Laboratory AI/imals (1975) 9, 61-68.
61
GROWTH OF NOSEMA CUNICULI IN ESTABLISHED CELL LINES by
T. WALLER National Veterinary Institute, S-104 05 Stockholm
50, Sweden
SUMMARY
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 flOm Nosemainfected canine kidney cells during the 10th to 17th week aftel 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. Nosematosis (encephalitozoonosis) caused by the protozoan Nosema cuniculi (Encephalitozoon cuniculi) has been reported to attack several different animal species and man. It has often been found in laboratory animals, causing evaluation problems when these animals are used in experiments. Most of the reports dealing with the subject have been summarized by Petri (J 969) and Shadduck & Pakes (1971). Nosema-infected animals mostly show no or very slight clinical symptoms and it is therefore essential to have a diagnostic procedure for tracing and eradicating the disease in different stocks of laboratory animals. Immunological methods for clinical diagnosis of nosematosis have been described (Chalupsky, Bedrnik & Vavra, 1971; Cox, Walden & Nairn, 1972; Pakes, Shadduck & Olsen, 1972). A potential diagnostic method is the skin test (Pakes et al., 1972) which requires large amounts of antigen. The first report of propagation of Nosema cuniculi in cell culture was published by Shadduck (I 969) in rabbit choroid plexus celI cultures (RCP). Since then, growth of this organism in other cell types have been demonstrated (Bismanis, 1970; Bedrnik & Vavra, 1972; Huldt & Waller, 1974). It appears that the best method so far is that involving the Rep cells, but no information about the number of parasites produced in the celI cultures has been reported.
Downloaded from lan.sagepub.com by guest on March 28, 2015
62
T. WALLER
As the RCP cell cultures are very laborious to use, it was considered value to find a simple procedure for large-scale production of nosema and a simple method to determine the number of organisms. In reach this target, several cell lines and cell strains were tested and of nosema organisms was calculated. MATERIALS
of great antigen, order to the yield
AND METHODS
Strain of Nosema cuniculi The Nosema cuniculi strain used originated from a spontaneously Nosemainfected rabbit. For isolation of the parasite, rabbit brain was homogenized and diluted to 1/2 in Eagle's minimal essential medium (Eagle MEM cellculture medium). 0,3-0,5 ml of the suspension was injected intraperitoneally in mice of a NMRI strain known to be free from Nosema cuniculi infections. After 5-8 weeks, when maximal abdominal swelling was observed, ascites fluid containing Nosema organisms was collected from the mice with a sterile syringe and inoculated on monolayer cell cultures of rabbit kidney cells (RK 13). All Nosema organisms used in the present work were originally cultured in RK ] 3 cells. Identification
of Nosema cuniculi
Tissue sections, smears and cell cultures on cover slips were fixed in 10% formalin and Gram-stained or stained according to Wright & Craighead (1922). The Nosema spores were ovoid with a diameter of ] ·5-2·5 !lm, Gram-positive and stained red with the method of Wright & Craighead. These characteristics correspond to the morphology and staining reactions for Nosema cuniculi (M0ller, ] 968). Selection of cell cultures 8 commercially available established cell lines and 2 cell strains established at the National Veterinary Institute, Stockholm, were tested for their ability to support growth of Nosema cuniculi. Each monolayer cell culture was grown on cover slips in 10 Leighton tubes (FBG-Trident Ltd, Herringham Road, London SE7 8NN, u.K.) which were inoculated with equal amounts of newly harvested Nosema spores. The infection rates of the cells were tested on day 3, 4, 5, 6 and ] 0 after inoculation by examin'ing 2 cover slips on each occasion. The cultures were fixed in 10% formalin and stained according to Wright & Craighead (1922). The number of intracellular Nosema colonies containing a minimum of 10 spores were registered on each cover slip (Fig. 3). The total number of spores in 10 adjacent colonies (Nt) and the number of free spores on the surface in 10 microscopic fields (Nf) were counted (Table 1).
Downloaded from lan.sagepub.com by guest on March 28, 2015
GROWTH
OF NOSEMA
63
CUNICULI
Table 1. Patterns of Nosema cuniculi propagation in monolayer cell cultures of bovine kidney (BK), feline lung (FL), and rabbit kidney (RK 13) grown on cover slips in Leighton tubes. Nt total number of Nosema organisms in 10 adjacent intracellular colonies. Nt number of free spores in 10 microscopic fields.
FL
Cell culture
BK
RK13
Day
Nt
Nt
Nt
Nt
Nt
N(
3 4 5
520 530 1415 1440 1380
13 19 21 35 31
270 330 710 440 890
2 7 7 9 19
290 350 410 580 850
6 9 7 23 15
6
10
Cell cultures and cell culture mediums 4 different cell types which supported growth of Nosema cuniculi are included in this report. Established cell lines of canine kidney cells (MDCK), feline lung cells (FL) and rabbit kidney cells (RK 13) were obtained from Flow Laboratories, Victoria Park, Heatherhouse Road, Irvine, KA12 8NB, U.K., while secondary bovine kidney cell cultures (BK) were from the National
Veterinary Institute, Stockholm.
The growth medium for BK, MDCK and
RK 13 cells was medium 199 with 5 % foetal calf serum and the maintenance medium was 199 with 1 % foetal calf serum. The growth medium for FL cells was Eagle MEM with 5 % foetal calf serum and as maintenance medium Eagle MEM with 1 % foetal calf serum was used. Penicillin, streptomycin sulphate and tylosin were supplemented to all cell culture mediums at the concentrations of 100 i.u., 300 I1g and ]0 I1g per mt respectively. Production and harvesting of Nosema organisms Newly trypsinated monolayer cell cultures of FL and MDCK cells were inoculated with 50900 to lOO 000 Nosema spores per em 2 in plastic flasks (Falcon Plastics, 1950 Williams Drive, Oxnard, California 93030, U.S.A.) with a bottom area of 75 em 2. If the cell mat decreased to cover only about 30 % of the bottom surface, new cells were added to the flask. Cell culture fluid from the Nosema-infected cell cultures was harvested twice a week and replaced with maintenance medium. The number of Nosema organisms in the harvested fluid was determined as described below. Estimation of the number of Nosema organisms The number of nosema organisms in harvested cell culture fluid of infected cell cultures was determined according to the principles of Prescott & Breed (J 910) for counting cells in milk. Using a micropipette, 0·01 ml of the flUid was placed on a slide and spread over an area of 1 em 2. Duplicate pre para-
Downloaded from lan.sagepub.com by guest on March 28, 2015
T. WALLER
64
tions were made from each harvest. The slides were allowed to dry on a level surface and were then Gram stained. The counting was carried out under oil immersion using a magnification of 1000 X. The diameter and area of the microscopic field were O'J 6 mm and 0,02 mm 2 respectively. The microscope 1 x J 00 (Little & Plastridge, 1946). On each speci0·00020 men 40 microscopic fields were counted, giving a working factor (WF) of factor (MF) was
12500 (WF= MF).
The Nosema concentration
per mt of fluid (Nc) was cat-
40 culated from the formula organisms counted.
Nc=WF
X
n, where n=the
number
of Nosema
RESULTS
Growth patterns of Nosema cuniculi in different cell cultures All cell lines and cell strains tested were found to support growth of Nosema cuniculi. When grown in Leighton tubes and plastic flasks the Nosema organisms appeared in intracellular colonies and as free spores on the surface of the monolayer cell cultures (Figs 1 and 2).
Fig. 1. 8-week-old cat-lung (FL) cell cultures grown in plastic flasks. Left Nosema-infected. Right control. Phase contrast. Line represents 50 Ilm.
Downloaded from lan.sagepub.com by guest on March 28, 2015
GROWTH OF NOSEMA
CUNICULI
65
Fig. 2. 17-week-old dog-kidney (MOCK) cell cultures grown in plastic flasks. Left Nosemainfected. Right control. Phase contrast. Line represents 50 J.lm.
3 cell cultures, bovine kidney (BK), feline lung (FL) and rabbit kidney (RK 13). have been selected to show the patterns of Nosema propagation when grown in Leighton tubes. Most colonies were found in the FL cells with 780 colonies on day 3 after inoculation and with a maximum of 2500 colonies on day 10 (Fig. 3). The largest colonies (Nt) were found ill· the FL cells with a mean of about ]400 spores per 10 colonies on days 5, 6 and IO (Table I). Fewer and smaller colonies were found in the BK and RK I3 cells. The largest number of free spores (Nf) were found in the FL cells with 13-35 spores per 10 microscopic fields (Table I). The corresponding numbers in the BK and RK 13 cells were 2-]9 and 6-23 respectively. Production of Nosema cuniculi 2 cell lines, FL and MDCK, were selected to obtain a method for largescale production of Nosema organisms. The weekly output of Nosema spores from each cell line is represented by the mean value of 3 identically handled, plastic flasks (Fig. 4). By the 6th week the harvest of Nosema spores from the FL cells had increased to 1·6 X 10 7 and from week 8 to week 17 the number of harvested organisms kept between 1·1 X 10 7 and 6,3 X 10 7. The yield from the MDCK cells was increasing at week 8 and during the 10th and 17th
Downloaded from lan.sagepub.com by guest on March 28, 2015
66
T. WALLER
2500
FL
2000
1500 I/)
Q)
'c0
••••
....... RK 13
'0
BK ••••••••
u
.0
, ------ ---x
1000
,: ,: ,: ,: ,: ,: ,x::. , , .: , , .f' x---x .: ..... .
800
600
400
200
00 •••• 0
o
2
3
4
5 6 Days
7
8
9
10
Fig. 3. Total number of intracellular Nosema colonies in monolayer cell cultures of bovine kidney (BK), feline lung (FL), and rabbit kidney (RK 13) grown on cover slips in Leighton tubes.
Downloaded from lan.sagepub.com by guest on March 28, 2015
GROWTH OF NOSEMA
CUNICULI
67
weeks the values varied between 4·1 X 10 7 and 9,9 x 10 7 Nosema organisms per flask. The FL cells had to be supplemented with new cells about once or twice a month (see Materials and Methods). There was no need to add cells to the MDCK cells during a period of 4 months .
,•
-.....•'
1\ I
~
,~ 1Il
•.. o
f
III
\ \
\.Fl
,,
,
c.
\.
••••
I
(/) 107
I
,\
\,
, !\...,
..
V
106 :.cOo_--.._
...•.... _
2
•... , ---._
4
....•..., _.--
6
...•.., _.--
...•.., _....-~
8
10
_
12
14
16
Weeks Fig. 4. Weekly output of Nosema cuniculi spores from infected monolayer cell cultures of feline lung (FL) and canine kidney (MOCK) grown in plastic flasks with a bottom area of7Scm2• DISCUSSION
The results of this study show that Nosema cuniculi can infect cultured cells originating from several animal species. This is contradictory to the results of Shadduck & Pakes (1971) who tested several cell cultures, not specified, in which they did not obtain propagation of the parasite. These contradictory results might be explained by the choice of cell cultures. However, the present study has shown that all cell lines and cell strains tested became infected when they were inoculated with large numbers of newly harvested spores. It was also found that it takes a considerable time for the infection to spread in the cell cultures. Therefore it must be possible to maintain a successful
Downloaded from lan.sagepub.com by guest on March 28, 2015
68
T. WALLER
cell line for several weeks, to give the infection the opportunity to increase to an optimal level. The results indicate that MDCK is a suitable cell line for propagation of large amounts of Nosema organisms when the method described above is used, thereby furnishing the quantity of Nosema spores needed to perform skin tests according to Pakes et al. (1972). An equilibrium was obtained between the infection and the MDCK cells where the destroyed cells were continuously and autonomously replaced by new cells. The FL cells are an alternative to the MDCK cells for production of large amounts of Nosema organisms, but they were more laborious to manage as new cells had to be added to the cultures at certain intervals. The differences in this respect between the FL and MDCK cells might be due to the split ratio of the cells which is low for the FL cell~ and high for the MDCK cells. The method of counting the Nosema organisms described was chosen since it is simple to perform and has the advantage over celloscope counting that each single organism counted is identified and the harvests can be determined without previous purification. ACKNOWLEDGEMENT
This work was supported by grants 3646 and 3940 from the Swedish Medical Research Council.
REFERENCES Bedrnik, P. & Vavra, J. (1972). Further observations on the maintenance of Encephalitozoon cuniculi in tissue culture. Journal of Protozoology 19, Suppl. 75. Bismanis, J. E. (1970). Detection of latent murine nosematosis and growth of Nosema cuniculi in cell cultures. Canadian Journal of Microbiology 16, 237-242. Chalupsky, J., Bedrnik, P. & Vavra, J. (1971). The indirect fluorescent antibody test for Nosema cuniculi. Journal of Protozoology 18, Suppl. 177. Cox, J. C., Walden, N. B. & Nairn, R. C. (1972). Presumptive diagnosis of Nosema cuniculi in rabbits by immunofluorescence. Research in Veterinary Science 13, 595-597. Huldt, G. & Waller, T. (1974). Accidental nosematosis in mice with impaired immunological competence. Acta pathologica et microbiologica scandinavica Section B 82, 451-452. Little, R. B. & Plastridge, W. N. (1946). Bovine mastitis, pp. 504-505. New York & London: McGraw-Hill. M0ller, T. (1968). A survey on toxoplasmosis and encephalitozoonosis in laboratory animals. Zeitschrift fiir Versuchstierkunde 10, 27-38. Pakes, S. P., Shadduck, J. A. & Olsen, R. G. (1972). A diagnostic skin test for encephalitozoonosis (nosematosis) in rabbits. Laboratory Animal Science 22, 870-877. Petri, M. (1969). Studies on Nosema cuniculi found in transplantable ascites tumours with a survey of microsporidiosis in mammals. Acta pathologica et microbiologica scandinavica Suppl. 204. Prescott, S. C. & Breed, R. S. (1910). The determination of the number of body cells in milk by a direct method. Journal of Infectious Diseases 7, 632-640. Shadduck, J. A. (1969). Nosema cuniculi: in vitro isolation. Science, New York 166,516-517. Shadduck, J. A. & Pakes, S. P. (1971). Encephalitozoonosis (nosematosis) and toxoplasmosis. American Journal of Pathology 64, 657-674. Wright, H. J. & Craighead, E. M. (1922). Infectious motor paralysis in young rabbits. Journal of Experimental Medicine 36, 135-140.
Downloaded from lan.sagepub.com by guest on March 28, 2015
61
GROWTH OF NOSEMA CUNICULI IN ESTABLISHED CELL LINES by
T. WALLER National Veterinary Institute, S-104 05 Stockholm
50, Sweden
SUMMARY
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 flOm Nosemainfected canine kidney cells during the 10th to 17th week aftel 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. Nosematosis (encephalitozoonosis) caused by the protozoan Nosema cuniculi (Encephalitozoon cuniculi) has been reported to attack several different animal species and man. It has often been found in laboratory animals, causing evaluation problems when these animals are used in experiments. Most of the reports dealing with the subject have been summarized by Petri (J 969) and Shadduck & Pakes (1971). Nosema-infected animals mostly show no or very slight clinical symptoms and it is therefore essential to have a diagnostic procedure for tracing and eradicating the disease in different stocks of laboratory animals. Immunological methods for clinical diagnosis of nosematosis have been described (Chalupsky, Bedrnik & Vavra, 1971; Cox, Walden & Nairn, 1972; Pakes, Shadduck & Olsen, 1972). A potential diagnostic method is the skin test (Pakes et al., 1972) which requires large amounts of antigen. The first report of propagation of Nosema cuniculi in cell culture was published by Shadduck (I 969) in rabbit choroid plexus celI cultures (RCP). Since then, growth of this organism in other cell types have been demonstrated (Bismanis, 1970; Bedrnik & Vavra, 1972; Huldt & Waller, 1974). It appears that the best method so far is that involving the Rep cells, but no information about the number of parasites produced in the celI cultures has been reported.
Downloaded from lan.sagepub.com by guest on March 28, 2015
62
T. WALLER
As the RCP cell cultures are very laborious to use, it was considered value to find a simple procedure for large-scale production of nosema and a simple method to determine the number of organisms. In reach this target, several cell lines and cell strains were tested and of nosema organisms was calculated. MATERIALS
of great antigen, order to the yield
AND METHODS
Strain of Nosema cuniculi The Nosema cuniculi strain used originated from a spontaneously Nosemainfected rabbit. For isolation of the parasite, rabbit brain was homogenized and diluted to 1/2 in Eagle's minimal essential medium (Eagle MEM cellculture medium). 0,3-0,5 ml of the suspension was injected intraperitoneally in mice of a NMRI strain known to be free from Nosema cuniculi infections. After 5-8 weeks, when maximal abdominal swelling was observed, ascites fluid containing Nosema organisms was collected from the mice with a sterile syringe and inoculated on monolayer cell cultures of rabbit kidney cells (RK 13). All Nosema organisms used in the present work were originally cultured in RK ] 3 cells. Identification
of Nosema cuniculi
Tissue sections, smears and cell cultures on cover slips were fixed in 10% formalin and Gram-stained or stained according to Wright & Craighead (1922). The Nosema spores were ovoid with a diameter of ] ·5-2·5 !lm, Gram-positive and stained red with the method of Wright & Craighead. These characteristics correspond to the morphology and staining reactions for Nosema cuniculi (M0ller, ] 968). Selection of cell cultures 8 commercially available established cell lines and 2 cell strains established at the National Veterinary Institute, Stockholm, were tested for their ability to support growth of Nosema cuniculi. Each monolayer cell culture was grown on cover slips in 10 Leighton tubes (FBG-Trident Ltd, Herringham Road, London SE7 8NN, u.K.) which were inoculated with equal amounts of newly harvested Nosema spores. The infection rates of the cells were tested on day 3, 4, 5, 6 and ] 0 after inoculation by examin'ing 2 cover slips on each occasion. The cultures were fixed in 10% formalin and stained according to Wright & Craighead (1922). The number of intracellular Nosema colonies containing a minimum of 10 spores were registered on each cover slip (Fig. 3). The total number of spores in 10 adjacent colonies (Nt) and the number of free spores on the surface in 10 microscopic fields (Nf) were counted (Table 1).
Downloaded from lan.sagepub.com by guest on March 28, 2015
GROWTH
OF NOSEMA
63
CUNICULI
Table 1. Patterns of Nosema cuniculi propagation in monolayer cell cultures of bovine kidney (BK), feline lung (FL), and rabbit kidney (RK 13) grown on cover slips in Leighton tubes. Nt total number of Nosema organisms in 10 adjacent intracellular colonies. Nt number of free spores in 10 microscopic fields.
FL
Cell culture
BK
RK13
Day
Nt
Nt
Nt
Nt
Nt
N(
3 4 5
520 530 1415 1440 1380
13 19 21 35 31
270 330 710 440 890
2 7 7 9 19
290 350 410 580 850
6 9 7 23 15
6
10
Cell cultures and cell culture mediums 4 different cell types which supported growth of Nosema cuniculi are included in this report. Established cell lines of canine kidney cells (MDCK), feline lung cells (FL) and rabbit kidney cells (RK 13) were obtained from Flow Laboratories, Victoria Park, Heatherhouse Road, Irvine, KA12 8NB, U.K., while secondary bovine kidney cell cultures (BK) were from the National
Veterinary Institute, Stockholm.
The growth medium for BK, MDCK and
RK 13 cells was medium 199 with 5 % foetal calf serum and the maintenance medium was 199 with 1 % foetal calf serum. The growth medium for FL cells was Eagle MEM with 5 % foetal calf serum and as maintenance medium Eagle MEM with 1 % foetal calf serum was used. Penicillin, streptomycin sulphate and tylosin were supplemented to all cell culture mediums at the concentrations of 100 i.u., 300 I1g and ]0 I1g per mt respectively. Production and harvesting of Nosema organisms Newly trypsinated monolayer cell cultures of FL and MDCK cells were inoculated with 50900 to lOO 000 Nosema spores per em 2 in plastic flasks (Falcon Plastics, 1950 Williams Drive, Oxnard, California 93030, U.S.A.) with a bottom area of 75 em 2. If the cell mat decreased to cover only about 30 % of the bottom surface, new cells were added to the flask. Cell culture fluid from the Nosema-infected cell cultures was harvested twice a week and replaced with maintenance medium. The number of Nosema organisms in the harvested fluid was determined as described below. Estimation of the number of Nosema organisms The number of nosema organisms in harvested cell culture fluid of infected cell cultures was determined according to the principles of Prescott & Breed (J 910) for counting cells in milk. Using a micropipette, 0·01 ml of the flUid was placed on a slide and spread over an area of 1 em 2. Duplicate pre para-
Downloaded from lan.sagepub.com by guest on March 28, 2015
T. WALLER
64
tions were made from each harvest. The slides were allowed to dry on a level surface and were then Gram stained. The counting was carried out under oil immersion using a magnification of 1000 X. The diameter and area of the microscopic field were O'J 6 mm and 0,02 mm 2 respectively. The microscope 1 x J 00 (Little & Plastridge, 1946). On each speci0·00020 men 40 microscopic fields were counted, giving a working factor (WF) of factor (MF) was
12500 (WF= MF).
The Nosema concentration
per mt of fluid (Nc) was cat-
40 culated from the formula organisms counted.
Nc=WF
X
n, where n=the
number
of Nosema
RESULTS
Growth patterns of Nosema cuniculi in different cell cultures All cell lines and cell strains tested were found to support growth of Nosema cuniculi. When grown in Leighton tubes and plastic flasks the Nosema organisms appeared in intracellular colonies and as free spores on the surface of the monolayer cell cultures (Figs 1 and 2).
Fig. 1. 8-week-old cat-lung (FL) cell cultures grown in plastic flasks. Left Nosema-infected. Right control. Phase contrast. Line represents 50 Ilm.
Downloaded from lan.sagepub.com by guest on March 28, 2015
GROWTH OF NOSEMA
CUNICULI
65
Fig. 2. 17-week-old dog-kidney (MOCK) cell cultures grown in plastic flasks. Left Nosemainfected. Right control. Phase contrast. Line represents 50 J.lm.
3 cell cultures, bovine kidney (BK), feline lung (FL) and rabbit kidney (RK 13). have been selected to show the patterns of Nosema propagation when grown in Leighton tubes. Most colonies were found in the FL cells with 780 colonies on day 3 after inoculation and with a maximum of 2500 colonies on day 10 (Fig. 3). The largest colonies (Nt) were found ill· the FL cells with a mean of about ]400 spores per 10 colonies on days 5, 6 and IO (Table I). Fewer and smaller colonies were found in the BK and RK I3 cells. The largest number of free spores (Nf) were found in the FL cells with 13-35 spores per 10 microscopic fields (Table I). The corresponding numbers in the BK and RK 13 cells were 2-]9 and 6-23 respectively. Production of Nosema cuniculi 2 cell lines, FL and MDCK, were selected to obtain a method for largescale production of Nosema organisms. The weekly output of Nosema spores from each cell line is represented by the mean value of 3 identically handled, plastic flasks (Fig. 4). By the 6th week the harvest of Nosema spores from the FL cells had increased to 1·6 X 10 7 and from week 8 to week 17 the number of harvested organisms kept between 1·1 X 10 7 and 6,3 X 10 7. The yield from the MDCK cells was increasing at week 8 and during the 10th and 17th
Downloaded from lan.sagepub.com by guest on March 28, 2015
66
T. WALLER
2500
FL
2000
1500 I/)
Q)
'c0
••••
....... RK 13
'0
BK ••••••••
u
.0
, ------ ---x
1000
,: ,: ,: ,: ,: ,: ,x::. , , .: , , .f' x---x .: ..... .
800
600
400
200
00 •••• 0
o
2
3
4
5 6 Days
7
8
9
10
Fig. 3. Total number of intracellular Nosema colonies in monolayer cell cultures of bovine kidney (BK), feline lung (FL), and rabbit kidney (RK 13) grown on cover slips in Leighton tubes.
Downloaded from lan.sagepub.com by guest on March 28, 2015
GROWTH OF NOSEMA
CUNICULI
67
weeks the values varied between 4·1 X 10 7 and 9,9 x 10 7 Nosema organisms per flask. The FL cells had to be supplemented with new cells about once or twice a month (see Materials and Methods). There was no need to add cells to the MDCK cells during a period of 4 months .
,•
-.....•'
1\ I
~
,~ 1Il
•.. o
f
III
\ \
\.Fl
,,
,
c.
\.
••••
I
(/) 107
I
,\
\,
, !\...,
..
V
106 :.cOo_--.._
...•.... _
2
•... , ---._
4
....•..., _.--
6
...•.., _.--
...•.., _....-~
8
10
_
12
14
16
Weeks Fig. 4. Weekly output of Nosema cuniculi spores from infected monolayer cell cultures of feline lung (FL) and canine kidney (MOCK) grown in plastic flasks with a bottom area of7Scm2• DISCUSSION
The results of this study show that Nosema cuniculi can infect cultured cells originating from several animal species. This is contradictory to the results of Shadduck & Pakes (1971) who tested several cell cultures, not specified, in which they did not obtain propagation of the parasite. These contradictory results might be explained by the choice of cell cultures. However, the present study has shown that all cell lines and cell strains tested became infected when they were inoculated with large numbers of newly harvested spores. It was also found that it takes a considerable time for the infection to spread in the cell cultures. Therefore it must be possible to maintain a successful
Downloaded from lan.sagepub.com by guest on March 28, 2015
68
T. WALLER
cell line for several weeks, to give the infection the opportunity to increase to an optimal level. The results indicate that MDCK is a suitable cell line for propagation of large amounts of Nosema organisms when the method described above is used, thereby furnishing the quantity of Nosema spores needed to perform skin tests according to Pakes et al. (1972). An equilibrium was obtained between the infection and the MDCK cells where the destroyed cells were continuously and autonomously replaced by new cells. The FL cells are an alternative to the MDCK cells for production of large amounts of Nosema organisms, but they were more laborious to manage as new cells had to be added to the cultures at certain intervals. The differences in this respect between the FL and MDCK cells might be due to the split ratio of the cells which is low for the FL cell~ and high for the MDCK cells. The method of counting the Nosema organisms described was chosen since it is simple to perform and has the advantage over celloscope counting that each single organism counted is identified and the harvests can be determined without previous purification. ACKNOWLEDGEMENT
This work was supported by grants 3646 and 3940 from the Swedish Medical Research Council.
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