies in epithelial cells from live and intestinal tract.
Viral agents such as those described by Kraft ( 1962) are found in the intestinal contents and reportedly can be transmitted directly to susceptible animals through this medium. Various types of bacteria, coliforms, Proteus sp., and pseudomonads have been found in separate outbreaks of the disease.
Epidemiologically the disease usually occurs in cycles. In certain affected mouse colonies, diarrheal disease is rarely seen until the fall and winter months. Our studies suggest that lower humidity and less fresh outside air are involved in seasonal recurrence rather than length of day and seasonal variation in quality of diet.
Colonies in which diarrheal disease is enzootic may show longer periods between epizootic outbreaks when moved to new quarters. During an outbreak the disease can be controlled by the use of broad-spectrum antibiotics such as the oxytetracyclines or tetracyclines administered in the drinking water. Therapeutic doses are administered over a 2-week period to all animals in a breeding colony. The preferable medication period is during the last third of pregnancy. This treatment is repeated in 2 weeks. Such therapy results in marked increase in the numbers of healthy-looking mice weaned and a decrease in the incidence of diarrhea. Similar effects are produced by the use of air-filtering material placed around each breeding cage or by the use of aluminum foil covering 50 per cent or more of open cage surfaces. The effects shown by cage modification (air filtering, foil covers) may be due to decreased aerosol dose levels. The use of certain types of highly absorbent bedding material also results in a higher recovery rate and a decrease in diarrheal disease. All of these control measures singly or in combination result initially in marked improvement of colony health, but usually over a period of months the disease reappears. Also, the effects of combining such control measures are not additive but only as good as the best measure used alone. Other effective control and prevention measures entail a variety of steps to isolate each breeding pen completely.
Much remains to be learned of the causes of infantile diarrheal disease, but the diarrhea should be considered as an accompaniment of certain diseases of suckling mice and not as a disease entity in itself.
BACTERIAL DISEASES
The spontaneously occurring or latently present bacterial diseases of mice are numerous, and the mouse is also susceptible to experimental infection by many pathogens from other species. Because of the small size of the animal, its habits, and the complexity of husbandry methods applied, it is extremely difficult to evaluate the health of a single animal by the usual clinical methods. Instead, an epidemiological approach is necessary. The population or a percentage thereof is observed for common factors which can be related to the various signs of morbidity and to mortality rates ( Shope, 1964).
Salmonellosis
Infection by Salmonella sp. is one of the more common types of bacteriological infections of mice ( Habermann and Williams, 1958a; Lane-Petter, 1963). The most commonly found strain is Salmonella typhimurium. However, mice are highly susceptible to infection by most of the Salmonella sp. organisms and other strains have been reported as responsible for epizootics or as latently infecting a few mice in a colony ( Wetmore and Hoag, 1960; Hoag et al., 1964a). The degree of virulence is dependent largely upon the dose and route of infection and the strain of host mouse as well as upon the inherent virulence of the organism itself. Salmonellosis is characterized initially by diarrhea or soft stools, sudden deaths, anorexia, and cachexia. Large number of animals are often lost not by death, but by the culling of underweight and poor-looking mice. The initial outbreak of salmonellosis soon subsides into a chronic form of the disease wherein the organisms are shed in fecal material. The adult carriers often appear healthy, but suckling and growing mice show great variation in body weight and rates of gain. Occasional breeding pairs are affected by attacks of mild diarrhea evidenced by soft, light-colored fecal pellets. Cull rates are high chiefly because of variations in weight and the poor appearance of weanling mice. At necropsy only a few animals show the classic "white spotted livers." Many will show enlarged spleens, but no gross necropsy findings are pathognomonic. Spleen, liver, and both large and small intestine are the tissues of choice for bacteriological examination. Tissues should be separately cultured for the presence of the organisms. In moribund or sick-looking animals the liver and spleen will yield Salmonella, whereas the chronically infected cases will usually yield organisms from the intestinal tract only.
For the isolation of Salmonella sp. enrichment media, preferably Tetrathionate Broth (Difco Laboratories), should be inoculated with a quantity of minced or ground tissues and the media incubated at 37°C for 72 hours ( Hoag and Rogers, 1961). During this period subcultures to Brilliant Green Agar (Difco) are incubated at 37°C for 48 hours before being discarded as negative for salmonella growths. Serological identification of suspect bacterial colonies is then carried out with polyvalent or group-specific antisera. Specific identification is dependent upon further serological techniques.
The disease is not controllable by any known therapeutic measures. Broad-spectrum antibiotics such as tetracycline or oxytetracycline seem to be of some use in epizootics in increasing the numbers of survivors, but they will not cure chronically infected animals. Vaccines of various types have been found to be highly effective against clinical signs and mortality from the disease but do not prevent chronic infection and carrier states from developing ( Hoag et. al., 1964b). We have demonstrated that three differently prepared S. typhimurium vaccines were effective against as many as 100 LD50 challenge doses of S. typhimurium but all of the surviving animals, although appearing perfectly healthy, were shedding organisms in fecal material and had infected livers and spleens as long as 6 weeks after the challenge.
The disease is controlled by elimination of infected or carrier stocks. Several (at least two and preferably six) weekly fecal tests should be performed before concluding that an animal is free of infection, since intermittent and low-level shedders are commonly encountered in a chronically infected colony. Sanitation is an important part of preventing spread of the disease, since an important mode of transmission is by contact with contaminated objects. Food supplies and bedding material should be treated as potential sources of infection. Occasional parathyphoid carriers are found among mouse handlers, but are not important sources of animal infection.
Pasteurellosis
Pasteurellosis due to Pasteurella pseudotuberculosis, P. septica, or P. pneumotropica may occur spontaneously in laboratory mice ( Sellers et al., 1961; Tuffery, 1958; Hoag et al., 1962). The latter two organisms may be found in various tissues of apparently normal mice. Latently infected animals subjected to various types of stress (radiation, abrupt temperature changes) will often produce signs of acute disease: anorexia, lassitude, sudden deaths. The livers of animals infected with P. pseudotuberculosis present multiple focal abscesses. P. pneumotropica has been reported in outbreaks of pneumonia in mice and has also been found in normal-appearing lung tissue ( Jawetz, 1948; Jawetz and Baker, 1948). In other instances this organism has been isolated from the brain, uterus, testes, liver, or spleen of normal-appearing mice. In rare instances septicemic deaths have been attributed to P. pneumotropica. Pasteurellosis is probably a stress-induced disease and the causative organism most commonly a latent infective one.
The pasteurella organism is susceptible to the tetracycline or oxytetracycline types of broad-spectrum antibiotics but is usually resistant to penicillin. Outbreaks in which signs of disease are thought to be caused by pasteurella organisms (on the basis of isolations from organ tissue) may be treated by administering the effective antibiotics in the drinking water. Response to the drugs is immediate and favorable.
Pseudomonas infections
Infection with Pseudomonas aeruginosa is common in laboratory mouse colonies ( Flynn, 1963a). No signs of infection are manifest unless animals are subjected to radiation or other types of stress (cortisone, etc.) ( Flynn, 1963a; Wensinck et al. 1957; Verder and Rosenthal, 1961). Such treatment results in rapid onset of septicemic disease (anorexia, listlessness) and high mortality. At necropsy the organisms are easily cultured from all organs, particularly liver and spleen. Although not important as a spontaneous disease-causing organism, P. aeruginosa is of major concern to investigators using mice in experiments employing radiation. The organism is shed in feces and urine from chronically infected animals. Nearly 100 per cent of the animals in a colony may become rapidly infected by contamination and recontamination of drinking water, caging equipment, and feeds. The infection can be controlled by hyperchlorination and acidification of water used for drinking and cleaning ( Hoag et al., 1965). Sanitation procedures must be of the highest efficiency and detectable carriers eliminated. Carrier detection should not serve as a criterion for culling until after control procedures such as water treatment and intensified sanitation have been inaugurated, since it has been shown that most fecal shedders of the organisms are only transiently infected and will seemingly spontaneously "recover" if not exposed to further doses of the organisms in contaminated water. After control procedures have been in effect for several weeks, a test-and-slaughter program should be started. Pens containing mice shedding the organism should be provided from the colony.
Antibiotics and other drugs are of no value in treating the infection. In one instance when oxytetracycline was being administered in drinking water to inbred mice, it was found that the organism grew more rapidly in water containing hypertherapeutic levels of the drug ( Hoag et al., 1965).
Klebsiella infections
Latent infections with capsulated lactose-fermenting bacilli, often loosely identified as Klebsiella group organisms, are not uncommon ( Wilson and Miles, 1964). Such organisms are occasionally isolated from lung tissue of normal-appearing mice. Sometimes the organism is found in the nasopharyngeal region or in various body wastes. In certain outbreaks of pneumonia in mice, Klebsiella sp. can be recovered from 100 per cent of the lungs of sick or moribund mice. The organisms recovered vary greatly in virulence. Rarely, the bacterium can be recovered from abscesses of lung or from other internal organs and cutaneous areas. Improved sanitation and environmental constancy are recommended control measures.
Tyzzer's disease
Disease caused by Bacillus piliformis has been described by several workers ( Fujiwara et al., 1963; Fujiwara et al., 1964; Saunders, 1958b). Most agree that the organism occurs in an infected colony as an intestinal saprophyte and that clinical disease occurs only after animals have been stressed, as by adverse nutrition or by the effects of stressors introduced during the course of an experiment. Onset of disease signs is abrupt with death occurring 2 to 3 days afterward. Diarrhea may occur as the major sing or may affect only a few of the sick animals. Anorexia and other signs of septicemia are manifest. Some immunity develops in recovered animals.
The condition may occur in mice of all ages, but the highest mortality rate is observed in 3- to 7-week-old animals. The most striking necropsy finding is the occurrence of numerous grayish-white spots (up to 2.5 mm in diameter) on the outer and cut surfaces of the liver. Mesenteric lymph nodes are usually enlarged and sometimes abscessed. Microscopically the organisms (long, thin, Gram-negative rods) are found intercellularly, surrounding the necrotic tissue areas, but are also seen in intact liver cells as well. the organisms have been reported as cultivatable in tissue culture, but cannot be grown on defined bacteriological media. Diagnosis is by histological sectioning and staining of tissues with subsequent demonstration of stained organisms.
Differences between mouse strains in susceptibility have been demonstrated, but it is emphasized that any incidence in a mouse colony represents a disease potential and infected colonies must therefore be considered as hazardous for other research. Little work has been done to demonstrate the effectiveness of therapy, although oxytetracycline has been of value ( Tuffery, 1958).
PPLO (Pleuropneumonia-like organisms)
Infections with organisms described as PPLO have been chiefly reported as causing catarrhal disease in mice ( Nelson, 1958). Some outbreaks of disease of the upper respiratory tract are characterized by sudden onset and involvement of a large percentage of a colony usually after chilling or overheating. Chattering and sniffling with variable nasal discharge are the observable signs of infection. Mortality rates are low, and recovery from the exudative catarrh is spontaneous. A few animals succumb to bronchopneumonia or may develop a chronic bronchiectatic pneumonia. In other types of upper respiratory disease caused by PPLO the onset is gradual and the outbreak appears enzootic in character, Because of the chronic nature and long course of the disease, the eventual involvement of large numbers of animals may give the appearance of a suddenly occurring epizootic disease, particularly if the mild symptoms in the early stages of the condition are unnoted. Labyrinthitis (manifested by disequilibrium) may occur in a few of the mice.
PPLO organisms are often found in animals from an apparently healthy colony and may be recovered from various tissues other than lung material ( Freundt, 1959). The pathogenic potential of latent infections is always manifest but has not been quantitatively evaluated.
Streptobacillus moniliformis
Infections with Streptobacillus moniliformis are usually latent. In certain outbreaks where clinical signs include lameness and swelling of joints and extremities (due to edema), the organism is readily isolated from blood and organs, including affected joints ( Wilson and Miles, 1964; Freundt, 1956). In other instances the organism is not found in the bacterial cell state, but is isolated with extreme difficulty in L form by PPLO culture techniques ( Freundt, 1959). Clinical signs are often confused with those seen in chronic ectromelia, and for this reason an early differential diagnosis is indicated ( Lane-Petter, 1963). Stress plays an important role in the activation of latent infection. In many outbreaks mice will arrive in apparently good health after exposure to adverse shipping conditions, but within 3 to 4 days 10 to 50 per cent will develop signs of the disease — swellings and ulcerations of feet and tail after the appearance of a sharp band of demarcation proximal to the swelling. Recovery is usually spontaneous, although severely affected animals are deformed because of deranged circulation to extremities with subsequent sloughing of the entire area distal to the necrotic demarcation band. In our experience antibiotics such as tetracycline and oxytetracycline seem to be useful in controlling outbreaks. In clinically affected animals these drugs serve to mitigate the severity of developing lesions.
PARASITIC DISEASES
Helminth infections
Mice may become infected with helminths from other species ( Haberman and Williams, 1958b). Outbreaks of infection with Taenia taeniformis can occur when mice are housed in the same area with cats. Infections with the various helminths rarely produce clinical signs and are only potentially important as producing unpredictable variables in animals used in research. Heavily infected animals are below norms in weight and may be anemic. Some mouse colonies may be infected with Hymenolopis nana (the dwarf tapeworm). The life cycle of this parasite does not involve any secondary hosts, with infection taking place directly from eggs excreted in feces. Upon necropsy, the tapeworm is easily observed through the walls of the unopened intestinal tract.
Mouse colonies are often infected with the oxyurids Syphacia obvelata and Aspiculuris tetraptera. These mouse pinworms have a direct life cycle and spread through a colony rapidly because of the large numbers of eggs excreted and the ease with which the eggs are airborne. There are no clinical signs of oxyuriasis. There may be some weight and growth variation between infected and noninfected animals, but other signs such as poor hair coat, etc., are not clear-cut. There is some evidence that oxyuriasis may contribute to rectal prolapse. Diagnosis can be made by examination of fecal material or the anal region for oxyurid eggs by the various flotation or contact-tape techniques. Pinworm infection may be eliminated by treatment with various drugs such as the piperazine compounds, usually most efficacious when administered via drinking water ( Hoag, 1961). Treatment must be accompanied by through cleaning of the room and caging equipment to remove the possibility of reinfection from egg-contaminated dust.
Arthropod infections
Lice (Polyplax serrata) and mites (Myobia musculi, Myocoptes musculinus, Myocoptes romboutsi, Radfordia affinis, and Psoregates simplex) are the chief ectoparasites of laboratory mice ( Flynn, 1963b). Their presence is manifested by hair loss and scratching, which may give rise to bacterially infected ulcerative wounds. It is not unusual for mice to die in a heavily infested colony as a result of these infected sores.
Control of ectoparasites is difficult. Various dusting powders containing DDT, methoxyclor, rotenone, or other parasiticides, and dips containing aramite or other materials have been used, but must be regularly or periodically applied. Bedding materials either treated with aromatic hydrocarbons (such as crude cedar wood oil) or naturally containing such materials (cedar wood shavings) are successful in suppressing infestations.
PROTOZOAN INFECTIONS
Several types of protozoan diseases have been reported in mice. Most are inapparent infections, noted only after synergistic activity with other agents such as viruses or after interference with immunological body defenses as by splenectomy, Control is usually by improving hygiene after elimination of infected animals or whole colonies.
Eperythrozoön coccoides occurs as a blood parasite in the form of disc-shaped structures on the surface of red blood cells. Splenectomy of infected mice results in a marked increase in the numbers of parasitized cells and onset of transient mild anemia. The presence of this parasite increases susceptibility to such infectious agents as the mouse hepatitis virus, lymphocytic choriomeningitis virus, and lactate dehydrogenase-elevating virus ( Seamer et al., 1961; Riley, 1964).
Hemobartonella muris is another red blood cell parasite activated in infected mice after splenectomy ( Griesemer, 1958). Infectivity is very low. The anemia produced is mild and transitory.
Eperythrozoön cuniculi causes mild febrile disease in mice, infects epithelial cells of the kidney papillae, and produces granulomatous lesions in brain tissue ( Yost, 1958). These organisms occur as 1.5- to 2.0-μ Gram-positive rods. Transmission is apparently by way of urine, which may contain large numbers of organisms.
Klosiella muris has been described as causing an infection of mouse kidney epithelial cells ( Dunn, 1949). Signs of disease are inapparent although, on necropsy, kidneys of infected animals may be surface-marked with varying numbers of tiny grayish-white foci. Transmission is by way of spore cysts released into the urine.
MYCOTIC INFECTIONS
Mycotic infections of mice usually produce a dermatitis. Such infections, which can be caused by any one of several Trichophyton or Microsporum sp., result in circumscribed encrusted areas with hair loss and are empirically called ringworm. The fungus from the lesions may be easily demonstrated by microscopic examination of skin scrapings ( La Touche, 1957). Control of the colony infection is difficult. Outbreaks are chiefly important as sources of human infection as most of these fungi cause similar lesions in man.
SUMMARY
The mouse is susceptible to an array of infectious agents. Many of these agents produce signs of disease and must be eliminated or controlled if only to maintain sizable mouse populations. On the other hand, because the mouse is so important as a biological yardstick for measurement of varied procedures, it becomes important as well to eliminate or control those infectious agents which do not produce observable disease, but which may affect the outcome of an experiment. The development of mouse colonies from caesarean-derived stock is an effective panacea for eliminating those disease agents which cannot penetrate the placental barriers. In another respect, it is important to study the synergistic or antagonistic effect of various disease agents either in combination with each other or with other factors so that information so derived may contribute toward man's understanding of his own disease problems. It would be unfortunate to eliminate completely all disease of mice before obtaining more complete knowledge of the etiology, epidemiology, and pathology of such naturally occurring conditions.
Mouse pox is a highly infectious disease to which all strains of mice are susceptible, some more than others. Ectromelia is an excellent example of a virus carried and spread by apparently healthy mice. There may be no signs of illness, yet active virus may be readily recovered from viscera by blind passage. The virus obviously not only persists and multiplies despite a certain suppression by antibody, but also is shed through the feces. The virus was first described in England, but has since been reported from continental Europe as well as the United States ( Saunders, 1958a; Trentin, 1953). It is from 100 to 150 μ in diameter, is destroyed by exposure to 55°C for 30 minutes, but withstands 50 per cent glycerol and 0.5 per cent phenol for several months at refrigerator temperature. Clinical aspects of the disease have been described in detail by Fenner ( 1949) and Trentin ( 1953). The disease is generalized, the virus multiplying in cells of the skin, lymph nodes, liver, spleen, and other organs. In all these tissues intracellular eosinophilic inclusion bodies develop. The acute form, occurring in previously unexposed mice, is characterized by visceral lesions, usually hepatic necroses, the animals dying within days without external signs of illness. Sometimes inconspicuous external primary lesions are present such as swollen eyelids or pocked noses. The disease usually spreads rapidly throughout a susceptible colony, killing 50 to 95 per cent of the mice within weeks. The surviving mice develop circulating antibodies and often show a chronic form of the disease characterized by necrosis of the extremities, gangrene, crusting and scarring of skin, and regenerative lesions of internal organs. The foot lesions must be distinguished from those caused by Streptobacillus moniliformis. The most reliable diagnostic test for ectromelia infection is hemagglutination inhibition (HI). Either vaccinia or ectromelia virus is used as a red blood cell-agglutinating antigen. Serum from mice recovered from acute mouse pox will inhibit such agglutination of blood cells ( Burnet and Boake, 1946; Briody, 1959). The test can be quickly and conveniently carried out with as little as 0.05 ml of serum. The virus is demonstrated by intraperitoneal inoculation of visceral suspensions into susceptible mice. These animals ordinarily die 4 to 6 days after inoculation and show the visible lesions of multiple focal necroses of the livers.
Mouse colonies can be protected from ectromelia by strict isolation to preclude contamination by infected mice and other materials. Once the disease is recognized, however, infected colonies should be destroyed and all presumably infected animals incinerated. Another preventative measure, applicable to valuable inbred stocks, is vaccination, either by scarification or intranasal instillation with vaccinia virus or with formolized liver suspensions from infected mice ( Shope, 1954). Although both Levaditi and IHD-T strains of vaccinia virus have been successfully employed, IHD-T is preferred because it is more immunogenic, does not induce HI antibodies, and will not interfere with the HI test for diagnosis of infection. It is therefore possible to distinguish between antibodies formed as a result of infection and those formed as a result of vaccination. Vaccinated mice may transfer the infection to unvaccinated cage mates but not to mice in other cages of the same room. DBA mice seem to be more resistant to contact infection than other stocks ( Briody, 1959).
Hepatoencephalitis group of viruses
Some viruses are capable of causing hepatitis, encephalitis, or both in suckling mice. These are designated as MHV, JHM, EHF 120, and H747 and are related by degrees of cross-immunity (complement-fixation, neutralization tests). All produce qualitatively similar histological lesions affecting mesothelial and endothelial surfaces. These viruses, particularly the neurotropic ones, have similar susceptible host species ranges and occur in latent form in mouse stocks. All are heat-labile and ether-sensitive and range in size from 80 to 120 μ. The MHV or mouse hepatitis virus gives rise to intranuclear inclusion bodies, but to produce active disease requires the synergistic action of Eperythrozoön coccoides, a red blood cell parasite. The link between the two agents is complex. References may be found in Gledhill and Niven ( 1955) and Gledhill ( 1962).
Neurotropic viruses causing spontaneous encephalitis
There are three groups of viruses causing spontaneous encephalitis with distinctions based on the type of pathological lesion. Theiler's FV, FA, FO, and GD-VII affect the gray matter primarily, producing lesions comparable to those found in poliomyelitis ( Theiler and Gard, 1940; Thompson et al., 1951). The second group includes the JHM virus which causes demyelinization ( Olitsky and Lee, 1955; Pappenheimer, 1958a). The third group, which does not cause demyelinization, consists of SK, the Columbia strain of Jungeblut, LCM or lymphatic choriomeningitis ( Traub, 1939; Haas, 1954), EK virus, herpes simplex, and eastern equine encephalitis.
Theiler's mouse encephalomyelitis. Spontaneous infections of the central nervous system due to this virus are considerably more prevalent in younger mice. Unless the disease is rapidly fatal, gradual flaccid paralysis, usually of the hind limbs, develops. Microscopically, anterior-horn lesions quite similar to those of human poliomyelitis (ganglionic cell destruction) are observed. However, there is no serological relationship between Theiler's virus and that of poliomyelitis. Theiler's virus is recoverable from the intestinal tract or central nervous system. Artificial infections are possible by virtually all routes, although the level of infective dose differs considerably. For a more detailed review see Maurer ( 1958a).
Lymphocytic choriomeningitis. Although mice of laboratory colonies may harbor this virus, the disease is carried in latent form with few animals showing clinical signs or dying. Clinical illness occurs only in young animals which may show photophobia, conjuctivitis, and convulsive leg movements. Tremors, spastic convulsions, and paralysis are observed in Artificial infections. Microscopically, leukocytic infiltration of the meninges, pleural exudation, and splenomegaly are found. Virus is detected by intracerebral inoculation of mice with sterile suspensions of brain, blood, or spleen from suspect animals. In view of the wide host range (mice, monkeys, dogs, guinea pigs, and man) LCM represents a hazard to both experimental animals and man. Aside from direct contact transmission, as by the urine of infected mice, the virus may also be spread by ectoparasites and mosquitoes. For a review see Maurer ( 1958b).
Pneumotropic viruses
A number of viruses have been implicated as causes of respiratory disease in mice. Among these are Nigg's pneumonitis ( Nigg and Eaton, 1944) and PVM or pneumonia virus of mice ( Mirick et al., 1952; Volkert and Horsfall, 1947).
K virus
K virus infection is also a respiratory disease. In suckling mice the clinical signs are labored breathing and early death. Large basophilic inclusion bodies are observed in greatly hypertrophied alveolar lining cells. On occasion, focal disseminated fatty liver dystrophy is found. Since the first description of the disease and isolation of the virus, the K virus has been isolated in several parts of the world, notably Australia ( Fisher and Kilham, 1953; Holt, 1959; Derrick and Pope, 1960). The virus presumably spreads by way of the urine and saliva, yet serological surveys of both laboratory and wild mouse colonies indicate that antibodies occur in only a small proportion of mice from infected colonies.
Livers of moribund suckling mice provide a source for a suitable complement-fixing antigen so that the virus may be detected by the presence of specific antibody, although intracerebral inoculation of newborn mice with suspect tissues is a more sensitive test. It has been suggested that the K virus may be generically related to polyoma in view of certain common biological properties such as size, stability, etc. (Kilham, cited by Rowe et al., 1962.
Salivary gland virus
This virus is ubiquitous in wild mouse populations, but has been observed in only a few laboratory colonies. Apparently the virus does not spread with ease ( Rowe et al., 1962) since even in infected colonies its incidence is 3 per cent or less. Virus isolation techniques must be used for laboratory diagnosis. Serological techniques are of little value, and inclusion bodies are rarely observed despite continual excretion of virus in saliva ( Rowe et al., 1962; Brodsky and Rowe, 1958). Two procedures (utilizing mouth swabs as a convenient source of virus) are generally employed: (1) isolation of the virus in mouse embryo tissue culture, and (2) inoculation of newborn mice. The disease may be suspected in suckling mice by their malnourished appearance and at necropsy by the gross yellow discoloration of the edges of the liver.
Thymic virus
The thymic agent has been discovered in as high as one-half the mice of one laboratory colony and is highly prevalent in wild mice. It is pathogenic only for newborn mice and is recognizable by the production of gross thymic necroses, visible about 2 weeks after inoculation. Direct virus isolation is the most reliable diagnostic means, although infected mice produce neutralizing antibodies in low titer. The salivary glands represent the best source of virus since naturally or artificially infected mice excrete the virus in saliva for periods of more than one year. In contrast to the mouse salivary gland virus, the thymic agent does not propagate in tissue culture ( Rowe and Capps, 1961).
Adenovirus
This virus regularly infects recently weaned mice after prolonged exposure to urine of carrier animals. Infection induces a good complement-fixing and neutralizing-antibody response. Spontaneous clinical disease rarely occurs. However, virus inoculation into suckling mice induces a fatal disease consisting in inflammation and necrosis of the heart, adrenals, and brown fat. Acidophilic intranuclear inclusion bodies occur in these and other tissues ( Hartley and Rowe, 1960). The virus produces cytopathic effects in mouse kidney tissue cultures. Fluids from such infected cultures contain an excellent complement-fixing antigen which reacts strongly with adenoviral antisera prepared in guinea pigs (but not with human, monkey, or dog antisera). Apparently the virus does not cross the placental barrier, since it has not been encountered in caesarian-derived colonies ( Rowe et al., 1962).
Reoviruses
The reovirus group is composed of three serotypes ( Sabin, 1959). Antibodies to reoviruses are assayed by hemagglutination inhibition, neutralization, or complement-fixation tests. Reo 3 virus can be detected by tissue-culture cytopathogenicity, inoculation of suckling mice, or mouse antibody production tests. The sera of some mice contain hemagglutination inhibitor which is undoubtedly nonspecific since it is commonly encountered even in specific-pathogen-free and germ-free mouse colonies.
Only Reo 3 is indigenous to mice and has been isolated from tissue suspensions containing Molony virus. The oncolytic virus of Nelson, obtained from a transplanted ascites tumor ( Nelson and Tarnowski, 1960) has been identified as Reo 3. Reo 2 has been encountered as a focal infection in several wild mouse populations which may have acquired infection by contact with other species, such as man and cattle ( Rowe et al., 1962).
Oncogenic and tumor-associated viruses
Gross ( 1961) has discussed the status of virus-induced neoplasms, including mouse leukemias as produced by cell-free leukemic filtrates, the induction of leukemia by filtrates of solid mouse tumors, and the filterable agent causing mouse mammary tumors (Bittner's milk factor). Sinkovics ( 1962) has reviewed viral leukemias in mice and oncogenic viruses are discussed in Chapters 27 and 28. We refer here only to mouse polyoma infection and the lactic dehydrogenase-stimulating viruses ( Gross, 1953; Stewart, 1953; Riley et al., 1960).
A detailed epidemiology of mouse polyoma infection has been developed after intensive studies. The classic techniques of virology for detection of antibodies reveal that mouse polyoma virus is widely disseminated among laboratory colonies and wild mouse populations. Focal reservoirs of infection are maintained by routes of transmission involving virus excretion in urine, feces, and saliva. Apparently the virus is not transferred transplacentally since mice derived by caesarean section for specific-pathogen-free or germ-free colonies have been negative to antibody tests. Polyoma virus (e.g., infected tissue culture fluid) induces multiple tumors when inoculated into newborn mice. Such tumors are of multicentric and multiple histological origins, with mixed tumors of the salivary glands most frequent. The virus is capable of producing tumors in other species as well.
From the epidemiology of polyoma virus infection several points are notable: (10 Resistance of infant mice is perfectly correlated with presence of maternal antibody titers; no tumors develop in offspring of positive mothers; (2) spontaneous parotid tumors are rarely found in naturally infected mouse colonies; (3) high antibody titers are present in milk of infected mice; and (4) maternally derived antibodies are presumably present in fetuses, since it has been shown that mice born of resistant mothers but not having access to immune mouse milk are resistant to polyoma infection.
Certain transmissible agents have been found to be associated with many transplanted and induced spontaneous tumors of mice ( Riley et al., 1960). These are manifested by biochemical response. Susceptible animals respond with five- to tenfold increases in serum lactic dehydrogenase (LDH) and by induction of other glycolytic enzymes after inoculation with plasma or organ extracts from tumor-bearing hosts. Natural transmission of the lactic dehydrogenase-stimulating agents(s) (LDH) occurs when normal mice are placed in the same cage with agent-infected mice or tumor-bearing mice. In most infected animals moderate splenomegaly occurs. Serum LDH elevation has been induced by inoculation with preparations of cells from infected mice ( Riley et al., 1961). Although there is a close correlation between LDH levels and the growth of several transplanted tumors, elevation is not directly related to the neoplastic process. There is evidence to indicate that the LDH factor is a virus, specifically of mouse origin, and that changes in the LDH level of a tumor-bearing animal depend on whether or not the LDH factor has become associated with that tumor ( Notkins et al., 1962). An increase in lactate dehydrogenase levels can also be induced by injection of several of the mouse hepatitis viruses. LDH virus may be eliminated as a contaminant of transplantable tumors by passage of such material through tissue culture. However, it has been reported that the agent can be propagated and maintained by serial passage on primary mouse embryo tissue-culture ( Yaffee, 1962). It can be inactivated by radiation and is unstable in the presence of ether ( Notkins et al., 1962).
Infantile diarrhea
Diarrheal disease of unweaned mice is a complex syndrome caused by a number of agents ( Pappenheimer, 1958b; Runner and Palm, 1953; Kraft, 1962). As is true for diarrheal disease in human infants, there is considerable evidence that no single pathogen is the causative agent ( Thomlinson, 1964; Erlandson et al., 1964; Altman, 1964; Payne, 1960; Barua et al., 1962). Rather, under certain environmental conditions (crowding, poor sanitation, improper nutrition, variable temperatures, too low or too high humidity, inadequate ventilation, etc.) ubiquitous viral or bacterial organisms may be incriminated as producing diarrheal or septicemic signs in unweaned mice. We find that host factors, such as strain of mouse and parity play a determining role in morbidity and degree of clinical signs.
The clinical signs of the syndrome are: slight-to-severe diarrhea in which fecal material ranges from bright yellow to light brown in color. Nursing mothers will sometimes very efficiently clean up the anal region of the affected animal so that only a slight "pasting up" condition is noted. Animals affected by diarrhea (usually 4 to 10 days of age) usually recover, but many are discarded as runts at weaning time. The diarrhea is often followed by constipation contributing to later mortality, Mortality more often occurs in animals not showing signs of diarrhea. Such deaths occur early, 1 to 2 days after onset of the disease in a litter. Survivors in these litters show varying degrees of diarrhea.
At necropsy, the only common finding is the presence of light colored and watery fecal material in the intestinal tract, with occasional bubbles of gas. Histologically the tissues demonstrate a mild catarrhal inflammation. Some workers have reported inclusion bodies in epithelial cells from live and intestinal tract.
Viral agents such as those described by Kraft ( 1962) are found in the intestinal contents and reportedly can be transmitted directly to susceptible animals through this medium. Various types of bacteria, coliforms, Proteus sp., and pseudomonads have been found in separate outbreaks of the disease.
Epidemiologically the disease usually occurs in cycles. In certain affected mouse colonies, diarrheal disease is rarely seen until the fall and winter months. Our studies suggest that lower humidity and less fresh outside air are involved in seasonal recurrence rather than length of day and seasonal variation in quality of diet.
Colonies in which diarrheal disease is enzootic may show longer periods between epizootic outbreaks when moved to new quarters. During an outbreak the disease can be controlled by the use of broad-spectrum antibiotics such as the oxytetracyclines or tetracyclines administered in the drinking water. Therapeutic doses are administered over a 2-week period to all animals in a breeding colony. The preferable medication period is during the last third of pregnancy. This treatment is repeated in 2 weeks. Such therapy results in marked increase in the numbers of healthy-looking mice weaned and a decrease in the incidence of diarrhea. Similar effects are produced by the use of air-filtering material placed around each breeding cage or by the use of aluminum foil covering 50 per cent or more of open cage surfaces. The effects shown by cage modification (air filtering, foil covers) may be due to decreased aerosol dose levels. The use of certain types of highly absorbent bedding material also results in a higher recovery rate and a decrease in diarrheal disease. All of these control measures singly or in combination result initially in marked improvement of colony health, but usually over a period of months the disease reappears. Also, the effects of combining such control measures are not additive but only as good as the best measure used alone. Other effective control and prevention measures entail a variety of steps to isolate each breeding pen completely.
Much remains to be learned of the causes of infantile diarrheal disease, but the diarrhea should be considered as an accompaniment of certain diseases of suckling mice and not as a disease entity in itself.
BACTERIAL DISEASES
The spontaneously occurring or latently present bacterial diseases of mice are numerous, and the mouse is also susceptible to experimental infection by many pathogens from other species. Because of the small size of the animal, its habits, and the complexity of husbandry methods applied, it is extremely difficult to evaluate the health of a single animal by the usual clinical methods. Instead, an epidemiological approach is necessary. The population or a percentage thereof is observed for common factors which can be related to the various signs of morbidity and to mortality rates ( Shope, 1964).
Salmonellosis
Infection by Salmonella sp. is one of the more common types of bacteriological infections of mice ( Habermann and Williams, 1958a; Lane-Petter, 1963). The most commonly found strain is Salmonella typhimurium. However, mice are highly susceptible to infection by most of the Salmonella sp. organisms and other strains have been reported as responsible for epizootics or as latently infecting a few mice in a colony ( Wetmore and Hoag, 1960; Hoag et al., 1964a). The degree of virulence is dependent largely upon the dose and route of infection and the strain of host mouse as well as upon the inherent virulence of the organism itself. Salmonellosis is characterized initially by diarrhea or soft stools, sudden deaths, anorexia, and cachexia. Large number of animals are often lost not by death, but by the culling of underweight and poor-looking mice. The initial outbreak of salmonellosis soon subsides into a chronic form of the disease wherein the organisms are shed in fecal material. The adult carriers often appear healthy, but suckling and growing mice show great variation in body weight and rates of gain. Occasional breeding pairs are affected by attacks of mild diarrhea evidenced by soft, light-colored fecal pellets. Cull rates are high chiefly because of variations in weight and the poor appearance of weanling mice. At necropsy only a few animals show the classic "white spotted livers." Many will show enlarged spleens, but no gross necropsy findings are pathognomonic. Spleen, liver, and both large and small intestine are the tissues of choice for bacteriological examination. Tissues should be separately cultured for the presence of the organisms. In moribund or sick-looking animals the liver and spleen will yield Salmonella, whereas the chronically infected cases will usually yield organisms from the intestinal tract only.
For the isolation of Salmonella sp. enrichment media, preferably Tetrathionate Broth (Difco Laboratories), should be inoculated with a quantity of minced or ground tissues and the media incubated at 37°C for 72 hours ( Hoag and Rogers, 1961). During this period subcultures to Brilliant Green Agar (Difco) are incubated at 37°C for 48 hours before being discarded as negative for salmonella growths. Serological identification of suspect bacterial colonies is then carried out with polyvalent or group-specific antisera. Specific identification is dependent upon further serological techniques.
The disease is not controllable by any known therapeutic measures. Broad-spectrum antibiotics such as tetracycline or oxytetracycline seem to be of some use in epizootics in increasing the numbers of survivors, but they will not cure chronically infected animals. Vaccines of various types have been found to be highly effective against clinical signs and mortality from the disease but do not prevent chronic infection and carrier states from developing ( Hoag et. al., 1964b). We have demonstrated that three differently prepared S. typhimurium vaccines were effective against as many as 100 LD50 challenge doses of S. typhimurium but all of the surviving animals, although appearing perfectly healthy, were shedding organisms in fecal material and had infected livers and spleens as long as 6 weeks after the challenge.
The disease is controlled by elimination of infected or carrier stocks. Several (at least two and preferably six) weekly fecal tests should be performed before concluding that an animal is free of infection, since intermittent and low-level shedders are commonly encountered in a chronically infected colony. Sanitation is an important part of preventing spread of the disease, since an important mode of transmission is by contact with contaminated objects. Food supplies and bedding material should be treated as potential sources of infection. Occasional parathyphoid carriers are found among mouse handlers, but are not important sources of animal infection.
Pasteurellosis
Pasteurellosis due to Pasteurella pseudotuberculosis, P. septica, or P. pneumotropica may occur spontaneously in laboratory mice ( Sellers et al., 1961; Tuffery, 1958; Hoag et al., 1962). The latter two organisms may be found in various tissues of apparently normal mice. Latently infected animals subjected to various types of stress (radiation, abrupt temperature changes) will often produce signs of acute disease: anorexia, lassitude, sudden deaths. The livers of animals infected with P. pseudotuberculosis present multiple focal abscesses. P. pneumotropica has been reported in outbreaks of pneumonia in mice and has also been found in normal-appearing lung tissue ( Jawetz, 1948; Jawetz and Baker, 1948). In other instances this organism has been isolated from the brain, uterus, testes, liver, or spleen of normal-appearing mice. In rare instances septicemic deaths have been attributed to P. pneumotropica. Pasteurellosis is probably a stress-induced disease and the causative organism most commonly a latent infective one.
The pasteurella organism is susceptible to the tetracycline or oxytetracycline types of broad-spectrum antibiotics but is usually resistant to penicillin. Outbreaks in which signs of disease are thought to be caused by pasteurella organisms (on the basis of isolations from organ tissue) may be treated by administering the effective antibiotics in the drinking water. Response to the drugs is immediate and favorable.
Pseudomonas infections
Infection with Pseudomonas aeruginosa is common in laboratory mouse colonies ( Flynn, 1963a). No signs of infection are manifest unless animals are subjected to radiation or other types of stress (cortisone, etc.) ( Flynn, 1963a; Wensinck et al. 1957; Verder and Rosenthal, 1961). Such treatment results in rapid onset of septicemic disease (anorexia, listlessness) and high mortality. At necropsy the organisms are easily cultured from all organs, particularly liver and spleen. Although not important as a spontaneous disease-causing organism, P. aeruginosa is of major concern to investigators using mice in experiments employing radiation. The organism is shed in feces and urine from chronically infected animals. Nearly 100 per cent of the animals in a colony may become rapidly infected by contamination and recontamination of drinking water, caging equipment, and feeds. The infection can be controlled by hyperchlorination and acidification of water used for drinking and cleaning ( Hoag et al., 1965). Sanitation procedures must be of the highest efficiency and detectable carriers eliminated. Carrier detection should not serve as a criterion for culling until after control procedures such as water treatment and intensified sanitation have been inaugurated, since it has been shown that most fecal shedders of the organisms are only transiently infected and will seemingly spontaneously "recover" if not exposed to further doses of the organisms in contaminated water. After control procedures have been in effect for several weeks, a test-and-slaughter program should be started. Pens containing mice shedding the organism should be provided from the colony.
Antibiotics and other drugs are of no value in treating the infection. In one instance when oxytetracycline was being administered in drinking water to inbred mice, it was found that the organism grew more rapidly in water containing hypertherapeutic levels of the drug ( Hoag et al., 1965).
Klebsiella infections
Latent infections with capsulated lactose-fermenting bacilli, often loosely identified as Klebsiella group organisms, are not uncommon ( Wilson and Miles, 1964). Such organisms are occasionally isolated from lung tissue of normal-appearing mice. Sometimes the organism is found in the nasopharyngeal region or in various body wastes. In certain outbreaks of pneumonia in mice, Klebsiella sp. can be recovered from 100 per cent of the lungs of sick or moribund mice. The organisms recovered vary greatly in virulence. Rarely, the bacterium can be recovered from abscesses of lung or from other internal organs and cutaneous areas. Improved sanitation and environmental constancy are recommended control measures.
Tyzzer's disease
Disease caused by Bacillus piliformis has been described by several workers ( Fujiwara et al., 1963; Fujiwara et al., 1964; Saunders, 1958b). Most agree that the organism occurs in an infected colony as an intestinal saprophyte and that clinical disease occurs only after animals have been stressed, as by adverse nutrition or by the effects of stressors introduced during the course of an experiment. Onset of disease signs is abrupt with death occurring 2 to 3 days afterward. Diarrhea may occur as the major sing or may affect only a few of the sick animals. Anorexia and other signs of septicemia are manifest. Some immunity develops in recovered animals.
The condition may occur in mice of all ages, but the highest mortality rate is observed in 3- to 7-week-old animals. The most striking necropsy finding is the occurrence of numerous grayish-white spots (up to 2.5 mm in diameter) on the outer and cut surfaces of the liver. Mesenteric lymph nodes are usually enlarged and sometimes abscessed. Microscopically the organisms (long, thin, Gram-negative rods) are found intercellularly, surrounding the necrotic tissue areas, but are also seen in intact liver cells as well. the organisms have been reported as cultivatable in tissue culture, but cannot be grown on defined bacteriological media. Diagnosis is by histological sectioning and staining of tissues with subsequent demonstration of stained organisms.
Differences between mouse strains in susceptibility have been demonstrated, but it is emphasized that any incidence in a mouse colony represents a disease potential and infected colonies must therefore be considered as hazardous for other research. Little work has been done to demonstrate the effectiveness of therapy, although oxytetracycline has been of value ( Tuffery, 1958).
PPLO (Pleuropneumonia-like organisms)
Infections with organisms described as PPLO have been chiefly reported as causing catarrhal disease in mice ( Nelson, 1958). Some outbreaks of disease of the upper respiratory tract are characterized by sudden onset and involvement of a large percentage of a colony usually after chilling or overheating. Chattering and sniffling with variable nasal discharge are the observable signs of infection. Mortality rates are low, and recovery from the exudative catarrh is spontaneous. A few animals succumb to bronchopneumonia or may develop a chronic bronchiectatic pneumonia. In other types of upper respiratory disease caused by PPLO the onset is gradual and the outbreak appears enzootic in character, Because of the chronic nature and long course of the disease, the eventual involvement of large numbers of animals may give the appearance of a suddenly occurring epizootic disease, particularly if the mild symptoms in the early stages of the condition are unnoted. Labyrinthitis (manifested by disequilibrium) may occur in a few of the mice.
PPLO organisms are often found in animals from an apparently healthy colony and may be recovered from various tissues other than lung material ( Freundt, 1959). The pathogenic potential of latent infections is always manifest but has not been quantitatively evaluated.
Streptobacillus moniliformis
Infections with Streptobacillus moniliformis are usually latent. In certain outbreaks where clinical signs include lameness and swelling of joints and extremities (due to edema), the organism is readily isolated from blood and organs, including affected joints ( Wilson and Miles, 1964; Freundt, 1956). In other instances the organism is not found in the bacterial cell state, but is isolated with extreme difficulty in L form by PPLO culture techniques ( Freundt, 1959). Clinical signs are often confused with those seen in chronic ectromelia, and for this reason an early differential diagnosis is indicated ( Lane-Petter, 1963). Stress plays an important role in the activation of latent infection. In many outbreaks mice will arrive in apparently good health after exposure to adverse shipping conditions, but within 3 to 4 days 10 to 50 per cent will develop signs of the disease — swellings and ulcerations of feet and tail after the appearance of a sharp band of demarcation proximal to the swelling. Recovery is usually spontaneous, although severely affected animals are deformed because of deranged circulation to extremities with subsequent sloughing of the entire area distal to the necrotic demarcation band. In our experience antibiotics such as tetracycline and oxytetracycline seem to be useful in controlling outbreaks. In clinically affected animals these drugs serve to mitigate the severity of developing lesions.
PARASITIC DISEASES
Helminth infections
Mice may become infected with helminths from other species ( Haberman and Williams, 1958b). Outbreaks of infection with Taenia taeniformis can occur when mice are housed in the same area with cats. Infections with the various helminths rarely produce clinical signs and are only potentially important as producing unpredictable variables in animals used in research. Heavily infected animals are below norms in weight and may be anemic. Some mouse colonies may be infected with Hymenolopis nana (the dwarf tapeworm). The life cycle of this parasite does not involve any secondary hosts, with infection taking place directly from eggs excreted in feces. Upon necropsy, the tapeworm is easily observed through the walls of the unopened intestinal tract.
Mouse colonies are often infected with the oxyurids Syphacia obvelata and Aspiculuris tetraptera. These mouse pinworms have a direct life cycle and spread through a colony rapidly because of the large numbers of eggs excreted and the ease with which the eggs are airborne. There are no clinical signs of oxyuriasis. There may be some weight and growth variation between infected and noninfected animals, but other signs such as poor hair coat, etc., are not clear-cut. There is some evidence that oxyuriasis may contribute to rectal prolapse. Diagnosis can be made by examination of fecal material or the anal region for oxyurid eggs by the various flotation or contact-tape techniques. Pinworm infection may be eliminated by treatment with various drugs such as the piperazine compounds, usually most efficacious when administered via drinking water ( Hoag, 1961). Treatment must be accompanied by through cleaning of the room and caging equipment to remove the possibility of reinfection from egg-contaminated dust.
Arthropod infections
Lice (Polyplax serrata) and mites (Myobia musculi, Myocoptes musculinus, Myocoptes romboutsi, Radfordia affinis, and Psoregates simplex) are the chief ectoparasites of laboratory mice ( Flynn, 1963b). Their presence is manifested by hair loss and scratching, which may give rise to bacterially infected ulcerative wounds. It is not unusual for mice to die in a heavily infested colony as a result of these infected sores.
Control of ectoparasites is difficult. Various dusting powders containing DDT, methoxyclor, rotenone, or other parasiticides, and dips containing aramite or other materials have been used, but must be regularly or periodically applied. Bedding materials either treated with aromatic hydrocarbons (such as crude cedar wood oil) or naturally containing such materials (cedar wood shavings) are successful in suppressing infestations.
PROTOZOAN INFECTIONS
Several types of protozoan diseases have been reported in mice. Most are inapparent infections, noted only after synergistic activity with other agents such as viruses or after interference with immunological body defenses as by splenectomy, Control is usually by improving hygiene after elimination of infected animals or whole colonies.
Eperythrozoön coccoides occurs as a blood parasite in the form of disc-shaped structures on the surface of red blood cells. Splenectomy of infected mice results in a marked increase in the numbers of parasitized cells and onset of transient mild anemia. The presence of this parasite increases susceptibility to such infectious agents as the mouse hepatitis virus, lymphocytic choriomeningitis virus, and lactate dehydrogenase-elevating virus ( Seamer et al., 1961; Riley, 1964).
Hemobartonella muris is another red blood cell parasite activated in infected mice after splenectomy ( Griesemer, 1958). Infectivity is very low. The anemia produced is mild and transitory.
Eperythrozoön cuniculi causes mild febrile disease in mice, infects epithelial cells of the kidney papillae, and produces granulomatous lesions in brain tissue ( Yost, 1958). These organisms occur as 1.5- to 2.0-μ Gram-positive rods. Transmission is apparently by way of urine, which may contain large numbers of organisms.
Klosiella muris has been described as causing an infection of mouse kidney epithelial cells ( Dunn, 1949). Signs of disease are inapparent although, on necropsy, kidneys of infected animals may be surface-marked with varying numbers of tiny grayish-white foci. Transmission is by way of spore cysts released into the urine.
MYCOTIC INFECTIONS
Mycotic infections of mice usually produce a dermatitis. Such infections, which can be caused by any one of several Trichophyton or Microsporum sp., result in circumscribed encrusted areas with hair loss and are empirically called ringworm. The fungus from the lesions may be easily demonstrated by microscopic examination of skin scrapings ( La Touche, 1957). Control of the colony infection is difficult. Outbreaks are chiefly important as sources of human infection as most of these fungi cause similar lesions in man.
SUMMARY
The mouse is susceptible to an array of infectious agents. Many of these agents produce signs of disease and must be eliminated or controlled if only to maintain sizable mouse populations. On the other hand, because the mouse is so important as a biological yardstick for measurement of varied procedures, it becomes important as well to eliminate or control those infectious agents which do not produce observable disease, but which may affect the outcome of an experiment. The development of mouse colonies from caesarean-derived stock is an effective panacea for eliminating those disease agents which cannot penetrate the placental barriers. In another respect, it is important to study the synergistic or antagonistic effect of various disease agents either in combination with each other or with other factors so that information so derived may contribute toward man's understanding of his own disease problems. It would be unfortunate to eliminate completely all disease of mice before obtaining more complete knowledge of the etiology, epidemiology, and pathology of such naturally occurring conditions.
