In vitro activity on Cryptosporidium parvum oocyst of ... - ResearchGate

<strong>In</strong> <strong>vitro</strong> <strong>activity</strong> on Cryptosporidium parvum
oocyst of different drugs with recognized
anticryptosporidial efficacy
J. A. CASTRO-HERMIDA 1,2 , I. PORS 1 , E. ARES-MAZAS 2 and C. CHARTIER 1
1
AFSSA site de NIORT , Laboratoire d’Etudes et de Recherches Caprines, 60 rue de Pied de Fond, B.P. 3081 - 79012 Niort Cedex, France.
2
Laboratorio de Parasitologia, Departamento de Microbiologia y Parasitologia, Facultad de Farmacia, Universidad de Santiago de Compostela, Avda. de Vigo s/n, 15782
Santiago de Compostela, La Coruña, Galicia, España.
Correspondance : Christophe Chartier E-mail : c.chartier@niort.afssa.fr
Tel. : (33) 5 49 79 61 28 Fax: (33) 5 49 79 42 19
SUMMARY
The in <strong>vitro</strong> <strong>activity</strong> of different drugs with recognized anticryptosporidial
efficacy was evaluated on Cryptosporidium parvum oocyst at the standard
doses used in the treatment of human and animals infections. The viability
of purified Cryptosporidium parvum oocysts, exposed for 30, 60, 90
and 120 min in 10 mL sterile distilled water to paromomycin (100 mg/kg
body weight), azithromycin (200 mg/kg body weight), halofuginone lactate
(120 µg/kg body weight), toltrazuril (30 mg/kg body weight), nitazoxanide
(200 mg/kg body weight) and two excipients widely used in pharmaceutical
technology of the group of the cyclodextrins: α-cyclodextrin (1 g/kg body
weight), β-cyclodextrin (1 g/kg body weight) and [α-cyclodextrin : β-cyclodextrin
(50%)] (1 g/kg body weight), was evaluated in <strong>vitro</strong> by inclusion or
exclusion of one fluorogenic vital dye and an excystation technique. Results
showed a significant decrease in the oocyst viability related to an increase
in exposure time for α-, β- and α-, β-cyclodextrins whereas no significant
evolution was seen with the other anticryptosporidial drugs. This effect was
mainly obtained during the first 30 minutes of exposure for both cyclodextrins.
KEY-WORDS : Cryptosporidium parvum, anticryptosporidial
drug, in <strong>vitro</strong> <strong>activity</strong>, oocyst, cyclodextrins.
RÉSUMÉ
Evaluation de l’activité in <strong>vitro</strong> sur l’oocyste de C. parvum de plusieurs
molécules ayant une efficacité vis-à-vis des cryptosporidies. Par J.-A.
CASTRO-HERMIDA, I. PORS, E. ARES-MAZAS et C. CHARTIER.
L’activité in <strong>vitro</strong> de plusieurs molécules ayant une efficacité anti-cryptosporidienne
avérée a été évaluée sur les oocystes de Cryptosporidium parvum
aux doses standards utilisées dans le traitement de la cryptosporiodiose
humaine ou animale. La viabilité d’oocystes purifiés de C. parvum exposés
pendant 30, 60, 90 et 120 mn dans 10 mL d’eau distillée stérile à la paromomycine
(100 mg/kg PV), l’azithromycine (200 mg/kg PV), le lactate
d’halofuginone (120 µg/kg PV), le toltrazuril (30 mg/kg PV), la nitazoxanide
(200 mg/kg PV) ou 2 excipients du groupe des cyclodextrines largement
utilisés dans la technologie pharmaceutique, l’ α-cyclodextrine (1g/kg PV),
la β-cyclodextrine (1g/kg PV) ou un mélange à partie égale de l’α-cyclodextrine
et de la β-cyclodextrine (1g/kg PV), a été évaluée in <strong>vitro</strong> par l’inclusion
ou l’exclusion d’une coloration vitale fluorogénique et par une technique
d’excystation. Les résultats ont montré une diminution significative
dans la viabilité des oocystes liée à l’augmentation du temps d’exposition à
l’ α-, la β- et l’α β-cyclodextrine tandis qu’aucune évolution significative
n’a été enregistrée avec les autres molécules à activité anti-cryptosporidienne.
Cette activité a été principalement obtenue durant les 30 premières
minutes d’exposition pour les 2 cyclodextrines.
MOTS-CLÉS : Cryptosporidium parvum, molécule anticryptosporidienne,
activité in <strong>vitro</strong>, oocyste, cyclodextrines.
<strong>In</strong>troduction
Cryptosporidium parvum is a protozoan parasite highly
infectious to man and animals. C.parvum oocysts excreted
with faeces of infected animals can be a source of infection
for man having great influence on public health in immunodeficient
but also immunocompetent host. The disease is
characterized by watery diarrhoea, dehydration and weight
loss. C. parvum has been reported as a common serious primary
cause of outbreaks of diarrhoea in ruminant neonates,
mainly calves, lambs, and goat kids, causing considerable
direct and indirect economic losses [9, 11, 35].
Although many chemotherapic antimicrobial compounds
have been tested for efficacy against cryptosporidiosis in
animals and humans, there is no clearly recognized, widely
accepted, or immediately available compound for prophylaxis
or therapy [10, 30]. Only a few aminoglycoside [7, 13,
22, 33], macrolide [16, 26, 29] and ionophore [3, 27] antibiotics
as well as halofuginone [8, 18, 23, 24, 32] have
shown detectable anticryptosporidial <strong>activity</strong>. Other results
obtained in chicken cryptosporidiosis [28] as well as field
observations made by veterinarians could indicate some <strong>activity</strong>
with toltrazuril. Lastly, more recent studies have given
encouraging results with nitazoxanide [1, 12, 19] and the
excipients widely used in pharmaceutical industry, α- and β-
cyclodextrin [4, 5, 6].
The aim of the present study was to compare the <strong>activity</strong> of
different drugs with demonstrated anticryptosporidial <strong>activity</strong>
in human or in animals and two cyclodextrins (α- and β-
cyclodextrin) on goat kid C. parvum oocysts through in <strong>vitro</strong>
techniques.
Materials and methods
PARASITE
C. parvum oocysts (4.5-5.0 x 4.5-5.0 µm) were collected
from a naturally infected Alpine neonatal kid. Previously,
faecal smears were examined microscopically and Heine’s
negative stain was used to enable identification of C. parvum
oocysts. The intensity of infection was estimated semiquantitatively
according to the average number of oocysts in 20
randomly selected fields at 1000x magnification. Finally, the
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454 CASTRO-HERMIDA (J.-A.) AND COLLABORATORS
selected faecal sample presented a severe intensity of infection
(>10 oocysts). Oocysts were concentrated and purified
as previously described by Lorenzo et al. [21]. Briefly, fecal
material was mixed with 0.04 M phosphate-buffered saline
(PBS), pH 7.2 and passed through sieves (mesh size 45 µm).
The resulting fluid was resuspended 2:1 (v/v) with diethyl
ether into 50 ml conical centrifuge tubes, shaken vigorously
for 30 s and centrifuged at 1000 g for 5 min. The top three
layers were decanted. This step was repeated until the sediment
was free of lipids. After several washings the sediment
was resuspended in 1 ml of PBS and overlaid on a Percoll®
(Sigma Chemical Co., St. Lousis, MO) discontinuous density
gradient consisting in four 2.5 ml layers with densities of
1.13, 1.09, 1.05 and 1.01 g/mL. The gradients were centrifuged
at 650 g for 15 min at 4°C and the oocysts were harvested
as a clearly defined band between 1.05 and 1.09 g/mL
Percoll® layer. The oocysts were washed three times in PBS
and resuspended 1:1 (v/v) in a 5% potassium dichromate
solution and stored at 4°C until viability assays were performed
(no later than 1 week). Previously to these assays,
oocysts were washed three times at 4°C in PBS in order to
remove the potassium dichromate solution. The number of
oocysts present in 1 mL of suspension was counted in a
modified Neubauer hemacytometer after mixing 0.2 ml with
0.8 ml of malachite green (malachite green, 0.16 g; distilled
water, 100 mL).
IN VITRO VIABILITY ASSAYS
Purified oocysts were exposed for 30, 60, 90 and 120 min
to paromomycin (100 mg/kg body weight, Sigma Chemical
Co., St. Lousis, MO), azithromycin (200 mg/kg body weight,
Zithromax®, Pfizer), halofuginone lactate (120 µg/kg body
weight, Halocur®, <strong>In</strong>tervet), toltrazuril (30 mg/kg body
weight, Baycox®, Bayer), nitazoxanide (200 mg/kg body
weight, Nodik®, Pharmanova, division Unipharm-
<strong>In</strong>ternational S.A.), α-cyclodextrin (1g/kg body weight,
Wacker-Chemie GmbH), β-cyclodextrin (1 g/kg body
weight, Roquette-Laisa, Spain) and [α-cyclodextrin : β-
cyclodextrin (50%)] (1 g/kg body weight). Each drug was
tested in 10 mL sterile distilled water at 20°C in a test tube (2
replicates : assay 1 and assay 2) and shaken (70 rpm) ; then,
the oocysts were washed twice and counted as described previously
Lorenzo et al. [21].
<strong>In</strong>clusion/exclusion of fluorogenic PI vital dye
This technique is based on the inclusion or exclusion of
propidium iodide (PI) fluorogenic vital dye and described
previously by Campbell et al. [2]. Briefly, approximately
2x10 6 treated and untreated oocysts were resuspended to 100
µL in Hanks balanced salt solution (HBSS) and incubated
simultaneously with 10 µL of PI working solution (1 mg/mL
in 0.1 M PBS, pH 7.2) at 37°C for the length of time optimal
for producing maximal dye uptake (2 h). Oocysts were
washed twice in HBSS before being observed by fluorescence
microscopy. Proportion of PI-positive (PI+: permeable
and dead) and PI-negative (PI-: impermeables and viable at
assay) oocysts were quantified by counting more than 100
oocysts in each sample.
Excystation technique
A technique offering approximately 98% of excystation in
untreated oocysts was used [31]. Treated and untreated
oocysts numbering 2x10 6 were resuspended in 10 mL of
0.9% NaCl and 0.5% (w/v) pepsin (62 units/mg solid) and 70
µL of concentrated HCl were added. The reactants were
mixed thoroughly prior to incubation at 37°C for 30 min.
After this preincubation treatment, the mixture was neutralized
with 2.2% HNaCO 3 . Then, 0.022 g of sodium taurocholate
and 0.004 g bovine trypsin were added and incubated at
37°C for 120 min and the suspension was fixed by addition
of 3% glutaraldehyde. Percentage excystation was calculated
by scanning 10 µL aliquots of suspension under phase
contrast microscopy and counting all the intact and empty
oocysts seen in a succession of random fields. Excystation
percentage was given by: (number of empty oocysts/total
oocysts)x100.
STATISTICAL ANALYSIS
The data were analyzed by a test of comparison of proportions
(Statitcf® Ver. 5) and a Analysis of Variance ANOVA
(Systat® Ver. 9.01 for Windows®).
Results
The results of in <strong>vitro</strong> viability assay for oocysts of C. parvum
treated with different drugs and two excipients of the
group of the cyclodextrins for different times (30, 60, 90 and
120 min) demonstrated a general trend with a decrease in the
percentage of excystation and an increase in the percentage
of dead oocysts (PI+), which was related to the increase in
exposure time for each of the evaluated products (Table I).
Nevertheless, the decrease of viability throughout the exposure
time was significant (P < 0.05) only with α-, β- and αβcyclodextrins.
<strong>In</strong> the control assays, the percentage of viable
oocysts, at the maximum of exposure time, remained 98%
and 99% for the inclusion/exclusion of PI fluorogenic vital
dye and excystation techniques respectively. The decrease in
oocyst viability with cyclodextrins was observed during the
first 30 minutes of exposure (P < 0.05).
Discussion
The results obtained in the present study demonstrated a
high proportion of nonviable oocysts ranging from 30 to 40 %
when in contact with α, β or both cyclodextrins. <strong>In</strong> contrast,
the viability of oocysts did not seem significantly affected by
the other anticryptosporidial drugs i.e., paromomycin, azithromycin,
halofuginone lactate, toltrazuril and nitazoxanide.
The mechanisms of inhibition of C. parvum infection by
the nonabsorbable cyclodextrins are still unknown. The
molecular structure of cyclodextrins, which approximates a
truncated cone or torus, generates a hydrophilic exterior surface
and a nonpolar cavity interior. As such, cyclodextrins
can interact with appropriately sized molecules to result in
the formation of inclusion complexes. Therefore, it is possible
that cyclodextrins interacted with hydrophobic mole-
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IN VITRO ACTIVITY ON CRYPTOSPORIDIUM PARVUM OOCYST 455
cules at the level of the oocyst wall. Moreover, the number of
glucose units in the cyclodextrin determines the size of the
hydrophobic pocket and, in turn, which lipids the cyclodextrin
solubilizes [15, 20, 25]. When both cyclodextrins were
mixed in equal proportion, the percentage of viable oocysts
at the maximum exposure time, was lower that with each
cyclodextrin separately although the differnce was not statistically
significant. It is thus possible that β-cyclodextrin
interacted with hydrophobic molecules different that β-
cyclodextrin at level oocyst wall. The anticryptosporidial
<strong>activity</strong> of the β-cyclodextrin has been documented in naturally
infected calves and lambs [4, 5] as well as more
recently for α-cyclodextrin in experimental cryptosporidiosis
in neonatal kids (unpublished data).
The PI vital dye is a valuable indicator of membrane integrity
since it cannot cross cell membranes unless compromised.
Once there are gaps in the membrane, PI enters the cell
and bind to nucleic acids [2, 14]. Our results showed that
oocysts wall were permeable to PI after exposure to both
cyclodextrins suggesting an alteration of the oocyst wall, and
consequently a decrease in their infectivity. Furthermore, a
good correlation was observed with the excystation technique
where we recorded a decrease in the percentages of
excystation and therefore lost of viability. Similarly,
Campbell et al. [2] considered as dead the PI+ oocysts,
which have sporozoites with disrupted or broken membranes
and have established a high correlation between
inclusion/exclusion vital dyes test and in <strong>vitro</strong> excystation
technique.
<strong>In</strong> this study, we only evaluated the in <strong>vitro</strong> <strong>activity</strong> of the
different drugs at the level of the oocyst wall. No significant
<strong>activity</strong> of paromomycin, azithromycin, halofuginone lactate,
toltrazuril or nitazoxanide has been demonstrated on the
oocyst. Obviously, the <strong>activity</strong> of these drugs on the other
exogenous or endogenous stages of the parasite has to be
reminded and could include the inhibition of DNA or protein
synthesis [10]. This explains the numerous reports on the
anticryptosporidial <strong>activity</strong> recorded in <strong>vitro</strong> and in vivo with
paromomycin [7, 13, 22, 33, 34] and in vivo with halofuginone
lactate [8, 18, 23, 24, 32]. These two drugs have shown
a
PI- : Impermeables and viable at assay
TABLE I : Comparative <strong>activity</strong> of different drugs with recognized anticryptosporidial <strong>activity</strong> and two cyclodextrins.
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456 CASTRO-HERMIDA (J.-A.) AND COLLABORATORS
a greater prophylactic than therapeutic <strong>activity</strong> suggesting an
effect on the early stages of the life cycle. On the other hand,
nitazoxanide, a nitrothiazolyl-salicylamide derivative, is
metabolised into its active metabolite, tizoxanide (desacetylnitazoxanide)
after oral administration [10] and its efficacy
against C. parvum was demonstrated in cell culture -inhibiting
the growth of sporozoites and oocysts of C. parvum-, in
animals and in human cryptosporidiosis [1, 12, 19].
The chemotherapy of the cryptosporidiosis remains a
major challenge. The main goal of modern antiparasitic chemotherapy
must be to bring the drug as directly to the target
pathogen as possible and to minimise potential side effects.
Drug chemistry alone can only provide partial answers.
Nevertheless, biochemical and pathophysiological aspects
among others will be a further key factor that must be carefully
scrutinised [10, 17].
Acknowledgements
We gratefully acknowledge to Wacker-Chemie GmbH and
Roquette-Laisa, Spain for freely providing us the α- and β-
cyclodextrin respectively.
This work was supported by Xunta de Galicia by means of
the Conselleria de Politica Agroalimentaria e
Desenvolvemento Rural (PGIDIT02RAG20301 PR) and
Post-Doctoral fellowship of the Conselleria de Presidencia e
Administracion Publica (PR409A2002/7-0).
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