Serodiagnosis of nosematosis by immunofluorescence using cell-culture-grown organisms

Laboratory Animals (1975) 9, 297-304. 297 SERODIAGNOSIS OF NOSEMATOSIS BY IMMUNOFLUORESCENCE USING CELL-CULTURE-GROWN ORGANISMS by J. C. COX and D. PYE Commonwealth Serum Laboratories, Poplar Road, Parkville, Victoria, 3052, Australia SUMMARY This simple procedure for the detection of serum antibodies to Nosema in rabbits and other host species incorporates indirect immunofluorescence and uses as antigen N. cuniculi isolated from urine and cultured in a human fibroblast-like cell line derived from foetal tongue. Examination of rabbit sera from 8 institutions indicated that no institution was free from Nosema infection. The prevalence of infection in 4 separate Australian rabbit colonies varied from 25 to 75 %. cuniculi Nosema (Encephalitozoon) cuniculi (Levaditi, Nicolau & Shoen, 1924; Lainson, Garnham, Killick-Kendrick & Bird, 1964) is an intracellular protozoan parasite infecting laboratory animals, many wild and domestic animals, and man. In rabbits, which are particularly susceptible, it causes a chronic infection characterized by loss of condition with occasional overt central nervous system involvement. On post-mortem examination, macroscopic and microscopic lesions are often encountered in many organs, especially kidney and brain. These lesions may seriously affect the interpretation of experimental results (Howell & Edington, 1968). Various diagnostic tests for nosematosis in the living animal have been employed including the development of ascites in mice following injection of infected material (Matsubayashi, Koike, Mikata, Takei & Hagiwara, 1959), staining of organisms in urine with histological (Goodman & Garner, 1972) and immunofluorescent reagents (Cox, Walden & Nairn, 1972), and a skin-test reaction (Pakes, Shadduck & Olsen, 1972). However, the development of a satisfactory serological test was dependent upon supplies of antigenically stable, tissue-culture-grown organisms. This paper extends the work of Shadduck (1969) and Bismanis (J 970) on in-vitro cultivation of Nosema cuniculi, and outlines procedures for the immunofluorescence detection of serum antibodies to N. cuniculi in which these organisms are used as antigen. 298 J. C. COX MATERIALS AND D. PYE AND METHODS Cell cultures Canine embryo cell cultures (Montrey, Shadduck & Pakes, ]973) were prepared from a 4-week embryo. The resulting fibroblast-like cells grew slow]y, 4 weeks being required to reach confluence. The human foetal lung cell line WT.38 (Hayflick, ] 965) was purchased from Cell Associates Inc., P.O.B. 4465, Stanford, California 94305, U.S.A., and the diploid human foetal tongue cell line, CSL 300, was derived at this Institution. All cell cultures were grown on Basa] Medium Eag]e (BME) (Eagle, ] 955) Confluent cultures were supplemented with] 0 % unheated foetal calf serum. maintained on Medium ]99 (Morgan, Morton & Parker, ]950) supplemented with 1 % or 10 % unheated foetal calf serum. Immunofluorescent FlTC infected conjugates rabbit IgG anti-No cuniculi, prepared from the serum of a naturallyrabbit which had an anti-No cuniculi immunofluorescence titre of 5000, was used at a concentration of 5 mg protein/m!. Its FITC:lgG molar ratio was 2·6 and it had been absorbed with homogenates of calf liver and stomach so that it did not stain norma] tissue-culture cells. This conjugate gave bright, specific staining of Nosema at a concentration below ] 00 J.lg protein/m\. FITC sheep IgG anti-rabbit immunoglobulin from the Commonwealth Serum Laboratories (CSL), FlTC sheep IgG anti-human immunoglobulin (CSL) and FlTC rabbit globu]in anti-bovine g]obulin (Mi]es Laboratories (Pentex) Inc., ] 127 Myrtle Street, Elkhart, Indiana 465]4, U.S.A.) were used In indirect immunofluorescence at a concentration of 2 mg protein/m\. Establishment of Nosema cuniculi in cell culture Urine specimens were collected by catheterization of young rabbits from a colony of high Nosema prevalence. They were prepared for staining by sandwich immunofluorescence using methods described previously (Cox et al., ] 972). Urine sediments found to contain Nosema were absorbed onto a monolayer of canine embryo cells for 1 h at 37°C, rinsed to remove precipitates, then incubated in maintenance medium containing] % serum. This medium was changed weekly and the sediment which resulted from its centrifugation at 1000 g for 10 min was examined by direct immunofluorescence for the presence of Nosema. Secondary Nosema infections in human fibroblasts were achieved by transferring to half-confluent bottles of these cells 2 ml of maintenance medium from infected canine embryo cells. The medium was 'examined for Nosema as already described for primary infections. Cells grown on coverslips were SERODIAGNOSIS OF NOSEMATOSIS 299 prepared for immunofluorescence examination by fixation in absolute ethanol for I min. N. cuniculi retained viability when stored for several months as infected fibroblast cells in 10 % dimethyl sulphoxide at -70°C. Detection of serum antibodies to Nosema cuniculi The indirect immunofluorescence technique as described by Nairn (1969) was used in the examination of sera for antibodies to N. cuniculi. Organisms liberated into culture medium from CSL 300 fibroblasts were centrifuged and resuspended in phosphate-buffered saline (PBS :O'OI-M sodium phosphate, O'145-M sodium chloride, pH 7· I) to a concentration of 10 5_106 free organisms! m!. Smears, 2-6 per slide, each 5 mm diameter and containing about 2 JlI of Nosema suspension were air-dried, gently heat-fixed and stored at 4°C over silica gel until ready for use. Sera were screened at 1 in 10 dilution in PBS and titrations were performed with doubling dilutions. The endpoint was defined arbitrarily as the highest dilution which showed clear bright staining. All conjugates used in the immunofluorescence detection of serum antibodies to Nosema were carefully evaluated on untreated smears at the working dilution to ensure total absence of staining. This was particularly important if the antiserum was prepared in rabbits, although other host species have been found to exhibit low anti-Nosema antibody levels. RESULTS Growth of Nosema cuniculi in cell culture The initial infection of canine embryo cells was achieved with a urine specimen which contained less than 1000 Nosema organisms. In the first 2 weeks no organisms were detected in the supernatant. However, in the 3rd week, a number of small groups of up to about 40 organisms per group, many with extruded polar filaments, were detected. At this stage, the cell sheet appeared microscopically the same as uninfected controls. These cells continued to release Nosema in considerable numbers for 2 months before all cells were destroyed. Most of the liberated organisms occurred singly and had extruded filament and terminal sporoplasm. A 2 ml sample of this culture medium was used to infect a bottle each of WI 38 and CSL 300 cells. Within 2 weeks organisms were detected both singly and in small groups in the culture fluid of the CSL 300 cells, and by 4 weeks they were being released in such numbers that culture medium appeared slightly opaque. Microscopically the cell sheet, by 4 weeks, contained many enlarged cells, often in groups, each having a cytoplasmic inclusion of up to 1000 organisms (Fig. 1). Organisms were detected in WI 38 cells after 3 weeks; subsequent growth was good but not as rapid as in CSL 300. 300 J. C. COX AND D. PYE Fig. 1. Human foetal tongue cell (CSL 300) which contains a large cytoplasmic inclusion crammed with Nosema cuniculi organisms. Direct immunofluorescent staining. Line represents 50 11m. Although N. cuniculi grew well when the maintenance medium contained I % foetal calf serum, the organisms were found most suitable as antigen in the immunofluorescence test when 10% serum was used. To achieve routine growth of the organism in CSL 300 cells, cells from heavily infected cultures were mixed with uninfected cells and fresh cultures were seeded. The Nosema multiplication rate under these circumstances was 10-100 fold per week. Serological investigations in rabbits The indirect immunofluorescence test was used to screen the sera of healthy, generally young rabbits from various Australian, English and American institutions. The results are presented in Table 1. A 'negative' reaction is defined as zero to trace staining of Nosema at a 1 in 10 dilution of serum, a 'doubtful' as weak but definite staining, and a 'positive' as bright staining. Fig. 2 shows several brightly fluorescing organisms with polar filaments and terminal sporoplasms, a typical reaction for a positive serum. Generally, positive sera had titres in excess of 100, and titres up to 5000 were encountered. The prevalence of Nosema infection in Australian rabbit-breeding institutions varied from 25 to 75 % and was comparable with figures based on histological examination reported for other institutions (Shadduck & Pakes, 1971). The urine of both seropositive and seronegative rabbits was examined for the presence of organisms. All 11 young seropositive rabbits tested daily over SERODIAGNOSIS OF NOSEMATOSIS 301 Table 1. Incidence of Nosema antibodies in laboratory rabbit sera. Source of sera *Commonwealth Australia Titr4 Serum Laboratories, Total p§ n d 16 7 68 (75 %) 91 7 16 (30%) 54 *Institute of Medical Science, Australia & Veterinary 31 *Monash Australia 14 5 (25 %) 20 16 9 (35 %) 26 2 7 10 University, *Keith Turnbull Australia Research Station, tlnstitutc A, England tlnstitutc B, England 3 4 3 10 tI nstitute C, U.S.A. 2 0 1 3 tlnstitute D, U.S.A. I 0 5 6 I *Sera obtained as individual bleeds. tPurchased as commercially available antisera. f.Negative no to trace staining at I in 10 dilution, doubtfid weak but definite, positive bright staining. § Average titre 1000 (100-5000). Fig. 2. Smear of Nosema cuniculi organisms stained by indirect immunofluorescence using a positive serum. Line represents 50 11m. 302 J. C. COX AND D. PYE 23 days for presence of organisms in the urine were found to excrete N. cuniculi. Excretion, however, was sporadic, the strongest excretor only shedding organisms in about half the samples tested whilst for several of the rabbits only 3 of the 23 samples were positive, and often only 3 or 4 organisms were detected per smear. Subsequent studies on seropositive rabbits have confirmed that positive detection will occur on an average of only 1 in every 3 samples tested. Conversely, no organisms have been detected in 60 urine specimens from 25 seronegative rabbits of a similar age. Other serological investigations Calf and foetal calf sera were tested for antibody levels to determine their suitability in tissue culture media. Of 16 adult bovine sera tested, 5 were negative, 9 doubtful and 2 positive with a titre around 40 to 50. Pools of foetal sera were negative, but pools of adult sera were found to be doubtful or positive-thus precluding their use. Serological screening of a small number of human sera was also undertaken in view of the established role of N. cuniculi as a human pathogen (Connor, Strano & Neafie, 1974): 3 of 4 persons who had been working with rabbits for several years showed weak antibody levels, whereas 20 control individuals were negative. Stability of antigen Smears of cell-culture-grown N. cuniculi were air-dried, heat-fixed and stored at 4°C over silica gel for 4 months. On subsequent use with positive control sera they were found to give the same titre and staining intensity as freshly prepared smears. This retention of antigenicity by dried smears of N. cuniculi makes it possible for a large number of identical diagnostic preparations to be made at the one time. Freezing of spore suspensions did not affect antigenicity but was a less suitable storage procedure because of the resultant dissociation of the filament from the spore body. DISCUSSION An essential prerequisite to a serological test for nosematosis was an adequate supply of reasonably pure antigen. N. cuniculi organisms can be obtained from urine or ascitic fluid but suffer the disadvantage of sometimes having host antibody attached to the cell surface (Cox et al., I972). The in-vitro cultivation of N. cuniculi reported by Shadduck (1969) presented a procedure for obtaining large quantities of antigenically stable organisms. In the isolation studies reported here, urine was selected as a preferred source of organisms because it could be obtained from a living host and examined for presence of Nosema before inoculation into cell culture. Indirect immunofluorescence was used to examine urine for N. cuniculi because of the risk that the organism SERODIAGNOSIS OF NOSEMATOSIS 303 would not be detected by direct immunofluorescence if antibody was already attached in vivo. The canine embryo cell line used for the primary isolation grew slowly and maintained satisfactorily over 2 months, giving optimum conditions both for the isolation and adaptation of N. cuniculi. Furthermore, on subsequent transfer of organisms to a secondary host cell line any transferred canine cells died out rapidly. When examining cell-culture supernatants for early signs of infection, groups of organisms and those with extruded filaments were considered most significant. This was based on experience gained from our unreported attempts to induce an infection in a number of established canine epithelial-like cell lines. In these experiments, infectivity and growth of N. cuniculi was poor and immunofluorescence examination revealed single spores without extruded filaments in the majority. The lack of success with these epithelial-like cell lines together with trends suggested by the results of Montrey et 01. (1973) prompted the trials in human fibroblast-like cell lines reported here. Rabbit sera examined generally fell into 2 distinct groups: negative, where no fluorescence was apparent (confirmation of the presence of Nosema in the smear, if desired, could only be obtained by dark-field examination with tungsten light); positive, with strong fluorescence to an average titre around 1000. Examination of the urine of rabbits from these 2 groups for the presence of N.cuniculi has shown an excellent correlation between the immunofluorescence diagnosis and infection. There was also a 3rd group of rabbits which gave weak staining at a dilution of I in 10. Seronegative rabbits sent to this Institution often converted to this doubtful state after some weeks in an environment of high Nosema prevalence, but did not proceed to positive. It seems possible that in many cases this immunological state may be induced by inhalation or ingestion of non-infective organisms resulting in a low-level immune response. The results reported for rabbit sera from England and U.S.A. (Table 1) do not give a reliable estimate of prevalence of infection, for it is unknown whether they were single bleeds or pools. However, the titres were sufficiently high to suggest that if they were pools a significant percentage of the contributing bleeds would have been positive. The primary reason for their inclusion in this report is to show that N. cuniculi in Australian, English and American rabbits have a common antigenicity. Because antibodies were demonstrated in pools of adult but not of foetal bovine sera, the latter was used in all cell-culture experiments. The large number of weak or doubtful reactions may, as in the case of rabbits, be due to sensitization with non-infective Nosema, although their source is hard to suggest. A smaller number of positive sera showed a low but definite titre and could possibly be associated with a subclinical infection. In view of the 304 J. C. COX AND D. PYE high incidence of infection in our own rabbit colony, a serological examination of the staff in the animal areas was undertaken: I negative and 3 doubtful results were recorded. The antibody response in the latter probably arose by inhalation, as suggested earlier for rabbits. Indirect immunofluorescence using human fibroblast grown N. cuniculi as antigen is a very convenient and seemingly reliable diagnostic procedure. This technique wiIl assist in the establishment and maintenance of Nosemafree animal colonies, is a valuable research tool in studies of this parasite, and gives a potentially rapid and reliable method for the diagnosis of nosematosis III man. ACKNOWLEDGEMENTS We wish to thank Dr H. Ward and Mr J. Edmonds for gifts of sera, and Dr S. K. Sutherland and Dr B. J. Feery for encouragement and advice. 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