THE IN VITRO EFFECTS OF CRYSTAL VIOLET
ON THE PATHOGENIC HAEMOFLAGELLATE CRYPTOBIA SALMOSITICA K A T Z , 1 9 5 1 (SARCOMASTIGOPHORA: KINETOPLASTIDA)
ARDELLI B.F.* & WOO P.T.K.*
S u m m a r y :
Crystal violet does not inhibit in vitro multiplication of a nonpathogenic strain of Cryptobia salmositica at low concentrations (0.01μM and 0 . 0 0 1 μM) but multiplication is inhibited at higher concentrations (≥ 0 . 0 5 μM). In contrast, the pathogenic strain of C. salmositica does not multiply in vitro when incubated with crystal violet (0.001 μM, 0 . 0 1 μM and 0 . 0 5 μM).
The infectivity of the pathogenic strain is significantly reduced after in vitro exposure to crystal violet. Crystal violet lyses C. salmositica (100.0 μM ) and causes lesions on mitochondrial and nuclear membranes of the parasite. Pathogenic strains of Cryptobia salmositica and C. bullocki are more susceptible to lysis after in vitro exposure to crystal violet than are nonpathogenic strains of Cryptobia salmositica and C. catostomi.
KEY W O R D S : crystal violet, toxicity, Cryptobia.
INTRODUCTION
C
ryptobia salmositica Katz, 1951 is a h a e m o flagellate that causes disease and mortality in e c o nomically important fishes in North America ( W o o & P o y n t o n , 1 9 9 5 ) . T h e clinical signs o f the disease include exophthalmia, splenomegaly, anaemia, general o e d e m a and abdominal distention with ascites ( W o o , 1 9 7 9 ) . Infection with the parasite results in high mortalities in natural infections and it is c o n s i d e r e d a lethal p a t h o g e n o f salmon in semi-natural and inten
sive culture facilities ( B o w e r & T h o m p s o n , 1 9 8 7 ) . Pacific salmon (Oncorhyncbus s p p . ) are a n a d r o m o u s fish s p e c i e s . Experimentally infected fish retain the infection and mortalities continue w h e n experimentally infected hosts are transferred from fresh-water to salt
water ( B o w e r & Margolis, 1 9 8 4 ; Li & W o o , 1 9 9 7 ) . Also, significant mortalities are associated with post- spawning r a i n b o w trout (Oncorhyncbus mykiss) and pre-spawning c h i n o o k salmon (Oncorhyncbus tshawyt- scha) ( W o o & Poynton, 1995). Adult Pacific salmon had
* Department of Zoology, University of Guelph, Guelph, Ontario, Canada, NIG 2W1.
Correspondence to: P.T.K. Woo.
Fax: (519) 767-1656 - Tel.: (519) 824-4120 email: [email protected]
Résumé : LES EFFETS IN VITRO DU CRISTAL VIOLET SUR L'HÉMOFLAGELLÉ C R Y P T O B I A SALMOSITICA KATZ, 1951 ( S A R C O M A S T I G O P H O R A : K I N E T O - PLASTIDA)
Le cristal violel, à de faibles concentrations 10.01 μM et
0.001 μM), n'empêche pas la multiplication in vitro de fa souche non pathogène de Cryptobia solmositica. Par contre, la souche pathogène de C. salmositica ne se multiplie pas in vitro lorsqu'elle est incubée avec du cristal violet ( 0 . 0 0 1 μM, 0.01 μM et 0.05 μM). L'infectivité de la souche pathogène est réduite significativement après une exposition au cristal violet. Le cristal violet lyse C. salmositica (100.0 μM) et cause des lésions aux membranes mitochondriales et nucléaires du parasite. Les souches pathogènes de Cryptobia salmositica et C. bullocki sont plus susceptibles à la lyse après une exposition au cristal violet que les souches non-pathogènes de Cryptobia salmositica et C. catostomi.
MOTS CLES : cristal violet, toxicité, Cryptobia.
detectable infections as early as five days after they returned to fresh-water and parasitaemias in m a n y fish w e r e very high w h e n they s p a w n e d ( B o w e r & Mar- golis, 1 9 8 4 ) .
O n e m e t h o d o f control is vaccination. T h e pathogenic- strain o f C. salmositica has b e e n attenuated and is used as a live v a c c i n e to protect fish against experimental cryptobiosis ( W o o & Li, 1 9 9 0 ; Ardelli etal., 1 9 9 4 ; L i &
W o o , 1 9 9 5 ; Sitja-Bobadilla & W o o , 1 9 9 4 ) . T h e strategy is to immunize presmolts as these fish would b e pro- tected while in fresh-water and w h e n they return from the marine environment ( W o o , 1 9 9 2 ) . Li & W o o ( 1 9 9 5 ) have s h o w n that vaccinated trout are protected for at least two years, and that protection is not affected w h e n vaccinated fish are transferred to salt-water (Li &
W o o , 1997). T h e r e is a n e e d for chemotherapy as there is about 5 0 - 6 0 % annual mortality in c h i n o o k , Onco- rhyncbus tsbawytscha, broodfish in the S o l e d u c k hat- chery in W a s h i n g t o n State, USA (L. P e c k personal c o m m u n i c a t i o n , 1 9 9 4 ) .
Low concentrations o f crystal violet ( 2 0 - 5 0 LIM) cause swelling o f the mitochondria and the uncoupling o f o x i d a t i v e p h o s p h o r y l a t i o n in Trypanosoma cruzi ( G a d e l h a et al., 1 9 8 9 ) . C. salmositica is phylogeneti- cally related to T. cruzi and it also has a large mito- chondria. T h e purpose o f the present study was to e x a - m i n e t h e in vitro e f f e c t s o f c r y s t a l v i o l e t o n Parasite, 1998, 5, 27-36
Mémoire 27
C. salmositica. This is part o f an o n g o i n g project o n the d e v e l o p m e n t o f c h e m o t h e r a p y against salmonid cryptobiosis.
MATERIALS AND METHODS
IN VITRO CULTURE OF CRYPTOBIA SPP.
A
c l o n e d strain o f p a t h o g e n i c Cryptobia salmositica was used to infect r a i n b o w trout held at 11°C. T h e strain w a s initially isolated from Piscicola salmositica and details o f the cloning o f the parasite and fish maintenance have b e e n described ear
lier ( W o o , 1 9 7 9 ) . B l o o d from an infected trout was aseptically inoculated into sterile culture flasks contai
ning Minimum Essential Medium (MEM) supplemented with Hank'S sails. L-glutamine, 25 mM Hepes buffer and 25 % heat-inactivated foetal bovine s e a i m , and cultured.
The nonpathogenic vaccine strain o f C. salmositica was c l o n e d and has b e e n maintained in MEM since 1 9 8 9 ( W o o & Li, 1 9 9 0 ) .
Cryptobia bullocki was originally isolated from the blood of a southern flounder, Paralichtbys lethostigma ( W o o
& T h o m a s , 1991). It has also b e e n maintained in MEM.
Cryptobia catostomi w a s isolated from the b l o o d o f a white s u c k e r Catostomus commersoni and maintained in TDL-15 medium supplemented with heat-inactivated white s u c k e r plasma (Li & W o o , 1 9 9 6 ) .
IN VITRO EFFECTS OF CRYSTAL VIOLET
Approximately 1,500 cultured individuals o f an aviru- lent strain o f C. salmositica (washed three times in cold b l o o d e d vertebrate Ringer's ( C B V R ) solution) in 25 μl of CBVR w e r e incubated for three hours at 11°C (in a microtitre plate) with 2 5 μl o f crystal violet at c o n c e n trations o f 0.1 μM, l . 0 μ M , 1 0 . 0 nm a n d 1 0 0 . 0 μM.
Control wells contained parasites incubated with CBVR, phosphate buffered saline (PBS) and MEM. T h e crystal violet was dissolved in P B S at pH 7.2. Each c o n c e n tration o f crystal violet was replicated 24 times. After three hours the wells w e r e e x a m i n e d using an inverted m i c r o s c o p e (ocular 10 X and objective 10 X ) for living parasites. T h e endpoint was the examination period w h e n there w a s an a b s e n c e o f living parasites.
LONG TERM EFFECTS OF CRYSTAL VIOLET ON PATHOGENIC AND NONPATHOGENIC STRAINS OF C. SAIMOSITICA
MEM and crystal violet w e r e added to a culture flask such that the final v o l u m e was 2 0 ml with a final c o n c e n t r a t i o n o f 0 . 0 μM ( G r o u p A and G r o u p E ; n = 1 0 / g r o u p ) , 0 . 0 5 μM ( G r o u p B a n d G r o u p F ; n = 1 0 / g r o u p ) , 0 . 0 1 μM ( G r o u p C a n d G r o u p G ; n = 1 0 / g r o u p ) and 0.001 μM ( G r o u p D and G r o u p H ;
n = 1 0 / g r o u p ) . A p p r o x i m a t e l y 2 5 0 , 0 0 0 p a t h o g e n i c ( G r o u p s A, B , C, D ) and n o n p a t h o g e n i c ( G r o u p s E, F, G, H ) C. salmositica w e r e inoculated aseptically into e a c h o f the culture flasks and incubated in a slanted position at 11°C. Flasks w e r e s a m p l e d ( 5 0 μl) every three days and the n u m b e r o f parasites determined using a h a e m a c y t o m e t e r (Archer, 1 9 6 5 ) .
INFECTIVITY TEST FOR ASSESSING
IN VITRO SENSITIVITY OF C. SALMOSITICA TO CRYSTAL VIOLET
B l o o d was withdrawn from the caudal vein o f an infected rainbow trout and diluted (with uninfected w h o l e trout b l o o d ) such that 25 μl o f b l o o d contained approximately 1,500 C. salmositica. Crystal violet in P B S (lOOixM-GroupJ, 200μM-Group K, and 5 0 0 μ M - G r o u p L ) w a s a d d e d to the b l o o d . Controls ( G r o u p I) w e r e w h o l e b l o o d with Cryptobia incubated without crystal violet. Experimental and control groups w e r e incu
bated for three hours at 11°C (in a microtitre plate) and then e x a m i n e d under an inverted m i c r o s c o p e (ocular 10 X and objective 10 X ) for living parasites.
After incubation, each, well in the microtitre plate (with 25 μl o f crystal violet and C. salmositica) w a s rinsed with 0.1 ml o f Alsever's solution and the c o n t e n t s ino
culated intraperitoneally into a juvenile r a i n b o w trout.
Fish w e r e bled (0.1 ml/fish) at o n e , two, three and four w e e k s postinoculation and parasitaemias w e r e deter
mined using a h a e m a c y t o m e t e r for high parasitaemias or the haematocrit centrifuge t e c h n i q u e for low para
sitaemias ( W o o , 1 9 6 9 ) .
The experiment was repeated. B l o o d was withdrawn from the caudal vein o f an infected rainbow trout (1,500 individuals o f Cryptobia in 25 μl o f b l o o d ) and treated as described earlier. Crystal violet ( 1 0 0 μM, 2 0 0 μM, 500 μM, 2,500 |μM, 5,000 |μM, 1 μM, 100 μM, 2 0 0 μ M , 300 mM and 1 M) was added to the blood. Controls were parasites incubated without crystal violet. Treat
ment and control groups w e r e incubated for three hours at 11°C. After incubation, the contents o f e a c h well were inoculated into a juvenile rainbow trout. Fish w e r e bled at o n e and two w e e k s postinoculation and parasitae
mias determined as described earlier.
i n VITRO EFFECTS OF CRYSTAL VIOLET ON PATHOGENIC AND NONPATHOGENIC CRYPTOBIA SPP.
T h r e e s p e c i e s o f Cryptobia ( p a t h o g e n i c and n o n p a t h o g e n i c strains o f C. salmositica; n o n p a t h o g e n i c C. cato
stomi; p a t h o g e n i c C. bullocki) w e r e used. Approxi
mately 1,500 individuals o f e a c h strain from culture ( w a s h e d three times in C B V R ) in 2 5 |d o f CBVR w e r e incubated (three hours at 11°C) with 0.1 μM, l . 0 μ M , 10.0 μM and 1 0 0 . 0 μM o f crystal violet in a microtitre plate kept o n ice. Controls w e r e parasites incubated
28 Mémoire Parasite, 1998. 5, 27-36
in PBS, CBVR. M E M and TDL-15 without crystal violet.
After incubation, the microtitre plates w e r e e x a m i n e d using an inverted m i c r o s c o p e (ocular 10 X and o b j e c tive 10 X ) for living parasites.
PREPARATION AND FIXATION OF C. SALMOSITICA FOR TRANSMISSION ELECTRON MICROSCOPY
Pathogenic C. salmositica w e r e c o n c e n t r a t e d directly from culture flasks by centrifugation and w a s h e d in CBVR. Parasites ( 1 . 0 X 1 06) w e r e incubated at 11°C in crystal violet (0.1 liM, 1.0 liM and 10.0 liM) and fixed in 2 . 5 % glutaraldehyde in 0.1 M sodium cacodylate at 15 minutes intervals for 2 4 0 minutes. Postfixation was in 2 % o s m i u m tetroxide. S a m p l e s w e r e serially dehy
drated to 7 0 % ethanol and then treated with 0 . 5 % p- p h e n y l e n e d i a m i n e (Ledingham & Simpson, 1 9 7 2 ) in 7 0 % ethanol. Dehydration was c o n t i n u e d to 1 0 0 % ethanol and samples w e r e e m b e d d e d in Spurr's resin and polymerized.
Ultrathin sections corresponding to a gold interference c o l o u r w e r e cut using a Reichert m i c r o t o m e e q u i p p e d with a diamond knife. Ribbons w e r e collected from the water surface o n c l e a n e d , u n c o a t e d c o p p e r m e s h grids and allowed to dry for 6 0 minutes. Sections attached to grids w e r e stained using saturated uranyl acetate (7.7
% ) and a c e t o n e ( 1 : 1 ) for five minutes and lead citrate for o n e minute. Sections w e r e e x a m i n e d using a J E O L 100CX electron m i c r o s c o p e operating at 8 0 kV.
STATISTICAL ANALYSIS
Data were analyzed using the Statistical Analysis System ( S A S I n s t i t u t e I n c . , 1 9 8 5 ) . A n a l y s i s o f V a r i a n c e (ANOVA) was used to determine significant differences in the n u m b e r o f parasites b e t w e e n control and e x p e rimental groups. Results w e r e c o n s i d e r e d significant if p =£0.05.
RESULTS
EFFECTS OF CRYSTAL VIOLET ON CRYPTOBIA SALMOSITICA
P
a t h o g e n i c and n o n p a t h o g e n i c strains o f C. salmositica w e r e still active after three hours in wells which c o n t a i n e d 0.1 μM o f crystal violet but w e r e lysed at concentrations o f 1.0 |xM, 10.0 μM and 100.0 μM. Parasites remained active in control wells with P B S , MEM or CBVR.
Crystal violet inhibited multiplication o f the pathoge
nic strain o f C. salmositica in 0.05 μM ( G r o u p B ) , 0.01 μM ( G r o u p C ) and 0.001 μM ( G r o u p D ) and most o f the parasites w e r e lysed in 12 days. T h e parasites in the controls ( G r o u p A) without crystal violet multi
plied readily. Significant differences w e r e detected bet
w e e n the n u m b e r o f parasites in control and treatment groups b e t w e e n 25-53 days after incubation in crystal violet (Fig. 1).
T h e n o n p a t h o g e n i c strain o f C. salmositica in 0.05 μM ( G r o u p F ) o f crystal violet did not multiply and the parasites w e r e lysed, w h i l e i n c u b a t i o n in 0.01 μM
Fig. 1. - Long term effects of crystal violet on a pathogenic strain of Crypiobia saltnositica. • Group A: not incubated in c r y s t l a l vioet;
V Group B: incubated in 0.05 μM of crystal violet; • Group C: incu
bated in 0.01 μM of crystal violet; • Group D: incubated in 0.001 μM of crystal violet.
Fig. 2 - Long term effects of crystal violet on a nonpathogenic strain of Cryptobia saltnositica. • Group E: not incubated in crystal violet;
V Group F: incubated in 0.05 μM of crystal violet; T Group G: incu
bated in 0.01 μM of crystal violet; • Group H: incubated in 0.001 μM of crystal violet.
Parasite, 1998, 5, 27-36
Mémoire 29
( G r o u p G ) and 0 . 0 0 1 μM ( G r o u p H ) o f crystal violet did not inhibit in vitro multiplication. N o n p a t h o g e n i c C. salmositica in 0.001 μM o f crystal violet multiplied m o r e readily than controls ( G r o u p E ) at 4 5 - 5 5 days postincubation (Fig. 2 ) .
INFECTIVITY OF C. SALMOSITICA
AFTER IN VITRO EXPOSURE TO CRYSTAL VIOLET IN FISH BLOOD
Incubation o f the parasite in w h o l e b l o o d with crystal violet reduced its infectivity in fish. Parasitaemias in fish inoculated with crystal violet e x p o s e d parasites w e r e significantly l o w e r than t h o s e i n f e c t e d with n o n - e x p o s e d parasites. T h e n u m b e r s o f parasites w e r e significantly l o w e r in G r o u p L ( 5 0 0 μM) at t h r e e ( p = 0 . 0 0 2 2 ) and four (p = 0 . 0 0 1 3 ) w e e k s postinocula- tion, in G r o u p K ( 2 0 0 μM) at three (p = 0 . 0 4 0 0 ) w e e k s p o s t i n o c u l a t i o n , and in G r o u p J ( 1 0 0 μM) at four ( p = 0 . 0 3 6 2 ) w e e k s postinoculation ( T a b l e I ) .
Low c o n c e n t r a t i o n s ( ≤ 2 , 5 0 0 μM) o f crystal violet r e d u c e d the infectivity o f the parasite, as s o m e fish w e r e infected a n d parasitaemias w e r e significantly l o w e r than controls. At high concentrations ( 2 , 5 0 0 μM-
1 M) o f crystal violet, the parasite was not infective as n o parasites w e r e detected in fish ( T a b l e I I ) .
EFFECTS OF CRYSTAL VIOLET ON CRYPTOBIA SPP.
Crystal violet was more toxic to pathogenic (pathogenic- strain o f C. salmositica and C. bullocki) than to nonpa
thogenic ( n o n p a t h o g e n i c strain o f C. salmositica and C.
catostomi) Cryptobia spp. At low concentrations (0.1 μM) o f crystal violet, all Cryptobia spp. w e r e active while at high concentrations ( 1 0 0 μM) o f crystal violet, only the
W e e k 1 W e e k 2 W e e k 3 W e e k 4
Group I 1/101 8/10 10/10 10/10
(infected 3.752 39,063 1,382,450
controls) ( 3 2 . 3 4 6 )3 (1,314,123)
Group J 2/10 9/10 7/10 10/10
(100 (iM) 1 30,357 626,250
(17,466)) (511,141)
Group K 0/10 7/10 7/10 9/10
(200 μM) 1.86 17,857 768,035
(9,835) (466,918)
Group I. 0/10 4/10 9/10 8/10
(500 \IM) 1 3 89,062
(1.6) (147,514)
1 Number of fish with detectable infection/number of fish inocu
lated (using the haematocrit centrifuge technique).
2 Mean parasitemia in infected fish.
3 Standard deviation.
Table I. - Infectivity of Cryptobia salmositica after in vitro exposure to crystal violet in fish blood.
30
C o n c e n t r a t i o n
( m o l e s / L ) 3 h o u r s1 W e e k l2 W e e k 22
0 μM 10/10 10/10 10/10
(31.9)-1 (254,950)
100 μM 10/10 10/10 10/10
(11.9) (ND)4
200 μM 10/10 8/10 10/10
(7.0) (ND)
500 μM 10/10 8/10 10/10
(4.9) (77,500)
2,500 μM 0/10 2/10 2/10
(1.4) (0.2)
5,000 μM 0/10 0/10 0/10
(0) (0)
1 Number of wells containing living parasites/number of replicates.
2 Number of fish with detectable infection/total number of fish ino
culated.
3 Mean parasitaemia.
4 Parasitaemia not determined.
Table II. - Effects of crystal violet on pathogenic Cryptobia salmo- sitica in fish blood and infectivity after exposure.
nonpathogenic parasites w e r e motile. Parasites in the medium with n o crystal violet w e r e active (Table III).
TARGET OF CRYSTAL VIOLET TOXICITY
Pathogenic C. salmositica had a large kinetoplast-mito- chondria c o m p l e x which contained a c o m p a c t mass o f kinetoplast DNA. T h e mitochondria was b o u n d b y a d o u b l e m e m b r a n e and it had m a n y cristae (Fig. 3 ) . After e x p o s u r e to crystal violet, the first m o r p h o l o g i c a l c h a n g e w a s t h e c o n d e n s a t i o n o f kinetoplast DNA (Fig. 4 ) . Fifteen minutes after e x p o s u r e the c o n d e n s a tion b e g a n as distinct linear masses o f DNA distributed throughout the kinetoplast (Fig. 4 ) and continued until the DNA had condensed into a single mass (Figs. 5 and 7 ) . After 4 5 minutes, v a c u o l e s b e g a n to form in the kine
toplast, and often p u s h e d the cristae to the outer e d g e s o f the mitochondria (Fig. 6 ) . At higher c o n c e n trations, the cristae often a p p e a r e d distorted (Fig. 8 ) and the kinetoplast was fragmented and swollen (Fig. 9 ) after l o n g e r e x p o s u r e to crystal violet.
The mitochondrial m e m b r a n e s w e r e characterized by a double m e m b r a n e which had a trilaminar appearance (Figs. 10 and 1 1 ) . After incubation in 1.0 nm o f crystal violet for 3 0 minutes, the outer m e m b r a n e w a s dis
torted and w a s n o l o n g e r discernible as having a tri
laminar a p p e a r a n c e (Figs. 12 and 1 3 ) with e x t e n s i v e swelling o f the kinetoplast.
The nucleus was b o u n d by a d o u b l e m e m b r a n e and had d e n s e chromatin (Fig. 14). After incubation in 1.0 μM o f crystal violet for 120 minutes the outer nuclear m e m b r a n e b e g a n to swell and was separated from the inner m e m b r a n e (Figs. 15 and 1 6 ) .
Mémoire Parasite, 1998, 5, 27-36
Figs. 3-9. - Fig. 3. Transmission electron micrograph of Cryptobia salmositica without exposure to crystal violet, k: kinetoplast; m: mito
chondria x 28,000. Fig. 4. Electron micrograph of Cryptobia salmositica showing condensation of kinetoplast DNA after exposure to 1.0 |iM of crystal violet for 30 minutes x 30,000. Fig. 5. Electron micrograph of Cryptobia salmositica showing condensation of kinetoplast DNA after exposure to 10.0U.M of crystal violet for IS minutes x 16,000. Fig. 6. Electron micrograph of Cryptobia salmositica after exposure to 1.0 NM of crystal violet for 45 minutes. Notice the vacuole formation in the kinetoplast region x 52 000. Fig. 7. Electron micrograph of Cryp
tobia salmositica showing fragmentation of the kinetoplast DNA x 26,000. Fig. 8. Electron micrograph of Cryptobia salmositica after expo
sure to 10.0 μM of crystal violet for 120 minutes. Note the distorted shape of the cristae x 30,000. k: kinetoplast. Fig. 9- Electron micro
graph of Cryptobia salmositica after exposure to 10.0 μM of crystal violet for 120 minutes. Note the distorted kinetoplast. k: kinetoplast x 26,000.
Parasite, 1998, 5, 27-36
Mémoire 31
Figs. 10-13. - Fig. 10. Electron micrograph of Cryptobia salmositica without exposure to crystal violet. Note the compact kinetoplast (k), well developed cristae, and the double membrane surrounding the mitochondria x 57,000. Fig. 11. Enlargement of Fig. 10 showing the double membrane x 87,000. Fig. 12. Electron micrograph of Cryptobia salmositica incubated in 1.0 μM of crystal violet for 30 minutes. Note the swollen kinetoplast x 57,000. Fig. 13. Enlargement of Fig. 12 showing the distortion of the double membrane surrounding the mito
chondria (m) x 87,000.
32 Mémoire Parasite, 1998, 5, 27-36
Figs. 14-16. - Fig. 14. Electron micrograph of Cryptobia salmositica incubated in the absence of crystal violet. Note the double membrane surrounding the nucleus x 47,000. Fig. 15. Electron micrograph of Cryptobia salmositica incubated in 1.0 μM of crystal violet for 120 minutes. Note the swelling of the outer nuclear membrane, n: nucleus x 40,000. Fig. 16. Electron micrograph of Cryptobia salmositica incubated in 1.0 of crystal violet for 120 minutes. Note the outer nuclear membrane beginning to swell (n) x 100.000.
Parasite, 1998, 5, 27-36
Mémoire 33
Crystal violet (μM)
C. salmositica (nonpathogenic strain)
C. salmositica (pathogenic strain)
(C. catostomi (nonpathogenic)
C. bullocki (pathogenic)
0.1 30/30' 30/30 30/30 30/30
1.0 30/30 0/30 30/30 25/30
10.0 30/30 0/30 3/30 23/30
100.0 8/30 0/30 3/30 0/30
PBS 5/5 5/5 5/5 5/5
culture media 5 5 5/5 5/5 5/5
1 Number of wells containing living parasites/number of replicates.
Table III. - The in vitro effects of crystal violet on Cryptobia spp.
DISCUSSION
I
n Brazil, crystal violet is used in bloodbanks to prevent transmission o f Trypanosoma cruzi via b l o o d transfusion ( G a d e l h a et al, 1 9 8 9 ) ; however, it has not b e e n used in c h e m o t h e r a p y . Since there is n o effective drug against C. salmositica ( W o o & Poynton,
1 9 9 5 ) , w e investigated the possibility that crystal violet may have similar effects against the pathogen.
Crystal violet was cryptobiacidal u n d e r in vitro condi
tions. At low c o n c e n t r a t i o n s (0.1 μM), C. salmositica w a s active, but was lysed at higher concentrations ( l 0 . 0 μ M - 1 0 0 . 0 μM). T h e r e w e r e differences in sus
ceptibility to crystal violet b e t w e e n the p a t h o g e n i c and n o n p a t h o g e n i c strains o f C. salmositica. L O W - concentrations (0.001 LlM and 0.01 JIM) o f crystal violet did not inhibit in vitro multiplication o f the n o n p a t h o g e n i c strain o f C. salmositica, while multiplication o f the p a t h o g e n i c strain was inhibited.
Free radicals produced metabolically are important in the toxicity o f a n u m b e r o f t r y p a n o c i d a l c o m p o u n d s ( D o c a m p o & Moreno, 1984). Either the free radical metabolites o f the trypanocidal agents themselves or the superoxide anion, which results from the reduction o f oxygen by the radicals, can initiate processes that lead to cell damage. Both T. cruzi cells and homogenates can enzymatically reduce crystal violet to a carbon-centred free radical ( D o c a m p o et al, 1983). Free radical for
mation may cause the toxicity o f crystal violet to T. cruzi.
Living cells are protected from the damaging effects o f free radicals by protective enzymes, which metabolise the radicals to harmless products, or by certain free radical scavenging c o m p o u n d s , termed antioxidants (e.g. superoxide dismutase, catalase and glutathione reductase) (Bryant & B e h m , 1 9 8 9 ) . If a cell's capacity to detoxify free radicals is e x c e e d e d , membrane damage will ultimately lead to cell lysis. Free radical d a m a g e to nucleic acids and proteins will also interfere with the cell's ability to g r o w and multiply (Bryant & B e h m , 1 9 8 9 ) . Thus, the a b s e n c e o f a protective enzyme, like catalase, would m a k e a parasite susceptible to lysis by free radicals. T h e pathogenic C. salmositica has unde
tectable catalase activities, while the n o n p a t h o g e n i c strain has detectable activities o f the e n z y m e (unpu
blished observations). Thus, w e suggest that pathogenic C. salmositica may b e more susceptible to crystal violet b e c a u s e it lacks catalase to protect it from free radicals formed b y the reduction o f crystal violet.
Parasitic protozoa have u n i q u e m e c h a n i s m s to avoid drug toxicity. Leishmania is thought to resist cytotoxic drugs by amplifying either target g e n e s w h i c h e n c o d e for m e m b r a n e proteins that function as ATP-dependent extrusion p u m p s or g e n e s involved in alternate meta
bolic pathways (Ouellette & P a p a d o p o u l o u , 1 9 9 3 ) . In s o m e cases, Leishmania is c a p a b l e o f avoiding toxi
city o f methotrexate and arsenite b y decreasing drug accumulation. If the drug is taken up, it may b e inac
tivated, excreted, modified and excreted, or routed into vacuoles. Furthermore, interaction o f the drug with the target m a y b e m a d e less effective b y increasing the level o f c o m p e t i n g substrates or b y altering the target to m a k e it less sensitive to the drug (Borst & O u e l lette, 1 9 9 5 ) . Amprolium inhibits the transport o f thia
mine across the cell m e m b r a n e o f Eimeria tenella, E. acervulina and E. maxima. T h e m e c h a n i s m o f resis
tance to this anticoccidial is thought to involve modi
fication o f a target receptor so that its sensitivity to inhi
bition is d e c r e a s e d ( C h a p m a n , 1 9 9 3 ) .
T h e infectivity o f p a t h o g e n i c C. salmositica in fish b l o o d w a s altered after in vitro e x p o s u r e to crystal violet. Parasitaemias w e r e significantly l o w e r e d in fish which received the inoculum e x p o s e d to crystal violet.
Infectivity o f T. b. brucei w a s prevented after incuba
tion for four hours in plasma from cattle treated with d i m i n a z e n e a c e t u r a t e . Similarly, i s o m e t a m i d i u m at 1 n g / m l was sufficient to c o m p l e t e l y prevent the infec
tivity o f the sensitive T. b. brucei stocks, T. h. evansi and T. vivax ( K a m i n s k y et al, 1 9 9 0 ) . In the present study, crystal violet might have r e d u c e d the n u m b e r o f C. salmositica in the inoculum or it reduced the mul
tiplication rate o f the parasite o r both, as control fish had significantly higher parasitaemias.
T h e m e c h a n i s m o f crystal violet toxicity is not unders
tood, but the results o f the electron m i c r o s c o p y study suggested that the target in C. salmositica is mainly the
34- Mémoire Parasite, 1998, 5, 27-36
mitochondria-kinetoplast c o m p l e x . In T. cruzi the dye causes swelling o f the mitochondria in trypomastigotes and epimastigotes with an uncoupling a n d inhibitory action o n oxidative phosphorylation. After incubating pathogenic C. salmositica in crystal violet the most consistent early morphological c h a n g e w a s c o n d e n s a tion o f kinetoplast DNA. Swelling o f the mitochondrial and nuclear m e m b r a n e s as well as disruption o f the kinetoplast w e r e detected. Pentamidine is thought to preferentially fragment t h e k i n e t o p l a s t r a t h e r than nuclear chromatin material in Trypanosoma brucei rho- desiense b l o o d forms (Macadam & Williamson, 1969).
This s a m e effect produced b y Berenil is thought to reflect the selective action o f Berenil o n kinetoplast DNA synthesis and o n the buoyant density o f trypanosome kinetoplast DNA (Newton & LePage, 1 9 6 7 ; 1968). T h e trypanocidal drug Isometamidium is k n o w n to linearize minicircle DNA in t h e kinetoplast o f t r y p a n o s o m e s ( R o b i n s o n & Gull, 1 9 9 1 ) and disruption o f the kineto
plast structure has b e e n implicated in its trypanocidal action (Chitambo etal, 1992). It is thought that the drug is effective due to an interaction o f isometamidium with DNA and a topoisomerase (Sutherland etal, 1991). T h e decantenation o f kinetoplast DNA a n d disruption o f the organelle suggest that the m e c h a n i s m o f crystal violet toxicity m a y b e similar to that o f isometamidium.
It is not clear h o w crystal violet enters cells. T h e para
site may actively uptake the dye, which leads to the cel
lular damage, o r as suggested b y Hoffman et al. ( 1 9 9 5 ) , crystal violet m a y bind actively to sites o n m e m b r a n e s , causing perturbation o f m e m b r a n e structure. T h e dis- niption o f the m e m b r a n e results in a cation intrusion accompanied by a phosphate translocation which causes swelling and dissipation o f m e m b r a n e potential leading to uncoupling o f oxidative phosphorylation. T h e damage to m e m b r a n e s in C. salmositica c a u s e d by crystal violet suggests that it actively binds to sites o n the m e m b r a n e and causes the distortion as indicated in the electron micrographs.
T h e results o f the in vitro study suggest that crystal violet m a y b e a potential therapeutic agent against salmonid cryptobiosis. In l o w dosage, the dye inhibits multipli
cation o f C. salmositica, alters infectivity o f the parasite, and causes lesions o n mitochondrial and nuclear m e m branes. T h e in vivo effective d o s e o f crystal violet has not b e e n determined, but o u r preliminary results sug
gest that the curative d o s e must b e at about 2 0 0 mM to eliminate the infection which may b e toxic to s o m e fish.
ACKNOWLEDGEMENTS
T
his study w a s supported b y grants from t h e National S c i e n c e a n d E n g i n e e r i n g R e s e a r c h Council o f Canada to P.T.K. W o o . W e thank Dr. Melissa Farquar a n d Lewis Melville for advice o nelectron m i c r o s c o p y , Dr. Larry Peterson for use o f equipment a n d facilities, and Louis T r e m b l a y for trans
lating the abstract.
REFERENCES
ARCHER R.K. Haematological techniques for use on animals, Blackwell Scientific Publications, Oxford, 1965.
ARDFLLI B.F., FORWARD G.M. & Woo P.T.K. Brook charr, Sal- velinus fontinalis, and cryptobiosis: a potential salmonid reservoir host for Cryptobia salmositica Katz, 1951. Journal of Fish Diseases, 1994, 17, 567-577.
BORST P. & OUELLETTE M. New mechanisms of drug resistance in parasitic protozoa. Annual Review of Microbiology, 1995, 49, 427-460.
BOWER S.M. & MARGOLIS L. Distribution of Cryptobia salmo
sitica, a haemoflagellate of fishes, in British Columbia and the seasonal pattern of infection in a coastal river. Cana
dian Journal of Zoology, 1984, 62, 2512-2518.
BOWER S.M. & THOMPSON A.B. Hatching of the Pacific salmon leech (Piscicola salmositica) from cocoons exposed to various treatments. Aquacnlture, 1987, 66, 1-8.
BRYANT C. & BFHM C. Biochemical Adaptation in Parasites, Chapman and Hall, London, 1989, 259.
CHAPMAN H.D. Resistance to anticoccidial drugs in fowl.
Parasitology Today, 1993, 9(5), 159-161.
CHITAMBO H., ARAKAWA A. & ONO T. In vivo assessment of drug sensitivity of African trypanosomes using the akine- toplast induction test. Research in Veterinary Science, 1992, 52, 243-249.
DOCAMPO R. & MORENO S.NJ. Free-radical intermediates in the antiparasitic action of drugs and phagocytic cells, in: Free Radicals in Biology. Vol. VI, Dryer W.A. (ed), Academic Press, Orlando, 1984, 243-288.
DOCAMPO R., MORENO S.NJ., MUNIZ R.P.A., CRUZ F.S. &
MASON R.R. Light-enhanced free radical formation and try
panocidal action of gentian violet (crystal violet). Science, 1983, 220, 1292-1294.
GADELHA F.R., MORENO S.N.J., DESOUZA W., CRUZ F.S. &
DOCAMPO R. The mitochondrion of Trypanosoma cruzi is a target of crystal violet toxicity. Molecular and Bioche
mical Parasitology, 1989, 34, 117-126.
HOFFMAN M.A., JANG J . , MORENO S.NJ. & DOCAMPO R. Inhibi
tion of protein synthesis and amino acid transport by crystal violet in Trypanosoma cruzi. Journal of Eukaryotic Microbiology, 1995, 42(3), 293-297.
KAMINSKY R., GUMM I.D., ZWEYGARTH E. & CHUMA F. A drug incubation infectivity test (DIIT) for assessing resistance in trypanosomes. Veterinary Parasitology, 1990, 34, 335-343- LEDINGHAM J.M. & SIMPSON P.O. The use of p-phenylamine
diamine in the block to enhance osmium staining for elec
tron microscopy. Stain Technology, 1972, 47, 239-243.
LI S. & Woo P.T.K. Efficacy of a live Cryptobia salmositica vaccine, and the mechanism of protection in vaccinated Oncorhynchus mykiss (Walbaum) against cryptobiosis.
Veterinary Immunology and Immunopathology, 1995, 48, 343-353.
Parasite, 1998, 5, 27-36
Mémoire 3 5
Li S. & Woo P.T.K. Effects of temperature and white sucker (Catostomus commersoni) serum supplement on the in vitro multiplication of Cryptobia catostomi in cell-free cul
ture medium. Parasitology Research, 1 9 9 6 , 82, 2 7 6 - 2 7 8 . Li S. & Woo P.T.K. Vaccination of rainbow trout, Onco-
rhyncbus mykiss (Walbaum) against cryptobiosis: efficacy of the vaccine in fresh and sea water. Journal of Fish Diseases, 1 9 9 7 , 20, 3 6 9 - 3 7 4 .
MACADAM R.F. & WILLIAMSON J . Lesions in the fine structure of Trypanosoma rhodesiense specifically associated with drug treatment. Transactions of the Royal Society for Tro
pical Medicine and Hygiene, 1 9 6 9 , 63, 4 2 1 .
NEWTON B.A. & LEPAGE R.W.F. Preferential inhibition of extra- nuclear deoxyribonucleic acid synthesis by the trypano- cide Berenil. Biochemistry Journal, 1 9 6 7 , 105, 5 0 . NEWTON B.A. & LEPAGE R.W.F. Interaction of Berenil with try-
panosome D N A . Transactions of the Royal Society for Tro
pical Medicine and Hygiene, 1 9 6 8 , 62, 1 3 1 .
OIIEUETTE M. & PAPADOPOULOI' B. Mechanisms of drug resistance in Leishmania. Parasitology Today, 1 9 9 3 , 9(5), 1 5 0 - 1 5 3 . ROBINSON D.R. & GULL K. Basal body movements as a mecha
nism for mitochondrial genome segregation in the trypa- nosome cell cycle, Nature (London), 1 9 9 1 , 352, 7 3 1 - 7 3 3 - SAS Institute Inc. SAS introductory guide for personal com
puters (version 6 ) , SAS Institute Inc., Carey, North Caro
lina, 1 9 8 5 .
SITJA-BOBADILLA A. & Woo P.T.K. An enzyme-linked immuno
sorbent assay (ELISA) for the detection of antibodies against the pathogenic haemoflagellate, Cryptobia salmositica Katz, and protection against cryptobiosis in juvenile rainbow troLit. Oncorbynchus mykiss (Walbaum) inoculated with a live vaccine. Journal of Fish Diseases, 1 9 9 4 , 17, 3 9 9 - 4 0 8 . SUTHERLAND I.A., PEREGRINE A.S., LONSDALE ECCLES J . D . & HOLMES P.H. Reduced accumulation of isometamidium by drug- resistant Trypanosoma congolaise, Parasitology, 1 9 9 1 , 103, 2 4 5 - 2 5 1 .
Woo P.T.K. Trypanosomes in amphibians and reptiles in sou
thern Ontario. Canadian Journal of Zoology, 1 9 6 9 , 47(5), 9 8 1 - 9 8 8 .
Woo P.T.K. Trypanoplasma salmositica: experimental infec
tions in rainbow trout, Salmo gairdneri. Experimental Parasitology; 1 9 7 9 , 47, 3 6 - 4 8 .
Woo P.T.K. Immunological responses of fish to parasitic orga
nisms. Annual Review of Fish Diseases, 1 9 9 2 , 2, 3 3 9 - 3 6 6 . Woo P.T.K. & Li S. In vitro attenuation of Cryptobia salmo
sitica and its use as a live vaccine against cryptobiosis in Oncorbynchus mykiss. Journal of Parasitology, 1 9 9 0 , 76, 7 5 2 - 7 5 5 .
Woo P.T.K. & POYNTON S.L. Diplomonadida, Kinetoplastida and Amoebida (Phylum Sarcomastigophora), IN. Fish Diseases and Disorders I. Protozoan and Metazoan Infections, Woo P.T.K. (ed), CAB International, Oxon UK, 2 7 - 9 6 .
Woo P.T.K. & THOMAS P.T. Comparative in vitro studies on virulent and avirulent strains of Cryptobia salmositica Katz, 1 9 5 1 (Sarcomastigophora. Kinetoplastida). Journal of Fish Diseases, 1 9 9 2 , 15, 2 6 1 - 2 6 6 .
Reçu le 2 2 mai 1 9 9 7 Accepté le 1 6 septembre 1 9 9 7
Parasite, 1998, 5, 27-36
3 6 Mémoire
