Vol. 21, No. 2
INFECTION AND IMMUNITY, Aug. 1978, p. 360-364 0019-9567/78/0021-0360$02.00/0 Copyright i 1978 American Society for Microbiology
Printed in U.S.A.
Encephalitozoon cuniculi Antibodies in a Specific-PathogenFree Rabbit Unit J. E. BYWATER* AND B. S. KELLETT Basel Institute for Immunology, CH-4005 Basel 5, Switzerland Received for publication 17 January 1978
We describe our discovery of Encephalitozoon cuniculi antibodies in a specificpathogen-free rabbit colony. Small-sized samples had failed to reveal the presence of infection with a prevalence of about 5%. Using an India ink immunoreaction test by which we were able to visualize both negative and positive reactions, we were able to undertake a 100% screen of the colony of more than 700 rabbits and to repeat this 4 weeks later when we had culled the positive reactors. By collating the results of those tests with the results of tests on previously collected samples, we have been able to discuss and observe age and sex susceptibilities and the mode of transmission of the naturally occurring disease.
Encephalitozoon cuniculi was first reported in 1922 (24), although it was 2 years later that the organism was described and named (14). The disease occurs throughout the world, affecting various species, and it is widespread in domestic rabbits (7, 20, 21). Histological lesions of E. cuniculi have been reported as being present in 55% of clinically normal rabbits, with an even higher prevalence in animals suffering from other infections (15), and there have been numerous reports concerning the widespread occurrence of the parasite in laboratory animals with its interference in the interpretation of experimental results (9). Rabbits appear to be particularly susceptible, and when symptoms are recognized they are usually characterized by loss of condition and occasional overt central nervous system involvement (5). However, the infection most frequently produces an inapparent and symptomless disease. This and the lack of an antemortem diagnostic test hindered the investigation of this parasite (11). The culturing of the organism (1, 6, 22) provided an antigen on which to base tests which could demonstrate circulating antibodies. A skin test (19) and immunofluorescence tests (2, 6) were developed and were followed by the more simple India ink immunoreaction (12, 23). The organism is included in the list of pathogens which the Gesellschaft fur Versuchtierkunde recommends to be excluded from specificpathogen-free (SPF) laboratory animals (8), but, somewhat surprisingly, it was omitted by the Medical Research Council Laboratory Animals Centre.in its manual series, no. 1, 2nd ed. (17). The infection can be transmitted intrauterinely and has been reported in Caesarian-derived barrier-maintained mice and rabbits (10, 11).
The purposes of this report are (i) to confirm the existence of the parasite in an SPF rabbit colony; (ii) to show that small sample size did not reveal a low incidence of the disease; (iii) to suggest that the organism was probably present in the original stock of the unit and does not necessarily indicate a breakdown of barrier maintenance; (iv) to show that by culling all positive reactors it may have been possible to establish an Encephalitozoon-free colony; and (v) to discuss incidence (familial, sexual, and age-related) and possible routes of infection, in particular, the importance of cross-infection within the colony.
360
MATERIALS AND METHODS Buildings. The animal unit, which is used both for experimentation and breeding, was completed in 1975. It is of a modern SPF design, with six animal rooms connected by a common corridor to facilities which include small laboratories and a central washing area. The total capacity of the unit is in excess of 1,000 rabbits. Cages. Stainless-steel cages were suspended in three tiers over troughs lined with siliconized paper. Water was supplied by an automatic system. Husbandry. The unit was operated as for barrier maintenance: the food was autoclaved, and all staff either changed clothing or donned protective outer garments before entering the unit. The usual working staff consists of two technicians who are responsible for all the husbandry, cleaning, and many of the experimental procedures (breeding, inoculations, and bleedings). Animals. The majority of the animals were Swiss hare rabbits (18); the foundation stock was supplied as SPF by the Institute for Biomedical Research Ltd., Fullinsdorf, Switzerland. Repeated tests have shown this source to be the equivalent of the Laboratory Animals Centre's four-star category (17). Although the larger part of our requirements have been met by in-
VOL. 21, 1978
361
E. CUNICULI IN SPF RABBITS
house breeding, deliveries of animals have continued from our supplier. In 1976 some Caesarian sections were performed to introduce the "bas mutant" (13), and in 1977 several ova transplants were performed to introduce the New Zealand white strain of rabbit. Diagnostic test. With the publication of the India ink immunoreaction for the diagnosis of E. cuniculi, this method was included in our routine quality-assurance laboratory investigations. Subsequently, an improvement on this method was evolved and has been used for all of the tests reported here (12). Equal, approximate 2-!1 volumes of heat-inactivated test serum, active India ink, and E. cuniculi antigen suspension were mixed together on a slide and covered immediately with a cover glass. After incubating for 5 min at room temperature, the reaction was examined microscopically using a xlOO oil objective. A positive reaction showed organisms having a dark border of attached carbon particles, and in the negative reaction the organisms were clearly visible, with an unstained cell membrane. Except where otherwise described, all of these tests were performed on a 1:25 dilution of serum, and the results are reported as either positive or negative. Serum samples. The main purpose of the animal unit is for the preparation of experimental antisera, and many sera samples from the various experiments had been collected and stored at -20'C. Some of these samples were still available to us. At approximately 3month intervals, animals were sacrificed to carry out the quality assurance investigations necessary for an SPF unit. In the first half of 1977, serum samples from groups of 10 animals were taken and screened for the presence of E. cuniculi antibodies. In August of that year, samples from 36 rabbits in an experiment were also examined for E. cuniculi antibodies. When these showed some positive reactions, every rabbit in the unit over 6 weeks of age, that is, 758 rabbits, were bled, and their sera were screened. All positive reactors were culled, and 1 month later further serum samples were collected from every animal (over 6 weeks old) and again tested.
bred in-house and those introduced into the colony as SPF which were bred by our supplier. Initially the prevalence was 4.66 and 8.94%, in in-house-bred and purchased animals, respectively, with an overall prevalence of 5.67%. This was reduced to 0.15% (represented by one rabbit) by culling. The occurrence of detectable antibodies relative to the sex of the animals is given in Table 2. These data include the results of the first total stock screen and a further 162 stored samples from rabbits which were dead but which had formerly been housed within the same animal unit. The prevalence was not sexually biased. A comparison of antibodies in rabbits relative to that of their dams is made in Table 3. The data in this table are limited to those dams of known antibody status at the time ofparturition, ie, previously collected and stored serum samples were available for testing. One seropositive dam had three litters, from which we had samples of 13 of her offspring; of these, 12 were seropositive and 1 remained seronegative. One seropositive dam had one litter of seven, all of which were seronegative. The other seronegative dam had a litter of only two, one of TABLE 2. Prevalence of antibodies relative to sex, including samples from an additional 162 rabbits tested posthumously Sex
a
TABLE 3. Prevalence of antibodies in offspring relative to that of dam at time ofparturition Dam Offspring
RESULTS
In the first sample 0/10 rabbits were positive; 3 months later, 0/10 were positive; and after 3 more months, 4/36 were positive. The results of the two samplings of the sera from the entire stock are recorded in Table 1. The reduction in colony size at the second screening was due to culling of the seropositive rabbits from the first screening and the termination of some experiments. A comparison was made between the prevalence in the animals
No. of rabbits seropositive/total
Male 22/404 (5.45) a Female 28/516 (5.43) Total 50/920 (5.44) Numbers in parentheses are percents.
Determination Seroposi- Seronegtive ative
Seroposi-
Seronega-
tive
tive
1 3a 13 (59)b 9 (41) 9 (1.9) 462 (98.1) 58c 2 a One dam produced 12 seropositive and 1 negative
offspring. b
Numbers in parentheses are percents. e Five dams produced 9 seropositive and 53 negative offspring.
TABLE 1. Prevalence of antibodies at first 100% sampling, and 4 weeks later (after culling the positives) No. seropositive Colony size Screen no.
1 2 a
Total
Bred by supplier
Bred in-house
758 670
179 143
579 537
Numbers in parentheses are percents.
Total 43 (5.67) a 1 (0.15)
Bred by supplier 16 (8.94) 1 (0.7)
Bred in-house 27 (4.66) 0
362
BYWATER AND KELLPTT
which was seropositive and the other seronegative. Five seronegative dams had 62 young, of which nine were seropositive. The 409 offspring of the remaining 53 seronegative dams were all negative. By chance the selection of seropositive and seronegative breeding females reflected the overall prevalence of the disease, and although it is not shown in any table, retrospective testing of the breeding males showed that these were all seronegative and remained so. A comparison of detectable antibodies with age of rabbits was made. In Table 4 we have compared age groups of those animals in the unit at the time of the first total stock screen, although we had to omit 62 animals which were not in-house bred and for which age could not be accurately determined. This showed no seropositive rabbits of less than 4 months of age. To check the significance of this observation, we tested all available stored samples from seropositive rabbits. These showed that, of 14 rabbits at 3 months of age, 7 were seronegative, and of a further 2 rabbits, 1 was seronegative at 4 months of age. Of the remaining samples, all were positive at 5 months or more, with the exception of two rabbits. These two acquired antibodies much later, i.e., at 9 months and between 19 and 24 months, respectively. These two animals had been used for experimentation and had been subjected to numerous inoculations and venesections. In addition to the results of the 1:25 dilutions of sera reported above, we have also titrated some of the seropositive samples. Unfortunately, the number was small and consisted of isolated samples collected between 4 and 24 months of age. Therefore, we have been unable to prepare a graph showing mean antibody titers related to age. We report these isolated observations concerning antibody titers below. The range of titers observed was similar to those reported for the immunofluorescence test TABLE 4. Prevalence of antibodies relative to age of animals in unit before culling of seropositivesa Age (mo)
No. of rabbits seropositive/total
12 a A total of 62 animals obtained from supplier is omitted because age was uncertain; these included 6
seropositives. b Only in-house bred. C Numbers in parentheses are percents.
INFECT. IMMUN.
(6) and the India ink inmunoreaction (23) (i.e., up to 1:5,120). The highest titers were observed in animals 5 and 6 months of age. All positive titers observed in animals of 12 months or older were 1:160 or greater. DISCUSSION The results presented were obtained when dealing, in a classical manner of diagnosis and culling, with a naturally occurring infection of E. cuniculi in a colony of Swiss hare rabbits. It was of more importance to eradicate the disease than to make a detailed study of the pathogen. However, we were able to examine a large number of samples, and the picture was not obscured as would have been the case with a high prevalence of the disease. Sampling. There has been much discussion as to a meaningful sample size or the frequency of testing. With destructive testing as would be necessary if histological examinations ofthe central nervous system were carried out, there is an obvious limit. With nondestructive testing, as with the examination of sera, even 100% sampling is possible, and the limitation must be economic, that is, economic with regard to technologists' time and laboratory requirement rather than to money. Factors influencing the choice of sample will include the overall size of the animal colony and the probable prevalence of disease. In retrospect, it is apparent that the size of our first two samples was insufficient to detect the low prevalence of E. cuniculi. In general, the larger the sample is, the more reliable it is. Many pathogens with a direct method of transmission spread rapidly, affecting the majority of the population, so that there is a high probability of detecting such infections from a small sample. But with organisms which do not spread rapidly, for example Listeria monocytogenes, detection of a low prevalence of infection is unlikely without an uneconomically large sample size (16). In the case of our SPF colony, after carrying out culling, the prevalence of the disease was less than 0.2%, and in the future could be, we hope, nil. We intend to carry out further 100% sampling, but there must be a limit to the frequency of this. Prevalence. The single rabbit which was positive in the second screen of the total colony was probably already infected, but failed to show demonstrable antibodies when first tested. The dramatic fall in prevalence after culling suggests a very low cross-infection rate. The higher prevalence of infection in animals supplied compared with that of in-house-bred animals is of doubtful
significance.
VOL. 21, 1978
Although E. cuniculi testing was only commenced this year, our routine quality-assurance screening of the previous 2 years had failed to reveal a change in other microflora either of the rabbits of our supplier or of the animals within the unit itself. Retrospective testing has shown that the prevalence of E. cuniculi has always been between 5 and 8%. We have discussed our findings with our supplier, and learn that tests for E. cuniculi had not been included in their own screening tests. Congenital transmission has been demonstrated (10), and we assume that this disease was endemic in our original foundation stock and had been perpetuated by further supplies and in-house breeding. There is an obvious high probability that seropositive offspring will develop from seropositive dams. Although the prevalence of seropositive offspring resulting from seronegative dams was very small, that is, less than 2%, these animals accounted for 40% of the infected animals bred in our colony, and this must be attributed to cross-infection. Offspring from seropositive dams may acquire infection in utero, but we have no evidence to suggest how frequently this may occur. Also, the young from these mothers will be exposed to the intermittent excretion of the organisms in the urine of their dams throughout the preweaning period. Studies in progress show low levels of antibodies (1:5 to 1:25) in this age group, which could also be passive immunity. The presence of maternally transferred immunity could account for some offspring failing to become infected in spite of their probable exposure to infected urine.
The offspring from seronegative dams cannot have received maternal antibodies, but neither will they be exposed to infection during the preweaning period. At the time of weaning there could be a high risk of exposure to infection through contact with the parasite, mechanically conveyed, by those who handle the animals. In postweaned animals it can be seen that, although we have samples from rabbits which at one time were negative and later developed to positive, we have no examples of animals with positive titers subsequently losing a detectable level of this antibody. We consider it most significant that we should have observed that fewer young animals showed antibodies, but that by 4 to 6 months of age the occurrence of antibodies was similar to that in older age groups. This suggests the establishment of the infection by this age. It will be seen that we failed to reveal a detectable antibody level in half of the animals at 3 months of age and this closely parallels the observations of Cox (3) who reported that in a group of 30 rabbits from an infected colony, 11
E. CUNICULI IN SPF RABBITS
363
were seropositive to the immunofluorescence test at 12 weeks of age and a further 6 had developed antibodies by the age of 15 weeks, with no further rabbits from this group becoming infected throughout a further 2 months, when his report was concluded. Cox attributed the antibodies in the first 11 rabbits to a chronic infection and the latter to the indication of a recent infection, but we consider that he could have been observing the natural pattern of the same disease. Susceptibility. This appears to vary with age, young animals being susceptible and animals over 5 months of age being resistant, and this argument is supported by observing that we have only two examples of older animals which developed antibodies. However, the individual caging of older animals must reduce exposure to infection. To offset this they have prolonged potential exposure. However, the argument of a reduced exposure cannot be made for the breeding animals. The breeding females are handled cumulatively to an amount equal to that of each of their litters, but the prevalence in our breeding females is no higher than other adult animals. The breeding males have definitely been exposed to infection and, in particular, four stud males were "used" many times on infected females; none of these males developed antibodies, and noninfected females subsequently mated to these males have also remained seronegative. Not only does this support the argument that adults are resistant to infection, but also it suggests that venereal infection does not occur, although this last observation is somewhat onesided, because we do not have experience of negative females being placed with positive males. Conclusion. We are not aware of any more evidence concerning the natural transmission of E. cuniculi since Flynn wrote that "little is known about the method of transmission" (7). We believe that our observations of a naturally occurring infection in rabbits supports the following statements. (i) There is no sex discrimination in susceptibility. Although there is evidence that congenital infection occurs, this route is not exclusive. (ii) Organisms excreted by an infected dam contribute greatly to the dissemination of the disease among her own, and unrelated, offspring. (iii) Cross-infection is a major factor in the spread of this disease and suggests that the oral route is important. There is an age susceptibility in rabbits, and infection usually occurs by 5 months; older animals are resistant. Venereal infection, at least from female to male, does not occur. (iv) Maternal antibodies can exist in preweaned animals; antibody levels ap-
364
BYWATER AND KELLETIINFECT. IMMUN.
pear at their highest at 5 to 6 months and do not fall below 1:160 throughout the life of infected animals. ACKNOWLEDGMENIS We are very pleased to acknowledge E. John and G. Waltert, the technicians responsible for the husbandry of the unit, because unwittingly they may have been responsible for the cross-contamination resulting in 40% of the incidence of infection. However, without their normally high standard of hygiene, this incidence could have been considerably higher. With their names we would couple that of E. Wagner in expressing our appreciation for carrying out the venesections. On several occasions these three technicians collected more than 300 blood samples in a working day.
LITERATURE CITED 1. Bismanis, J. E. 1970. Detection of latent murine nosematosis and growth of Nosema cuniculi in cell culture. Can. J. Microbiol. 16:237-242. 2. Chalupsky, J., R. Bedrnik, and J. Vavra. 1971. The indirect fluorescence antibody test for Nosema cuniculi. J. Protozool. 18 (Suppl):77. 3. Cox, J. C. 1977. Altered immune responsiveness associated with Encephalitozoon cuniculi infection in rabbits. Infect. Immun. 15:392-395. 4. Cox, J. C. 1977. Isolation of Encephalitozoon cuniculi from urine samples. Lab. Anim. 11:233-234. 5. Cox, J. C., and D. Pye. 1975. Serodiagnosis of nosematosis by immunofluorescence using cell-culture-grown organisms. Lab. Anim. 9:297-304. 6. Cox, J. C., N. B. Walden, and R. C. Nairn. 1972. Presumptive diagnosis of Nosema cuniculi in rabbits by immunofluorescence. Res. Vet. Sci. 13:595-597. 7. Flynn, R. J. 1973. Parasites of laboratory animals, p. 84. The Iowa State University Press, Ames. 8. Gesellschaft fur Versuchtierkunde. 1973. Gesellschaft fur Versuchtierkunde Liste von Nachweismethoden zur Uberprufung von SPF Versuchtieren auf Freisein von Erregern, no. 3. Basel. 9. Howell, J. McC., and N. Edington. 1968. The production of rabbits free from lesions associated with Encephalitozoon cuniculi. Lab. Anim. 2:143-148. 10. Hunt, R. D., N. W. King, and H. L. Foster. 1972. Encephalitizoonosis: evidence for vertical transmission. J. Infect. Dis. 126:212-214.
11. Innes, J. R. ML, W. Zeman, J. K. Frenkel, and Borner. 1962. Occult endemic encephalitozoonosis of the central nervous system of mice (Swiss-Bagg-O'Grady strain). J. Neuropathol. Exp. Neurol. 21:519-33. 12. Kellett, B. S., and J. E. C. Bywater. 1978. A modified India-ink immunoreaction for the detection of Encephalitozoonosis. Lab. Anim. 12:59-60. 13. Kelus, A. S., and S. Weiss. 1977. Variant strain of rabbit lacking immunoglobulin K polypeptide chain. Nature (London) 265:156-158. 14. Levaditi, C., S. Nicolau, and Schoen. 1924. L'etiologie de l'encephalite epizootique du lapin dana ses rapports avec l'etude experimental de l'encephalite lethargique du Encephalitozoon cuniculi. Ann. Inst. Pasteur Paris 1938:651. 15. McCartny, J. E. 1924. Brain lesions of the domestic rabbit. J. Exp. Med. 39:51. 16. Medical Research Council. 1971. Microbiological examination of laboratory animals for the purposes of accreditation, p. 2i-2ii Medical Research Council, Laboratory Animals Centre, Great Britain. 17. Medical Research Council. 1974. The accreditation and recognition schemes for suppliers of laboratory animals, manual 1, 2nd ed. Medical Research Council, Laboratory Animals Centre, Great Britain. 18. Medical Research Council. 1975. International index of laboratory animals, 3rd ed, p. 55. Medical Research Council, Laboratory Animal Centre, Great Britain. 19. Pakes, S. P., J. A. Shadduck, and R. G. Olsen. 1972. A diagnostic skin test for encephalitozoonosis (nosematosis) in rabbits. Lab. Anim. Sci. 22:870-877. 20. Petri, M. 1969. Studies on Nosema cuniculi found in transplantable ascites tumours with a survey of microsporidosis in mammals. Acta Pathol. Microbiol. Scand. Suppl., p. 204. 21. Shadduck, J. A., and S. P. Pakes. 1971. Encephalitozoonosis (nosematosis) and toxaplasmosis. Am. J. Pathol. 64:657-672. 22. Vavra, J., R. Bedrnik, and Cinathyl. 1972. Isolation and in vitro cultivation of the mammalian microsporidian Encephalitozoon cuniculi. Folia Parasitologica (Praha) 19:349-354. 23. Waller, T. 1977. The india-ink immunoreaction: a method for the rapid diagnosis of Encephalitizoonosis. Lab. Anim. 11:93-97. 24. Wright, J. H., and E. M. Craighead. 1922. Infectious motor paralysis in young rabbits. J. Exp. Med. 36:135.
INFECTION AND IMMUNITY, Aug. 1978, p. 360-364 0019-9567/78/0021-0360$02.00/0 Copyright i 1978 American Society for Microbiology
Printed in U.S.A.
Encephalitozoon cuniculi Antibodies in a Specific-PathogenFree Rabbit Unit J. E. BYWATER* AND B. S. KELLETT Basel Institute for Immunology, CH-4005 Basel 5, Switzerland Received for publication 17 January 1978
We describe our discovery of Encephalitozoon cuniculi antibodies in a specificpathogen-free rabbit colony. Small-sized samples had failed to reveal the presence of infection with a prevalence of about 5%. Using an India ink immunoreaction test by which we were able to visualize both negative and positive reactions, we were able to undertake a 100% screen of the colony of more than 700 rabbits and to repeat this 4 weeks later when we had culled the positive reactors. By collating the results of those tests with the results of tests on previously collected samples, we have been able to discuss and observe age and sex susceptibilities and the mode of transmission of the naturally occurring disease.
Encephalitozoon cuniculi was first reported in 1922 (24), although it was 2 years later that the organism was described and named (14). The disease occurs throughout the world, affecting various species, and it is widespread in domestic rabbits (7, 20, 21). Histological lesions of E. cuniculi have been reported as being present in 55% of clinically normal rabbits, with an even higher prevalence in animals suffering from other infections (15), and there have been numerous reports concerning the widespread occurrence of the parasite in laboratory animals with its interference in the interpretation of experimental results (9). Rabbits appear to be particularly susceptible, and when symptoms are recognized they are usually characterized by loss of condition and occasional overt central nervous system involvement (5). However, the infection most frequently produces an inapparent and symptomless disease. This and the lack of an antemortem diagnostic test hindered the investigation of this parasite (11). The culturing of the organism (1, 6, 22) provided an antigen on which to base tests which could demonstrate circulating antibodies. A skin test (19) and immunofluorescence tests (2, 6) were developed and were followed by the more simple India ink immunoreaction (12, 23). The organism is included in the list of pathogens which the Gesellschaft fur Versuchtierkunde recommends to be excluded from specificpathogen-free (SPF) laboratory animals (8), but, somewhat surprisingly, it was omitted by the Medical Research Council Laboratory Animals Centre.in its manual series, no. 1, 2nd ed. (17). The infection can be transmitted intrauterinely and has been reported in Caesarian-derived barrier-maintained mice and rabbits (10, 11).
The purposes of this report are (i) to confirm the existence of the parasite in an SPF rabbit colony; (ii) to show that small sample size did not reveal a low incidence of the disease; (iii) to suggest that the organism was probably present in the original stock of the unit and does not necessarily indicate a breakdown of barrier maintenance; (iv) to show that by culling all positive reactors it may have been possible to establish an Encephalitozoon-free colony; and (v) to discuss incidence (familial, sexual, and age-related) and possible routes of infection, in particular, the importance of cross-infection within the colony.
360
MATERIALS AND METHODS Buildings. The animal unit, which is used both for experimentation and breeding, was completed in 1975. It is of a modern SPF design, with six animal rooms connected by a common corridor to facilities which include small laboratories and a central washing area. The total capacity of the unit is in excess of 1,000 rabbits. Cages. Stainless-steel cages were suspended in three tiers over troughs lined with siliconized paper. Water was supplied by an automatic system. Husbandry. The unit was operated as for barrier maintenance: the food was autoclaved, and all staff either changed clothing or donned protective outer garments before entering the unit. The usual working staff consists of two technicians who are responsible for all the husbandry, cleaning, and many of the experimental procedures (breeding, inoculations, and bleedings). Animals. The majority of the animals were Swiss hare rabbits (18); the foundation stock was supplied as SPF by the Institute for Biomedical Research Ltd., Fullinsdorf, Switzerland. Repeated tests have shown this source to be the equivalent of the Laboratory Animals Centre's four-star category (17). Although the larger part of our requirements have been met by in-
VOL. 21, 1978
361
E. CUNICULI IN SPF RABBITS
house breeding, deliveries of animals have continued from our supplier. In 1976 some Caesarian sections were performed to introduce the "bas mutant" (13), and in 1977 several ova transplants were performed to introduce the New Zealand white strain of rabbit. Diagnostic test. With the publication of the India ink immunoreaction for the diagnosis of E. cuniculi, this method was included in our routine quality-assurance laboratory investigations. Subsequently, an improvement on this method was evolved and has been used for all of the tests reported here (12). Equal, approximate 2-!1 volumes of heat-inactivated test serum, active India ink, and E. cuniculi antigen suspension were mixed together on a slide and covered immediately with a cover glass. After incubating for 5 min at room temperature, the reaction was examined microscopically using a xlOO oil objective. A positive reaction showed organisms having a dark border of attached carbon particles, and in the negative reaction the organisms were clearly visible, with an unstained cell membrane. Except where otherwise described, all of these tests were performed on a 1:25 dilution of serum, and the results are reported as either positive or negative. Serum samples. The main purpose of the animal unit is for the preparation of experimental antisera, and many sera samples from the various experiments had been collected and stored at -20'C. Some of these samples were still available to us. At approximately 3month intervals, animals were sacrificed to carry out the quality assurance investigations necessary for an SPF unit. In the first half of 1977, serum samples from groups of 10 animals were taken and screened for the presence of E. cuniculi antibodies. In August of that year, samples from 36 rabbits in an experiment were also examined for E. cuniculi antibodies. When these showed some positive reactions, every rabbit in the unit over 6 weeks of age, that is, 758 rabbits, were bled, and their sera were screened. All positive reactors were culled, and 1 month later further serum samples were collected from every animal (over 6 weeks old) and again tested.
bred in-house and those introduced into the colony as SPF which were bred by our supplier. Initially the prevalence was 4.66 and 8.94%, in in-house-bred and purchased animals, respectively, with an overall prevalence of 5.67%. This was reduced to 0.15% (represented by one rabbit) by culling. The occurrence of detectable antibodies relative to the sex of the animals is given in Table 2. These data include the results of the first total stock screen and a further 162 stored samples from rabbits which were dead but which had formerly been housed within the same animal unit. The prevalence was not sexually biased. A comparison of antibodies in rabbits relative to that of their dams is made in Table 3. The data in this table are limited to those dams of known antibody status at the time ofparturition, ie, previously collected and stored serum samples were available for testing. One seropositive dam had three litters, from which we had samples of 13 of her offspring; of these, 12 were seropositive and 1 remained seronegative. One seropositive dam had one litter of seven, all of which were seronegative. The other seronegative dam had a litter of only two, one of TABLE 2. Prevalence of antibodies relative to sex, including samples from an additional 162 rabbits tested posthumously Sex
a
TABLE 3. Prevalence of antibodies in offspring relative to that of dam at time ofparturition Dam Offspring
RESULTS
In the first sample 0/10 rabbits were positive; 3 months later, 0/10 were positive; and after 3 more months, 4/36 were positive. The results of the two samplings of the sera from the entire stock are recorded in Table 1. The reduction in colony size at the second screening was due to culling of the seropositive rabbits from the first screening and the termination of some experiments. A comparison was made between the prevalence in the animals
No. of rabbits seropositive/total
Male 22/404 (5.45) a Female 28/516 (5.43) Total 50/920 (5.44) Numbers in parentheses are percents.
Determination Seroposi- Seronegtive ative
Seroposi-
Seronega-
tive
tive
1 3a 13 (59)b 9 (41) 9 (1.9) 462 (98.1) 58c 2 a One dam produced 12 seropositive and 1 negative
offspring. b
Numbers in parentheses are percents. e Five dams produced 9 seropositive and 53 negative offspring.
TABLE 1. Prevalence of antibodies at first 100% sampling, and 4 weeks later (after culling the positives) No. seropositive Colony size Screen no.
1 2 a
Total
Bred by supplier
Bred in-house
758 670
179 143
579 537
Numbers in parentheses are percents.
Total 43 (5.67) a 1 (0.15)
Bred by supplier 16 (8.94) 1 (0.7)
Bred in-house 27 (4.66) 0
362
BYWATER AND KELLPTT
which was seropositive and the other seronegative. Five seronegative dams had 62 young, of which nine were seropositive. The 409 offspring of the remaining 53 seronegative dams were all negative. By chance the selection of seropositive and seronegative breeding females reflected the overall prevalence of the disease, and although it is not shown in any table, retrospective testing of the breeding males showed that these were all seronegative and remained so. A comparison of detectable antibodies with age of rabbits was made. In Table 4 we have compared age groups of those animals in the unit at the time of the first total stock screen, although we had to omit 62 animals which were not in-house bred and for which age could not be accurately determined. This showed no seropositive rabbits of less than 4 months of age. To check the significance of this observation, we tested all available stored samples from seropositive rabbits. These showed that, of 14 rabbits at 3 months of age, 7 were seronegative, and of a further 2 rabbits, 1 was seronegative at 4 months of age. Of the remaining samples, all were positive at 5 months or more, with the exception of two rabbits. These two acquired antibodies much later, i.e., at 9 months and between 19 and 24 months, respectively. These two animals had been used for experimentation and had been subjected to numerous inoculations and venesections. In addition to the results of the 1:25 dilutions of sera reported above, we have also titrated some of the seropositive samples. Unfortunately, the number was small and consisted of isolated samples collected between 4 and 24 months of age. Therefore, we have been unable to prepare a graph showing mean antibody titers related to age. We report these isolated observations concerning antibody titers below. The range of titers observed was similar to those reported for the immunofluorescence test TABLE 4. Prevalence of antibodies relative to age of animals in unit before culling of seropositivesa Age (mo)
No. of rabbits seropositive/total
12 a A total of 62 animals obtained from supplier is omitted because age was uncertain; these included 6
seropositives. b Only in-house bred. C Numbers in parentheses are percents.
INFECT. IMMUN.
(6) and the India ink inmunoreaction (23) (i.e., up to 1:5,120). The highest titers were observed in animals 5 and 6 months of age. All positive titers observed in animals of 12 months or older were 1:160 or greater. DISCUSSION The results presented were obtained when dealing, in a classical manner of diagnosis and culling, with a naturally occurring infection of E. cuniculi in a colony of Swiss hare rabbits. It was of more importance to eradicate the disease than to make a detailed study of the pathogen. However, we were able to examine a large number of samples, and the picture was not obscured as would have been the case with a high prevalence of the disease. Sampling. There has been much discussion as to a meaningful sample size or the frequency of testing. With destructive testing as would be necessary if histological examinations ofthe central nervous system were carried out, there is an obvious limit. With nondestructive testing, as with the examination of sera, even 100% sampling is possible, and the limitation must be economic, that is, economic with regard to technologists' time and laboratory requirement rather than to money. Factors influencing the choice of sample will include the overall size of the animal colony and the probable prevalence of disease. In retrospect, it is apparent that the size of our first two samples was insufficient to detect the low prevalence of E. cuniculi. In general, the larger the sample is, the more reliable it is. Many pathogens with a direct method of transmission spread rapidly, affecting the majority of the population, so that there is a high probability of detecting such infections from a small sample. But with organisms which do not spread rapidly, for example Listeria monocytogenes, detection of a low prevalence of infection is unlikely without an uneconomically large sample size (16). In the case of our SPF colony, after carrying out culling, the prevalence of the disease was less than 0.2%, and in the future could be, we hope, nil. We intend to carry out further 100% sampling, but there must be a limit to the frequency of this. Prevalence. The single rabbit which was positive in the second screen of the total colony was probably already infected, but failed to show demonstrable antibodies when first tested. The dramatic fall in prevalence after culling suggests a very low cross-infection rate. The higher prevalence of infection in animals supplied compared with that of in-house-bred animals is of doubtful
significance.
VOL. 21, 1978
Although E. cuniculi testing was only commenced this year, our routine quality-assurance screening of the previous 2 years had failed to reveal a change in other microflora either of the rabbits of our supplier or of the animals within the unit itself. Retrospective testing has shown that the prevalence of E. cuniculi has always been between 5 and 8%. We have discussed our findings with our supplier, and learn that tests for E. cuniculi had not been included in their own screening tests. Congenital transmission has been demonstrated (10), and we assume that this disease was endemic in our original foundation stock and had been perpetuated by further supplies and in-house breeding. There is an obvious high probability that seropositive offspring will develop from seropositive dams. Although the prevalence of seropositive offspring resulting from seronegative dams was very small, that is, less than 2%, these animals accounted for 40% of the infected animals bred in our colony, and this must be attributed to cross-infection. Offspring from seropositive dams may acquire infection in utero, but we have no evidence to suggest how frequently this may occur. Also, the young from these mothers will be exposed to the intermittent excretion of the organisms in the urine of their dams throughout the preweaning period. Studies in progress show low levels of antibodies (1:5 to 1:25) in this age group, which could also be passive immunity. The presence of maternally transferred immunity could account for some offspring failing to become infected in spite of their probable exposure to infected urine.
The offspring from seronegative dams cannot have received maternal antibodies, but neither will they be exposed to infection during the preweaning period. At the time of weaning there could be a high risk of exposure to infection through contact with the parasite, mechanically conveyed, by those who handle the animals. In postweaned animals it can be seen that, although we have samples from rabbits which at one time were negative and later developed to positive, we have no examples of animals with positive titers subsequently losing a detectable level of this antibody. We consider it most significant that we should have observed that fewer young animals showed antibodies, but that by 4 to 6 months of age the occurrence of antibodies was similar to that in older age groups. This suggests the establishment of the infection by this age. It will be seen that we failed to reveal a detectable antibody level in half of the animals at 3 months of age and this closely parallels the observations of Cox (3) who reported that in a group of 30 rabbits from an infected colony, 11
E. CUNICULI IN SPF RABBITS
363
were seropositive to the immunofluorescence test at 12 weeks of age and a further 6 had developed antibodies by the age of 15 weeks, with no further rabbits from this group becoming infected throughout a further 2 months, when his report was concluded. Cox attributed the antibodies in the first 11 rabbits to a chronic infection and the latter to the indication of a recent infection, but we consider that he could have been observing the natural pattern of the same disease. Susceptibility. This appears to vary with age, young animals being susceptible and animals over 5 months of age being resistant, and this argument is supported by observing that we have only two examples of older animals which developed antibodies. However, the individual caging of older animals must reduce exposure to infection. To offset this they have prolonged potential exposure. However, the argument of a reduced exposure cannot be made for the breeding animals. The breeding females are handled cumulatively to an amount equal to that of each of their litters, but the prevalence in our breeding females is no higher than other adult animals. The breeding males have definitely been exposed to infection and, in particular, four stud males were "used" many times on infected females; none of these males developed antibodies, and noninfected females subsequently mated to these males have also remained seronegative. Not only does this support the argument that adults are resistant to infection, but also it suggests that venereal infection does not occur, although this last observation is somewhat onesided, because we do not have experience of negative females being placed with positive males. Conclusion. We are not aware of any more evidence concerning the natural transmission of E. cuniculi since Flynn wrote that "little is known about the method of transmission" (7). We believe that our observations of a naturally occurring infection in rabbits supports the following statements. (i) There is no sex discrimination in susceptibility. Although there is evidence that congenital infection occurs, this route is not exclusive. (ii) Organisms excreted by an infected dam contribute greatly to the dissemination of the disease among her own, and unrelated, offspring. (iii) Cross-infection is a major factor in the spread of this disease and suggests that the oral route is important. There is an age susceptibility in rabbits, and infection usually occurs by 5 months; older animals are resistant. Venereal infection, at least from female to male, does not occur. (iv) Maternal antibodies can exist in preweaned animals; antibody levels ap-
364
BYWATER AND KELLETIINFECT. IMMUN.
pear at their highest at 5 to 6 months and do not fall below 1:160 throughout the life of infected animals. ACKNOWLEDGMENIS We are very pleased to acknowledge E. John and G. Waltert, the technicians responsible for the husbandry of the unit, because unwittingly they may have been responsible for the cross-contamination resulting in 40% of the incidence of infection. However, without their normally high standard of hygiene, this incidence could have been considerably higher. With their names we would couple that of E. Wagner in expressing our appreciation for carrying out the venesections. On several occasions these three technicians collected more than 300 blood samples in a working day.
LITERATURE CITED 1. Bismanis, J. E. 1970. Detection of latent murine nosematosis and growth of Nosema cuniculi in cell culture. Can. J. Microbiol. 16:237-242. 2. Chalupsky, J., R. Bedrnik, and J. Vavra. 1971. The indirect fluorescence antibody test for Nosema cuniculi. J. Protozool. 18 (Suppl):77. 3. Cox, J. C. 1977. Altered immune responsiveness associated with Encephalitozoon cuniculi infection in rabbits. Infect. Immun. 15:392-395. 4. Cox, J. C. 1977. Isolation of Encephalitozoon cuniculi from urine samples. Lab. Anim. 11:233-234. 5. Cox, J. C., and D. Pye. 1975. Serodiagnosis of nosematosis by immunofluorescence using cell-culture-grown organisms. Lab. Anim. 9:297-304. 6. Cox, J. C., N. B. Walden, and R. C. Nairn. 1972. Presumptive diagnosis of Nosema cuniculi in rabbits by immunofluorescence. Res. Vet. Sci. 13:595-597. 7. Flynn, R. J. 1973. Parasites of laboratory animals, p. 84. The Iowa State University Press, Ames. 8. Gesellschaft fur Versuchtierkunde. 1973. Gesellschaft fur Versuchtierkunde Liste von Nachweismethoden zur Uberprufung von SPF Versuchtieren auf Freisein von Erregern, no. 3. Basel. 9. Howell, J. McC., and N. Edington. 1968. The production of rabbits free from lesions associated with Encephalitozoon cuniculi. Lab. Anim. 2:143-148. 10. Hunt, R. D., N. W. King, and H. L. Foster. 1972. Encephalitizoonosis: evidence for vertical transmission. J. Infect. Dis. 126:212-214.
11. Innes, J. R. ML, W. Zeman, J. K. Frenkel, and Borner. 1962. Occult endemic encephalitozoonosis of the central nervous system of mice (Swiss-Bagg-O'Grady strain). J. Neuropathol. Exp. Neurol. 21:519-33. 12. Kellett, B. S., and J. E. C. Bywater. 1978. A modified India-ink immunoreaction for the detection of Encephalitozoonosis. Lab. Anim. 12:59-60. 13. Kelus, A. S., and S. Weiss. 1977. Variant strain of rabbit lacking immunoglobulin K polypeptide chain. Nature (London) 265:156-158. 14. Levaditi, C., S. Nicolau, and Schoen. 1924. L'etiologie de l'encephalite epizootique du lapin dana ses rapports avec l'etude experimental de l'encephalite lethargique du Encephalitozoon cuniculi. Ann. Inst. Pasteur Paris 1938:651. 15. McCartny, J. E. 1924. Brain lesions of the domestic rabbit. J. Exp. Med. 39:51. 16. Medical Research Council. 1971. Microbiological examination of laboratory animals for the purposes of accreditation, p. 2i-2ii Medical Research Council, Laboratory Animals Centre, Great Britain. 17. Medical Research Council. 1974. The accreditation and recognition schemes for suppliers of laboratory animals, manual 1, 2nd ed. Medical Research Council, Laboratory Animals Centre, Great Britain. 18. Medical Research Council. 1975. International index of laboratory animals, 3rd ed, p. 55. Medical Research Council, Laboratory Animal Centre, Great Britain. 19. Pakes, S. P., J. A. Shadduck, and R. G. Olsen. 1972. A diagnostic skin test for encephalitozoonosis (nosematosis) in rabbits. Lab. Anim. Sci. 22:870-877. 20. Petri, M. 1969. Studies on Nosema cuniculi found in transplantable ascites tumours with a survey of microsporidosis in mammals. Acta Pathol. Microbiol. Scand. Suppl., p. 204. 21. Shadduck, J. A., and S. P. Pakes. 1971. Encephalitozoonosis (nosematosis) and toxaplasmosis. Am. J. Pathol. 64:657-672. 22. Vavra, J., R. Bedrnik, and Cinathyl. 1972. Isolation and in vitro cultivation of the mammalian microsporidian Encephalitozoon cuniculi. Folia Parasitologica (Praha) 19:349-354. 23. Waller, T. 1977. The india-ink immunoreaction: a method for the rapid diagnosis of Encephalitizoonosis. Lab. Anim. 11:93-97. 24. Wright, J. H., and E. M. Craighead. 1922. Infectious motor paralysis in young rabbits. J. Exp. Med. 36:135.
