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Dynamics of faecal egg counts in rabbits experimentally infected with Strongyloides papillosus
Veterinary Parasitology, 39 ( 1991 ) 3 3 3 - 3 3 6 Elsevier Science Publishers B.V., A m s t e r d a m
333
Short Communication Dynamics of faecal egg counts in rabbits experimentally infected with Strongyloides
papillosus N. Taira a, T.
M i n a m i a a n d J. S m i t a n o n b
aFirst Research Division of National Institute of Animal Health, Tsukuba, Ibaraki 305, Japan bNational Animal Health and Production Institute, Department of Livestock Development, Bangkok 10900, Thailand (Accepted 28 February 1991 )
ABSTRACT Taira, N., Minami, T. and Smitanon, J., 1991. Dynamics of faecal egg counts in rabbits experimentally infected with Strongyloides papillosus. Vet. Parasitol., 39: 333-336. An experiment using rabbits was carried out to determine basic data to maintain the calf strain of
Strongyloides papillosus (SPL). Rabbits treated with four different numbers of doses of dexamethasone were exposed to 50 000 or about 100 000 infective larvae of SPL (Himeji strain, 1988). As a result, faecal egg output was valid in individual rabbits, regardless of dexamethasone administration. Eggs appeared in the faeces 9 or 10 days after exposure, and increased to as much as 20 000-573 000 eggs per gram (EPG). Thirteen of 18 rabbits died (72%) 12-29 days after infection. Five surviving rabbits discharged eggs in their faeces for more than 1 month; however, the EPG value of these cases on the last day was comparatively low.
INTRODUCTION
Several researchers at the University of Warsaw have studied rabbits infected with Strongyloides papillosus (SPL) with respect to serial passage (Bezubik, 1962, 1965; Bezubik and Turner, 1964; Jaron, 1964; Stankiewicz, 1965 ), effect of sex and age (Chomicz, 1969, 1984), immunity (Sinski and Krzysztofowics, 1971; Stankiewicz et al., 1971 ) and behaviour (Nwaorgu and Connan, 1980). On the other hand, strongyloidiasis in calves has recently become a serious problem in Japan (Taira and Ura, 1991 ). To promote the study of this infection, it is important to maintain the strain of SPL using small animals such as rabbits. The present study was aimed at observing the duration of faecal egg output in rabbits exposed to SPL and also at evaluating the effect of dexamethasone (DEXA) treatment. 0 3 0 4 - 4 0 1 7 / 9 1 / $ 0 3 . 5 0 © 1991 Elsevier Science Publishers B.V. All rights reserved.
334
N. TAIRA ET AE.
MATERIALS AND METHODS
Rabbits and experimental design Eighteen male albino rabbits aged 3-5 months were reared individually in separate cages. They were injected subcutaneously with 0.1 ml kg-~ body weight day-~ of DEXA (20 mg/20 ml) during the experiment. Two or four rabbits were allotted to each of Groups A (rabbit Nos. 1 and 2), B (Nos. 3-6), C (Nos. 7-10), D (Nos. 11-14) and E (Nos. 15-18). The rabbits of Group A received DEXA for 7 days a week. Groups B, C and D received DEXA for 3 days, 2 days and 1 day a week, respectively. Group E did not receive DEXA.
Source of infective SPL larvae and the faecal culture The original SPL larvae were obtained from the faeces of a calf at a farm in Himeji, Hyogo prefecture (Himeji strain, 1988 ), and subsequently passaged in rabbits. In the culture procedure, a faecal suspension in water was sieved with a 100 mesh net and the filtrate was filtered with absorbent cotton wool layered 3 cm deep. The wet cotton wool containing SPL eggs was packed in a polyethylene tube, incubated at 25°C for 3 or 4 days, and kept at 20°C until use. The larvae used in the experimental infection were derived from rabbits (after 1-4 passages) and exposed within 7 days of culture.
Infection and faecal egg counts The number of infective larvae was estimated by two methods. One counted the larvae in the suspension before exposure; the other counted not the larvae, but the eggs in the suspension before larval culture. A total of 50 000 larvae by the former method, which was an accurate count, were exposed to rabbits Nos. 5-7 and No. 10. The other rabbits were exposed to an estimated 100 000 eggs/larvae by the latter method. The wet cotton wool containing the SPL larvae was placed in a bucket. The rabbits were then exposed by being placed in the bucket for 5 h. Faecal examination was carried out on each rabbit every day from the day of exposure to the time of death or killing. Fresh wet faeces were examined by the modified McMaster technique. The egg count (EPG) was determined from the number of eggs detected, multiplied by 200.
335
RABBITS INFECTED WITH STRONGYLOIDES PAPILLOSUS
RESULTS
Termination and faecal egg output
Thirteen of 18 infected rabbits (72%) died 12-29 days after exposure. All rabbits in Groups A and D died 14-27 days after exposure; however, five rabbits among Groups B, C and E survived (Fig. 1 ). Eggs appeared in the faeces 9 or 10 days after exposure in all rabbits. The highest EPG values in each rabbit were 189 600 (rabbit No. 1 ), 71 200 (No. 2), 234 200 (No. 3), 573 000 (No. 4), 25 600 (No. 5), 212 800 (No. 6), 20 000 (No. 7), 530 000 (No. 8), 430 000 (No. 9), 40 000 (No. 10), 496 000 (No. 11), 553 000 (No. 12), 530 000 (No. 13), 380 000 (No. 14), 217 000 (No. 15), 524 000 (No. 16), 330 400 (No. 17) and 168 000 (No. 18). Survivor rabbits, Nos. 5, 6, 10, 17 and 18, had been discharging the eggs for more
EPG [, OOO,000 100, 000 10,000 1,000
, ~ i(]4)~ 2(16)
i, 000,000 100,000 10,000
_
//.i:I I
.-"']4
12,1..-
......
3)
1,000 1,000, 000 lO0,000 10,000 1,000
Group B
,,x t<..." 9{29) ~ -.,.j-~ t. :~ .: %~, r'
t~.~
: .;- ~_L;. . ~ - - - " . . . . . . . . . . . . .
/i~..-'"~ I,~,"7( 12)
1,000, 000 100,000 tO,000
v
..,,.--.---,
8(2~)
5... lO "',
G,rou p C
",
i,$<~ ,; ..'..~ ........13(27)
il?(IYI
~'!,[
t:" j f1,~1(2o),
I, OOO 1,000,000 100, 000 10,000 1,000 100
Group A
~
,
-
t
Group D
16(24)
iroup ~' 18
X..
5
10
, i'"'-"','-'""'""y';
15 20 25 30 35 40 4-5 50 Days
after
exposure
Fig. 1. Dynamics of the egg output in rabbits exposed to Strongyloides papillosus, aRabbit No. with number of days after exposure when it died shown in parentheses.
336
N. TAIRA ET AL.
than 1 month after exposure. Rabbit No. 6 discharged the eggs for more than 52 days ( E P G = 10 100) after exposure. DISCUSSION
In some reports (Bezubik and Turner, 1964; Jaron, 1964), rabbits exposed to 100 000 or 200 000 SPL larvae showed a high EPG, e.g. 100 000-300 0000, and died 13-95 days after infection. These data essentially agree with the present findings. Differentiation of susceptibility among rabbits in each group was not clear. The results indicate that rabbits are highly susceptible to the Japanese calf strain of SPL used, regardless of DEXA administration; however, the egg output was valid in each infected rabbit exposed to the same number of larvae. Above all, we consider that two or three rabbits are needed for one passage and that it should be repeated every 2 or 3 weeks for the maintenance of SPL.
REFERENCES Bezubik, B., 1962. Investigation on the sheep strain ofStrongyloides papillosus II. Adaptability to abnormal hosts and changes in virulence after serial passages in rabbits. Acta Parasitol. Pol., 10: 321-346. Bezubik, B., 1965. Failure to establish infection in rats and guinea pigs exposed to the larvae of Strongyloides papillosus. Acta Parasitol. Pol., 8: 349-354. Bezubik, B. and Turner, H., 1964. Investigations on the sheep strain ofStrongyloides papillosus IV. The physiological and pathological blood picture of sheep and goats experimentally infected with S. papillosus after 5 or 7 serial passages through rabbits. Acta Parasitol. Pol., 12: 101-116. Chomicz, L., 1969. Further studies on the blood picture depending on sex and age of rabbits experimentally infected with Strongyloides papillosus. Acta Parasitol. Pol., 17: 47-54. Chomicz, L., 1984. The effect ofgonadectomy and sex hormones on the course of experimental strongyloidosis (sheep strain) in rabbits. I. Single infections. Acta Parasitol. Pol., 39: 149166. Jaron, W., 1964. Immunity reaction of the rabbit against sheep and rabbit strains of Strongyloides papillosus in repeated infections. Acta Parasitol. Pol., 12:133-149. Nwaorgu, O.C. and Connan, R.M., 1980. The migration of Strongyloides papillosus in rabbits following infection by the oral and subcutaneous routes. J. Helminthol., 54: 223-232. Sinski, E. and Krzysztofowics, M.M., 1971. The blood picture and immunological reactions in rabbits exposed to repeated doses of the sheep and rabbit strains ofStrongyloides papillosus. Acta Parasitol. Pol., 19: 427-444. Stankiewicz, M., 1965. The blood picture of rabbits experimentally infected with Strongyloides papillosus after 5 serial passages through rabbits. Acta Parasitol. Pol., 13: 355-365. Stankiewicz, M., Bezubik, B. and Sinski, E., 1971. The effect of UV-rays on the infective larvae of Strongyloides papillosus in the rabbit. III. Production of immunity by irradiated larvae. Acta Parasitol. Pol., 19: 327-336. Taira, N. and Ura, S., 1991. Sudden death in calves associated with Strongyloides papillosus infection. Vet. Parasitol., 39:313-319.
333
Short Communication Dynamics of faecal egg counts in rabbits experimentally infected with Strongyloides
papillosus N. Taira a, T.
M i n a m i a a n d J. S m i t a n o n b
aFirst Research Division of National Institute of Animal Health, Tsukuba, Ibaraki 305, Japan bNational Animal Health and Production Institute, Department of Livestock Development, Bangkok 10900, Thailand (Accepted 28 February 1991 )
ABSTRACT Taira, N., Minami, T. and Smitanon, J., 1991. Dynamics of faecal egg counts in rabbits experimentally infected with Strongyloides papillosus. Vet. Parasitol., 39: 333-336. An experiment using rabbits was carried out to determine basic data to maintain the calf strain of
Strongyloides papillosus (SPL). Rabbits treated with four different numbers of doses of dexamethasone were exposed to 50 000 or about 100 000 infective larvae of SPL (Himeji strain, 1988). As a result, faecal egg output was valid in individual rabbits, regardless of dexamethasone administration. Eggs appeared in the faeces 9 or 10 days after exposure, and increased to as much as 20 000-573 000 eggs per gram (EPG). Thirteen of 18 rabbits died (72%) 12-29 days after infection. Five surviving rabbits discharged eggs in their faeces for more than 1 month; however, the EPG value of these cases on the last day was comparatively low.
INTRODUCTION
Several researchers at the University of Warsaw have studied rabbits infected with Strongyloides papillosus (SPL) with respect to serial passage (Bezubik, 1962, 1965; Bezubik and Turner, 1964; Jaron, 1964; Stankiewicz, 1965 ), effect of sex and age (Chomicz, 1969, 1984), immunity (Sinski and Krzysztofowics, 1971; Stankiewicz et al., 1971 ) and behaviour (Nwaorgu and Connan, 1980). On the other hand, strongyloidiasis in calves has recently become a serious problem in Japan (Taira and Ura, 1991 ). To promote the study of this infection, it is important to maintain the strain of SPL using small animals such as rabbits. The present study was aimed at observing the duration of faecal egg output in rabbits exposed to SPL and also at evaluating the effect of dexamethasone (DEXA) treatment. 0 3 0 4 - 4 0 1 7 / 9 1 / $ 0 3 . 5 0 © 1991 Elsevier Science Publishers B.V. All rights reserved.
334
N. TAIRA ET AE.
MATERIALS AND METHODS
Rabbits and experimental design Eighteen male albino rabbits aged 3-5 months were reared individually in separate cages. They were injected subcutaneously with 0.1 ml kg-~ body weight day-~ of DEXA (20 mg/20 ml) during the experiment. Two or four rabbits were allotted to each of Groups A (rabbit Nos. 1 and 2), B (Nos. 3-6), C (Nos. 7-10), D (Nos. 11-14) and E (Nos. 15-18). The rabbits of Group A received DEXA for 7 days a week. Groups B, C and D received DEXA for 3 days, 2 days and 1 day a week, respectively. Group E did not receive DEXA.
Source of infective SPL larvae and the faecal culture The original SPL larvae were obtained from the faeces of a calf at a farm in Himeji, Hyogo prefecture (Himeji strain, 1988 ), and subsequently passaged in rabbits. In the culture procedure, a faecal suspension in water was sieved with a 100 mesh net and the filtrate was filtered with absorbent cotton wool layered 3 cm deep. The wet cotton wool containing SPL eggs was packed in a polyethylene tube, incubated at 25°C for 3 or 4 days, and kept at 20°C until use. The larvae used in the experimental infection were derived from rabbits (after 1-4 passages) and exposed within 7 days of culture.
Infection and faecal egg counts The number of infective larvae was estimated by two methods. One counted the larvae in the suspension before exposure; the other counted not the larvae, but the eggs in the suspension before larval culture. A total of 50 000 larvae by the former method, which was an accurate count, were exposed to rabbits Nos. 5-7 and No. 10. The other rabbits were exposed to an estimated 100 000 eggs/larvae by the latter method. The wet cotton wool containing the SPL larvae was placed in a bucket. The rabbits were then exposed by being placed in the bucket for 5 h. Faecal examination was carried out on each rabbit every day from the day of exposure to the time of death or killing. Fresh wet faeces were examined by the modified McMaster technique. The egg count (EPG) was determined from the number of eggs detected, multiplied by 200.
335
RABBITS INFECTED WITH STRONGYLOIDES PAPILLOSUS
RESULTS
Termination and faecal egg output
Thirteen of 18 infected rabbits (72%) died 12-29 days after exposure. All rabbits in Groups A and D died 14-27 days after exposure; however, five rabbits among Groups B, C and E survived (Fig. 1 ). Eggs appeared in the faeces 9 or 10 days after exposure in all rabbits. The highest EPG values in each rabbit were 189 600 (rabbit No. 1 ), 71 200 (No. 2), 234 200 (No. 3), 573 000 (No. 4), 25 600 (No. 5), 212 800 (No. 6), 20 000 (No. 7), 530 000 (No. 8), 430 000 (No. 9), 40 000 (No. 10), 496 000 (No. 11), 553 000 (No. 12), 530 000 (No. 13), 380 000 (No. 14), 217 000 (No. 15), 524 000 (No. 16), 330 400 (No. 17) and 168 000 (No. 18). Survivor rabbits, Nos. 5, 6, 10, 17 and 18, had been discharging the eggs for more
EPG [, OOO,000 100, 000 10,000 1,000
, ~ i(]4)~ 2(16)
i, 000,000 100,000 10,000
_
//.i:I I
.-"']4
12,1..-
......
3)
1,000 1,000, 000 lO0,000 10,000 1,000
Group B
,,x t<..." 9{29) ~ -.,.j-~ t. :~ .: %~, r'
t~.~
: .;- ~_L;. . ~ - - - " . . . . . . . . . . . . .
/i~..-'"~ I,~,"7( 12)
1,000, 000 100,000 tO,000
v
..,,.--.---,
8(2~)
5... lO "',
G,rou p C
",
i,$<~ ,; ..'..~ ........13(27)
il?(IYI
~'!,[
t:" j f1,~1(2o),
I, OOO 1,000,000 100, 000 10,000 1,000 100
Group A
~
,
-
t
Group D
16(24)
iroup ~' 18
X..
5
10
, i'"'-"','-'""'""y';
15 20 25 30 35 40 4-5 50 Days
after
exposure
Fig. 1. Dynamics of the egg output in rabbits exposed to Strongyloides papillosus, aRabbit No. with number of days after exposure when it died shown in parentheses.
336
N. TAIRA ET AL.
than 1 month after exposure. Rabbit No. 6 discharged the eggs for more than 52 days ( E P G = 10 100) after exposure. DISCUSSION
In some reports (Bezubik and Turner, 1964; Jaron, 1964), rabbits exposed to 100 000 or 200 000 SPL larvae showed a high EPG, e.g. 100 000-300 0000, and died 13-95 days after infection. These data essentially agree with the present findings. Differentiation of susceptibility among rabbits in each group was not clear. The results indicate that rabbits are highly susceptible to the Japanese calf strain of SPL used, regardless of DEXA administration; however, the egg output was valid in each infected rabbit exposed to the same number of larvae. Above all, we consider that two or three rabbits are needed for one passage and that it should be repeated every 2 or 3 weeks for the maintenance of SPL.
REFERENCES Bezubik, B., 1962. Investigation on the sheep strain ofStrongyloides papillosus II. Adaptability to abnormal hosts and changes in virulence after serial passages in rabbits. Acta Parasitol. Pol., 10: 321-346. Bezubik, B., 1965. Failure to establish infection in rats and guinea pigs exposed to the larvae of Strongyloides papillosus. Acta Parasitol. Pol., 8: 349-354. Bezubik, B. and Turner, H., 1964. Investigations on the sheep strain ofStrongyloides papillosus IV. The physiological and pathological blood picture of sheep and goats experimentally infected with S. papillosus after 5 or 7 serial passages through rabbits. Acta Parasitol. Pol., 12: 101-116. Chomicz, L., 1969. Further studies on the blood picture depending on sex and age of rabbits experimentally infected with Strongyloides papillosus. Acta Parasitol. Pol., 17: 47-54. Chomicz, L., 1984. The effect ofgonadectomy and sex hormones on the course of experimental strongyloidosis (sheep strain) in rabbits. I. Single infections. Acta Parasitol. Pol., 39: 149166. Jaron, W., 1964. Immunity reaction of the rabbit against sheep and rabbit strains of Strongyloides papillosus in repeated infections. Acta Parasitol. Pol., 12:133-149. Nwaorgu, O.C. and Connan, R.M., 1980. The migration of Strongyloides papillosus in rabbits following infection by the oral and subcutaneous routes. J. Helminthol., 54: 223-232. Sinski, E. and Krzysztofowics, M.M., 1971. The blood picture and immunological reactions in rabbits exposed to repeated doses of the sheep and rabbit strains ofStrongyloides papillosus. Acta Parasitol. Pol., 19: 427-444. Stankiewicz, M., 1965. The blood picture of rabbits experimentally infected with Strongyloides papillosus after 5 serial passages through rabbits. Acta Parasitol. Pol., 13: 355-365. Stankiewicz, M., Bezubik, B. and Sinski, E., 1971. The effect of UV-rays on the infective larvae of Strongyloides papillosus in the rabbit. III. Production of immunity by irradiated larvae. Acta Parasitol. Pol., 19: 327-336. Taira, N. and Ura, S., 1991. Sudden death in calves associated with Strongyloides papillosus infection. Vet. Parasitol., 39:313-319.