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Pregnant rabbits were serologically diagnosed as having been infected with Encephalitozoon cuniculi. At necropsy at 28 days of gestation, does, placentas and fetuses were found to be infected with E. cuniculi strain type I as evidenced by using the nested-polymerase chain reaction (PCR) technique, thereby confirming vertical transplacental transmission.

51:77 Pneumocystis carinii 51:80 Toxoplasma gondii 51:83 Encephalitozoon cuniculi 51:85 Oxyurina 51:87 Mites Introduction It is generally accepted that rodent pathogens may not only be hazardous for animals (and humans) but can severely influence the results of animal experiments. Microbiological standardization of laboratory animals is therefore of crucial importance. It has been known for decades that microorganisms may have an impact on their hosts in various ways. Many years ago, influences of microorganisms were detected on development and growth of tumours. It was shown by various authors that germ-free mice develop fewer tumours (lung, liver, mammary glands, uterus, ovary) after treatment with chemical carcinogens than conventionally housed animals IBurstein et al. 1970, Roe & Grant 1970, Schreiber et al. 1972). The importance of microorganisms as factors that may influence animal experiments has already been described in review articles more than 25 years ago (van der Waaij & van Bekkum 1967, Hanna et al. 1973, Baker et al. 1979). A first symposium dealing with this issue was held in 1971, and hitherto known influences of selected microorganisms were published afterwards IPakes & Benirschke 1971I.

In this study, we investigated the prevalence of infectious microorganisms (viruses, bacteria, fungi and eukaryotic parasites) in mice from different pet shops in Germany; such animals may compromise the hygienic integrity of laboratory animal vivaria if private pet holders act as unintended vectors of infections carried by them. House mice sold as pets or feed specimens were purchased from different pet shops and tested for a comprehensive panel of unwanted microorganisms. We found a number of microorganisms in these pet shop mice, the most prevalent of which were Helicobacter species (92.9%), mouse parvovirus (89.3%), mouse hepatitis virus (82.7%), Pasteurella pneumotropica (71.4%) and Syphacia species (57.1%). Several microorganisms (e.g. mouse parvovirus, Theiler's murine encephalomyelitis virus, pneumonia virus of mice, Encephalitozoon cuniculi, Clostridium piliforme) had considerably higher prevalences than those reported in similar studies on wild mice from North America, Europe or Australia. Our study shows that direct contact with pet shop mice may constitute a risk for laboratory animal vivaria if hygienic precautions are not taken. However, even relatively simple precautions seem effective enough to hold the risk at bay.

Between 1982 and 1987 sera from 4952 New Zealand White rabbits ( Oryctolagus cuniculus) obtained from a single commercial supplier were tested for the presence of antibodies to Encephalitozoon cuniculi. A commercially available carbon immunoassay test kit was used. Initially 32·9% of the rabbits were seropositive with the number progressively decreasing to 2·3% by 1987. The reason for the significant decline in the incidence of infection was most likely due to a selection process for breeding stock instituted by the supplier based upon productivity, posture and weight of each animal.

Summary Spores of Encephalitozoon cuniculi were exposed to various temperatures or to disinfectants, and their infectivity was then tested on monolayer cultures of canine kidney cells. The maximum survival time for spores suspended in medium 199 was 1 day at –20°C, 98 days at 4°C, 6 days at 22°C, and 2 days at 37°C. Only 2·5% survived 30 min at 56°C. Boiling for 5 min or autoclaving at 120°C for 10 min killed all spores. Dry spores survived less than a week at 4°C but at least 4 weeks at 22°C. Exposure for 30 min to recommended working concentrations of 9 of the 11 disinfectants tested killed all spores. The growth-inhibition effect of 7 antibiotics and chemotherapeutics was studied on canine kidney cell culture inoculated with E. cuniculi. None could completely inhibit growth. The most effective was chloroquine phosphate which, at a concentration of 12·5 mg per 1000 ml culture medium and during a test period of 8 weeks, reduced the harvest of E. cuniculi to 31% of that from inoculated, untreated cultures.

Summary Sera from 4 generations of a family ofrabbits having a natural Encephalitozoon cuniculi infection were investigated. Antibodies were demonstrated in a litter of newborn rabbits and in another litter II days old. Histological examination of the brains and kidneys of these animals failed to reveal lesions attributable to the disease. Sera from a further litter of 2 rabbits, taken at weekly intervals from 4 to 42 weeks of age, revealed a low initial antibody titre which regressed to an undetectable level. After an interval, titres rose and then plateaued to the end of the period of observation. Encephalitozoon cuniculi infections in the dams of all the litters were confirmed by antibody assay and by histology. Parallel titration of samples treated and not treated with 2-mercaptoethanol showed that the india-ink immunoreaction demonstrates only immunoglobulin G. There is evidence of in utero transmission of the parasite Encephalitozoon cuniculi in rabbits (Hunt, King & Foster, 1972) and mice (Innes, Zeman, Frenkel & Borners, 1962), but observations made during our screening of a large rabbit colony for antibodies suggested that horizontal transmission was an important mode of infection (Bywater & Kellett, 1978a). Further observation confirmed reports that there is a greater probability that infected animals will be derived from infected rather than non-infected dams although litters from infected mothers may include otT

Encephalitozoon cuniculi infection (nosemiasis) was serologically diagnosed in hysterectomy-derived gnotobiotically reared guineapigs. The probability of vertical transplacental transmission is discussed.

Summary Rabbit antibodies against Encephalitozoon cuniculi were detected in an enzyme immunoassay procedure in which antigen was grown and used in situ. The test appeared to be more sensitive than the indirect immunofluorescence test with which it was compared, but gave essentially the same results for the 64 sera evaluated. This procedure will allow any laboratory with cell-culture facilities to produce a diagnostic antigen without the need for antigen processing. It is simple and reliable, and does not require specialized equipment or microscopic assessment. Various serodiagnostic procedures for Encephalitozoon cuniculi infection have been reported over recent years. These include immunofluorescence (Chalupsky, Vavra & Bedrnik, 1973; Cox & Pye, 1975), the india-ink immunoreaction (Waller, 1977), immunoperoxidase (Gannon, 1978), indirect microagglutination (Shadduck & Geroulo, 1979) and complement fixation (Wosu, Olsen, Shadduck, Koestner & Pakes, 1977). All of these tests have certain problems associated with their use. The complement-fixation test requires antigen to be further processed after harvesting, a step that can cause variation between batches of antigen. Neither the complement-fixation test nor the india-ink immunoreaction are able to measure separately the various immunoglobulin classes, achieved when a doubleantibody diagnostic procedure is used. Additionally, the underlying principle of the india-ink immunoreaction is as yet unexplained, and recently several reports have also questionedthe reliabilityof the technique (Shadduck & Geroulo, 1979; Chalupsky, Vavra & Bedrnik, 1979). However, all the doubleantibody tests described require microscopic evaluation with concomitant subjectivity. This can lead to poor reliability in inexperienced hands. We have now applied the enzyme immunoassay (EIA) test to the diagnosis of encephalitozoonosis. Antigen was grown and used in situ in a manner similar to that described recently for influenza virus by Bishai & Galli (1977). The technique therefore has immunological precision without requiring antigen processing or microscopic evaluation.

Parenteral administration of Encephalitozoon cuniculi induced an antibody response within 7-11 days. Peroral administration was less effective since only 2 of 6 animals showed seroconversion; they became seropositive within 14-21 days. Sera from animals which became seropositive had high antibody titres during the whole test period. Immune sera from 3 animals were fractionated by gel filtration. With the india-ink immunoreaction test, antibodies to E. cuniculi were found only in the 7S fractions, while the indirect fluorescent-antibody test detected them in fractions 19S and 7S. The 7S fractions were identified as IgG and the 19S fractions as IgM. A program for eradication of encephalitozoonosis, based on these results, is discussed.

Summary A survey of the histopathological lesions associated with Encephalitozoon cuniculi infection was undertaken in naturally infected rats. 13 of 130 Wistar rats were infected at an early age. Distinctive cerebra-spinal granulomata were predominant, with or without renal and/or hepatic lesions. Encephalitozoon cuniculi is a protozoan of the phylum Microsporidia (Sprague, 1977) and was first discovered by Levaditi, Nicolau & Schoen (1923) in rabbits. There are fewer reports of the parasite occurring in other mammals (Shadduck & Pakes, 1971; Jortner & Percy, 1978; Wilson, 1979), but it is commonest in rats (Attwood & Sutton, 1965). Surveys recently carried out by Gannon (1980) show that all conventional rabbit colonies are infected by this intracellular parasite to a varying degree and that rodents such as rats, mice and guinea pigs living in contact with infected rabbits are susceptible to infection. During a routine toxicity study of tissues supplied by an outside organization, cerebrospinal, hepatic and renal granulomata due to E. cuniculi were seen in 13 of 130 Wistar rats and it was felt, therefore, to be of interest to describe such lesions occurring in a colony of relatively young rats. The animals were conventionally bred and obtained from a commercial Belgian supplier. Materials and methods 130 Caesarian-derived, conventionally-bred Wistar rats 6 weeks old were used in a short-term (13 weeks) toxicity study. The rats were housed in groups of 5, in galvanized metal cages and given free access to food and water. The laboratory temperature was 22 ± 1°C, and the relative humidity 45-55%. The light: dark cycle was 12: 12 h. Details of husbandry and housing prior to the experimental period are not available. Equal numbers of male and female rats were used. At termination they were killed by carbon dioxide asphyxiation. At post-mortem examination various *On leave from Baghdad University, Department of Pathology.

Lesions produced by Encephalitozoon cuniculi are common in the central nervous system of laboratory rabbits, and may affect experimental results. Post-mortem examination often does disclosed that three manifested typical lesions, while the other seven did not. Of the fourteen young born to the three does in which lesions were discovered, four (29 per cent) were free from obvious evidence of the disease. Of the fifty young born to the seven lesion-free does, forty-seven (94 per cent) were also free from such lesions.

Sera collected from both naturally and artificially infected rabbits were found to show excellent correlation when examined for the presence of Encephalitozoon cuniculi antibodies using the immunoperoxidase and immunofluorescence tests. Out of 85 randomly selected rabbits, 21 were found to be serologically positive using both the tests. However, lesions which could be attributed to E. cuniculi infection were only demonstrated in 16.

Antibodies to Encephalitozoon cuniculi were demonstrated in 24 of 29 rabbit antisera and serum products from 10 suppliers.

An investigation into the transmission of Encephalito-zoon cuniculi was undertaken in both naturally- and experimentally-infected rabbits. Only 2 nurslings were found with rising antibody titres at 8-10 weeks of age, when infection could have been caused by environmental contamination.

Of 38 animal colonies serologically examined for Encephalitozoon cuniculi, 1 mouse, 2 rat and 4 guineapig colonies were positive. A further survey showed that the prevalence within mouse, rat, guineapig and rabbit colonies varied between 25 and 95%. Guineapigs housed with infected rabbits are at a greater risk of being infected than those housed separately. Nephritis was a common feature, but cerebral granulomata were not seen.

Summary In this in vitro infectivity assay for Encephalitozoon cuniculi, lesions due to the organism appeared as macroscopically distinct foci. The number of such foci was used as a direct measure of the number of infectious units in the original sample. The expected correlation between focus-forming units and 50% infectious doses was observed in limit dilution experiments.

Sera from 37 rabbits were assayed for antibodies against Encephalitozoon cuniculi (Nosema cuniculi) by the india-ink immunoreaction and the indirect fluorescent antibody tests: all animals seropositive to the former were also positive to the latter test. 27 of the rabbits were also tested for skin hypersensitivity and then autopsied. Animals positive to the skin test were also positive to the serological tests. At autopsy 18 of 22 rabbits positive in the immunological tests showed lesions typical of encephalitozoonosis. Sera from 200 rabbits originating from 6 institutes were assayed by the india-ink test: seropositive rabbits were found from all institutes (9·1 to 81·9% incidence), with serum titres ranging from 1:125 to 1:5000. The india-ink test appears to be a rapid and convenient method for diagnosis of encephalitozoonosis in rabbits.

A humoral and cellular response was detected in C57BL mice 14-21 days after experimental infection with Encephalitozoon cuniculi. In athymic ( nu/nu) mice a severely limited cellular response and no antibodies were detected, but masses of spores were found in the liver and other viscera. These spores were probably responsible for the death of the nude mice. Antibodies in C57BL mice remained at high levels for at least 17 months after infection

The indirect india-ink immunoreaction and the india-ink immunoreaction inhibition tests are described and results are compared with those obtained by the indirect fluorescence antibody test. Titrations of seropositive samples were made by 2 methods and the titres obtained were similar. Some advantages of the india-ink immunoreaction as a diagnostic test are discussed.

Rabbits ( Oryctolagus cuniculus) of non-dwarf (Group A) and dwarf (Group B) strains showing a common clinical sign of head tilt (torticollis) were examined. With 1 exception, all rabbits of group A had otitis and empyema of either one or both middle ears. Pasteurella multocida was isolated from pus and from the nose of all but 1 of these rabbits, and on occasion was also isolated from the brain. By contrast in all dwarf rabbits the presence of the protozoan Encephalitozoon cuniculi was confirmed both histologically and serologically. This parasite did not affect the ears of the animals but rather the central nervous system. We assume that the different exposure to both agents, rather than the degree of susceptibility, was responsible for the differences found between the 2 types of rabbits.

Growth patterns of Nosema cuniculi ( Encephalitozoon cuniculi) in cell cultures of bovine kidney, canine kidney, feline lung, and rabbit kidney were studied. All cell cultures used were easy to manage and the last 3 are commercially-available established cell lines. The dog kidney cells were the most suitable for large-scale production of Nosema. When grown in plastic flasks with a bottom area of 75 cm2, the weekly yield from Nosema-infected canine kidney cells during the 10th to 17th week after inoculation was between 4·1 x 107 and 9·9 x 107 spores per flask. An equilibrium was obtained between the Nosema infection and the kidney cells during this time. A simple method for estimating the numbel of harvested spores is also described.

Encephalitozoon cuniculi was isolated from the urine of infected rabbits using human and canine tissue cultures. The organism was isolated from 7 of 11 contaminated urines from seropositive animals. The advantages of urine over tissue as a source of E. cuniculi are that it is obtainable from living animals, can be examined for the presence of organisms, and is essentially free of cells likely to overgrow the tissue cultures used for isolation.