J. Parasitol., 90(2), 2004, pp. 405–407
᭧ American Society of Parasitologists 2004
Infection by Anisakid Nematodes Contracaecum spp. in the Mayan Cichlid Fish ‘Cichlasoma (Nandopsis)’ urophthalmus (Gu ¨ nther 1862) Gaddy T. Bergmann and Philip J. Motta, Department of Biology, University of South Florida, 4202 East Fowler Avenue, SCA 110, Tampa, Florida 33620. e-mail: [email protected]
Larval nematodes that parasitize the Mayan cichlid fish
fection by Contracaecum spp. in ‘C. (Nandopsis)’ urophthalmus differs
‘Cichlasoma (Nandopsis)’ urophthalmus (Gu¨nther 1862) in southern
among habitats or through ontogeny. Because Contracaecum spp. par-
Florida were identified as Contracaecum spp. (Nematoda: Anisakidae,
asitize both marine and freshwater fishes, it was hypothesized that nei-
Anisakinae). The objective of this study was to determine whether in-
ther prevalence nor infection intensity of the nematodes would differ
fection intensity and prevalence of these parasites differ between a
significantly between adult ‘C. (Nandopsis)’ urophthalmus from a
brackish water and freshwater habitat or through ontogeny in the fresh-
brackish water site (BWS) and a freshwater site (FWS). Finally, on the
water habitat only. The nematodes were removed from the abdominal
basis of the notion that Contracaecum spp. might accumulate in ‘C.
cavity of the fishes and counted. Infection intensity was compared be-
(Nandopsis)’ urophthalmus with time, it was hypothesized that both
tween habitats using analysis of covariance and evaluated through on-
prevalence and infection intensity would increase significantly through
togeny using Spearman rank order correlation. Prevalence was com-
ontogeny at the FWS where juvenile fishes were collected.
pared between habitats and between adults and juveniles from the fresh-
The 2 sites chosen for this study, one of which is a brackish water
water habitat using a z-test. Although infection intensity did not differ
habitat and the other a freshwater, are part of the canal system of south-
between habitats, infection prevalence was greater at the freshwater site
ern Florida. The first site was located approximately at the intersection
(FWS). Both the prevalence and intensity of nematode infection in-
of US-41, the Tamiami Trail, and State Route 29. This is the site of
creased through ontogeny at the FWS, and no nematode was found in
intersection of the Tamiami Canal and the Main Barron River Canal in
fishes that were smaller than 93 mm standard length. Thus, the parasites
Carnestown, Collier County (25Њ54Ј41ЉN,81Њ21Ј53ЉW), at the western
appear to accumulate during the lifetime of the fishes.
border of the Big Cypress National Preserve. This region of the Tam-iami Canal passes through brackish water and consists mostly of saltmarsh and some mangrove stands and naturally experiences seasonal
Native to the Atlantic slope of tropical Central America (Miller,
saltwater influx from Chocoloskee Bay. Data from a nearby water mon-
1966), the Mayan cichlid ‘Cichlasoma (Nandopsis)’ urophthalmus
itoring station on the Tamiami Canal (25Њ57Ј40ЉN,81Њ30Ј60ЉW) are used
(Gu¨nther 1862) was first recorded in Florida in 1983 from the Ever-
in this study as indicators of the salinity fluctuations at this first collec-
glades National Park (Loftus, 1987). It is referred to in this study as
tion site. In this region, salinity fluctuates seasonally from 0.00 to 34.30
‘C. (Nandopsis)’ urophthalmus in accord with the convention for tax-
ppt. Sampling at this site took place on 29 April 2000 and 27 May
onomically undetermined cichlids (Stiassny, 1991). The means by
2000. The second collection site was within the Big Cypress National
which ‘C. (Nandopsis)’ urophthalmus was introduced into Florida are
Preserve in the canal along State Route 94 (Loop Road), east of Trail
unknown (Loftus, 1987), but it is likely that it was released by private
City, Monroe County (25Њ45Ј35ЉN,81Њ0Ј9ЉW). This area is a freshwater
aquarists. This species is euryhaline and can survive in a range of sa-
cypress swamp, and sampling took place on a monthly basis from 24
linity from 0 to 40 ppt (Martinez-Palacios and Ross, 1992; Martinez-
June 2000 to 19 October 2000. Data from a nearby water monitoring
Palacios et al., 1993). ‘Cichlasoma (Nandopsis)’ urophthalmus is sus-
station on the Tamiami Canal (25Њ50Ј35ЉN,80Њ55Ј4ЉW) verify that this
ceptible to infection by parasites such as nematodes (roundworms), di-
is a FWS, with salinity ranging from 0.00 to 0.29 ppt only.
genean trematodes, and poecilostomatoid copepods (Vidal-Martı´nez et
Fishes were collected from the BWS and the FWS using seine and
al., 1994; Salgado-Maldonado and Kennedy, 1997). In the present study,
dip nets, hook and line angling, and minnow traps. Samples were
‘C. (Nandopsis)’ urophthalmus was found to have larval Contracaecum
taken on a monthly basis from 29 April 2000 to 19 October 2000 to
spp. (Nematoda: Anisakidae, Anisakinae) (E. C. Greiner, pers. comm.)
minimize the confounding effect of changing seasons; 257 ‘C. (Nan-
in the coelom. The species of Contracaecum infects ‘C. (Nandopsis)’
dopsis)’ urophthalmus were collected for this study. Fifty-three
urophthalmus in Mexico (Vidal-Martı´nez et al., 1994; Salgado-Maldon-
adults were collected from the brackish water population. Sixty-eight
ado and Kennedy, 1997). Lament (1999) found parasitic nematodes in
adults and 136 juveniles were collected from the freshwater popu-
the visceral cavity of ‘C. (Nandopsis)’ urophthalmus collected in the
lation. Only adult ‘C. (Nandopsis)’ urophthalmus were considered
Everglades region but did not identify them. The nematode described
when comparing the brackish and freshwater habitats. All individ-
in Lament (1999) may also be Contracaecum spp.
uals greater than 102 mm standard length (SL) were considered to
Contracaecum spp. are widely distributed parasitic nematodes. Be-
be adults (Caso Cha´vez et al., 1986; Martinez-Palacios and Ross,
sides ‘C. (Nandopsis)’ urophthalmus, they have also been recovered
1992; Martinez-Palacios et al., 1993; Faunce and Lorenz, 2000). It
from other cichlids and many other fishes and aquatic birds (Deardorff
was presumed that the 2 groups sampled are not subpopulations ca-
and Overstreet, 1980; Banks and Ashley, 2000; Machado et al., 2000).
pable of intermixing because ‘C. (Nandopsis)’ urophthalmus is phil-
Anisakids, such as Contracaecum spp., usually infect the alimentary
opatric or site tenacious (Caso Cha´vez et al., 1986; Salgado-Mal-
tract of their definitive hosts, which are generally marine mammals.
donado and Kennedy, 1997; Faunce and Lorenz, 2000).
Marine crustaceans, cephalopod mollusks, or fishes often serve as in-
Fishes were killed with an overdose of tricaine methanesulfonate
termediate or paratenic (transport) hosts. Anisakids are not found in the
(MS-222). They were then injected with 10% buffered formalin and
flesh of fishes, they infect the visceral organs instead. Thus, these par-
placed on ice for preservation. On return to the laboratory, the fishes
asites are not usually harmful to the fish they infect (Deardorff and
were cut from vent to pelvic girdle. All parasitic nematodes found in
Overstreet, 1980; Bush et al., 2001). In experimental infections of ju-
the coelomic cavity were removed and counted. The parasites were then
venile and adult chickens, mallard ducklings, mice, hamsters, and rats,
fixed in 10% buffered formalin and stored in 70% ethanol. Twelve
Contracaecum multipapillatum was not harmful. However, in marine
nematodes from both the brackish and freshwater populations were
birds, C. multipapillatum can infect the proventriculus portion of the
identified by E. C. Greiner, Department of Pathobiology, College of
stomach and cause lesions. This parasite can also cause granuloma in
Veterinary Medicine, University of Florida, Gainesville, Florida. The
dogs that eat fish. Contracaecum multipapillatum is also capable of
fact that only 1 site was used for each habitat type means that there is
infecting domestic cats (Deardorff and Overstreet, 1980; Vidal-Martı´nez
no replication for each treatment. Therefore, statistical power is low,
et al., 1994). Anisakids are dangerous when ingested by humans, caus-
and the results of the analysis presented in this study must be interpreted
ing a condition known as anisakiasis, which causes violent abdominal
with caution. Parasite infection was quantified using both prevalence
pain, nausea, and vomiting (Bush et al., 2001; Plath et al., 2001).
(the proportion of the host population infected) and intensity (the num-
The goal of this study was to determine whether the pattern of in-
ber of parasites per host). Descriptive statistics were computed using
THE JOURNAL OF PARASITOLOGY, VOL. 90, NO. 2, APRIL 2004
the arithmetic mean as the measure of central tendency and the standard
dopsis)’ urophthalmus is also detritivorous (Caso Cha´vez et al., 1986;
deviation as the measure of data dispersion. Because nematodes are
Martinez-Palacios and Ross, 1988; Bergmann, 2002). In the detritivo-
individual organisms, descriptive statistics are reported in this study
rous striped mullet, Mugil cephalus, fish size is likewise positively cor-
using whole numbers. Prevalence was compared between sites and be-
related with the intensity of infection by Contracaecum spp. and other
tween adults and juveniles at FWS using a z-test with an alpha level of
parasites combined (Valles-Rios et al., 2000). Thus, detritivory may lead
0.05. Comparison of infection intensity between sites was accomplished
to an increase in infection intensity through ontogeny, perhaps because
using analysis of covariance (ANCOVA), with SL as covariate and an
of repeated exposure to the parasite. Further investigation would be
alpha value of 0.05. Both the number of nematodes and the SL were
necessary to determine whether predation, detritivory, or other factors
square-root transformed for analysis. Infection intensity was examined
are the primary sources of infection by Contracaecum spp. in ‘C. (Nan-
through ontogeny at FWS using Spearman rank order correlation on
untransformed data (Zar, 1996). The statistical programs used were
The Department of Biology at the University of South Florida pro-
SigmaStat 2.03 and SYSTAT 10.0 (SPSS Science).
vided generous support. We thank S. S. Bell, E. D. McCoy, W. F. Font,
Contracaecum spp. found in the visceral cavity of ‘C. (Nandopsis)’
and anonymous reviewers for their contributions to this publication. We
urophthalmus ranged from approximately 5 to 20 mm in length. They
are grateful to E. C. Greiner for identifying the parasitic nematodes
were less than 1 mm in cross-sectional diameter, light pink in color, and
found within the Mayan cichlids. We also thank S. Connor, J. Hill, S.
generally found in a coiled position, although some were extended.
Huskey, J. Lament, B. Loftus, and K. O. Winemiller for their guidance.
These larvae were probably all in the L3 stage (B. G. Chitwood and
We are grateful to K. Dugan of Collier County for providing us with
M. B. Chitwood, 1974; E. R. Noble and G. A. Noble, 1982; Olsen,
water quality data. Finally, we acknowledge A. Cockburn, S. Jacobson,
1986), and only these larvae were considered in this analysis. Of the
D. Klemm, J. Krebs, H. Porter, and M. Robeson for their critical tech-
56 fish collected from BWS, 22 contained Contracaecum spp. (none
nical assistance. The contents of this paper are from a thesis submitted
among the 3 juvenile specimens, and all 22 among the 53 adults). This
to the University of South Florida by G.T.B. in partial fulfillment of the
is equivalent to 39.3% of all BWS fish sampled or 41.5% of the adults.
requirements for an M.S. degree in zoology. This research was con-
Of the 204 fishes collected at FWS, 47 were infected with the nema-
ducted with approval by the University of South Florida Institutional
todes (3 among the 136 juveniles and 44 among the 68 adults). This is
Animal Care and Use Committee, file 1714.
equivalent to 23.0% of all FWS fishes or 2.2% of juveniles and 64.7%of adults. The z-tests revealed that prevalence of infection was signifi-cantly higher in FWS adults than in either BWS adults (z ϭ 2.359, PLITERATURE CITED
ϭ 0.018) or FWS juveniles (z ϭ 9.850, P Ͻ 0.001).
Infection intensity per individual fish at BWS ranged from 1 to 16
ANKS, S. M., AND D. C. ASHLEY. 2000. Observations on the internal
helminth parasite fauna of largemouth bass, Micropterus salmoides,
nematodes, whereas it ranged from 1 to 43 nematodes at FWS. If the
from Smithville Reservoir, Missouri. Journal of Freshwater Ecol-
individual fish that contained 43 nematodes is discarded from the anal-
ogy 15: 299–306.
ysis, infection intensity at FWS ranged from 1 to 6 nematodes. Meaninfection intensity among adults at BWS was 2 Ϯ 4 worms/fish. At
BERGMANN, G. T. 2002. Trophic ecomorphology of the nonindigenous
FWS, mean infection intensity was 2 Ϯ 5 worms/fish for adults and 0
Mayan cichlid ‘Cichlasoma (Nandopsis)’ uropthalmus (Gu¨nther,
Ϯ 0 worms/fish for juveniles. ANCOVA indicates that infection inten-
1862) in southern Florida. M.S. Thesis. University of South Flor-
sity is not significantly different between sites (F ϭ 2.621, P ϭ 0.108).
Nematode infection showed an increase through ontogeny at FWS.
BUSH, A. O., J. C. FERNA´NDEZ, G. W. ESCH, AND J. R. SEED. 2001.
Very small fish showed no evidence of infection. Ninety-three milli-
Parasitism: The diversity and ecology of animal parasites. Cam-
meters was the first SL at which infection was recorded. Beyond this
bridge University Press, New York, 566 p.
length, there was no clear relationship between SL and infection inten-
CASO CHA´VEZ, M., A. YA´N˜EZ-ARANCIBIA, AND A. L. LARA-DOMı´NGUEZ.
sity as the fish continued to grow. A Spearman rank order correlation
1986. Biologia, ecologia y dinamica de poblaciones de Cichlasoma
was performed to evaluate Contracaecum spp. infection through ontog-
urophthalmus (Gunter) (Pisces: Cichlidae) en habitat de Thalassia
eny. Two separate tests were performed, 1 including the outlying data
testudinum y Rhizophora mangle, Laguna de Terminos, sur del Gol-
point of 43 nematodes in the coelom of 1 fish and 1 excluding that data
fo de Mexico. Biotica 11: 79–111.
point. If the outlier was included, there was a significant and positive
CHITWOOD, B. G., AND M. B. CHITWOOD. 1974. Introduction to Nema-
correlation between number of nematodes in the coelom and SL (R2 ϭ
tology. University Park Press, Baltimore, Maryland, 334 p.
0.612, P Ͻ 0.001). If the outlier was excluded, a similar result was
DEARDORFF, T. L., AND R. M. OVERSTREET. 1980. Contracaecum mul-
obtained (R2 ϭ 0.605, P Ͻ 0.001). tipapillatum (ϭC. robustum) from fishes and birds in the northern
The broad ranges of Contracaecum spp. appear to be naturally oc-
Gulf of Mexico. Journal of Parasitology 66: 853–856.
curring, probably because of migratory birds (Deardorff and Overstreet,
FAUNCE, C. H., AND J. J. LORENZ. 2000. Reproductive biology of the
1980). It is presently unclear whether ‘C. (Nandopsis)’ urophthalmus
introduced Mayan cichlid, Cichlasoma urophthalmus, within an es-
in Florida are infected by a Contracaecum spp. from Florida, from
tuarine mangrove habitat of southern Florida. Environmental Bi-
Central America, or by species from both locations (Hoffman and Schu-
ology of Fishes 58: 215–225.
bert, 1984; Lament, 1999). This study shows that although infection
HOFFMAN, G. L., AND G. SCHUBERT. 1984. Some parasites of exotic
intensity among adults did not differ significantly between sites, adults
fishes. In Distribution, biology, and management of exotic fishes,
at FWS exhibited a significantly higher prevalence of infection by Con-
W. R. Courtenay Jr. and J. R. Stauffer Jr. (eds.). The Johns Hopkins
tracaecum spp. than adults at BWS. The cause for this higher preva-
University Press, Baltimore, Maryland, p. 233–261.
lence of infection at FWS could be attributed to a greater density of
LAMENT, J. J. 1999. Life history variation in introduced populations of the
fishes at this site, but this remains to be determined. This study also
Mayan cichlid, Cichlasoma urophthalmus in the Everglades. Ph.D.
shows that smaller ‘C. (Nandopsis)’ urophthalmus harbors fewer para-
Dissertation, University of Miami, Coral Gables, Florida, 153 p.
sites than do larger individuals. The smallest infected fish from BWS
LOFTUS, W. F. 1987. Possible establishment of the Mayan cichlid, Cich-
was 115 mm SL, and the smallest infected fish from FWS was 93 mm
lasoma urophthalmus (Gu¨nther) (Pisces: Cichlidae), in Everglades
SL. However, there is no clear relationship between size and infection
National Park, Florida. Florida Scientist 50: 1–6.
intensity beyond the minimum age of infection.
MACHADO, P. M., S. C. DE ALMEIDA, G. C. PAVANELLI, AND R. M. TAK-
Larger individuals may harbor more parasites because they have had
EMTO. 2000. Ecological aspects of endohelminths parasitizing Cich-
more time to accumulate them. ‘Cichlasoma (Nandopsis)’ urophthalmusla monoculus Spix, 1831 (Perciformes: Cichlidae) in the Parana
are carnivorous, feeding on decapod crustaceans such as crayfish Pro-
River near Porto Rico, State of Parana, Brazil. Comparative Para-
cambarus sp. and shrimp Palaemonetes paludosus. They also prey on
sitology 67: 210–217.
fish such as mosquitofish Gambusia holbrooki, sailfin mollies Poecilia
MARTINEZ-PALACIOS, C. A., C. CHAVEZ-SANCHEZ, AND M. A. OLVERA
latipinna, and juvenile ‘C. (Nandopsis)’ urophthalmus (Caso Cha´vez et
NOVOA. 1993. The potential for culture of the American Cichlidae
al., 1986; Martinez-Palacios and Ross, 1988; Bergmann, 2002). Con-
with emphasis on Cichlasoma urophthalmus. Recent Advances in
suming these prey items could have led to postcyclic parasitic trans-
Aquaculture 4: 193–232.
mission (Scott, 1954; Smith and Wootten, 1975). However, ‘C. (Nan-
———, AND L. G. ROSS. 1988. The feeding ecology of the Central
American cichlid Cichlasoma urophthalmus (Gunther). Journal of
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search Board of Canada 11: 894–900.
———, AND ———. 1992. The reproductive biology and growth of
SMITH, J. W., AND R. WOOTTEN. 1975. Experimental studies on the mi-
the Central American cichlid Cichlasoma urophthalmus (Gu¨nther).
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᭧ American Society of Parasitologists 2004
Ex Vivo Anthelmintic Activity of Albendazole-Sulphoxide Enantiomers F. Bola´s-Ferna´ndez, S. Rama-In ˜ iguez, and J. J. Torrado*, Departamento de Parasitologı´a, Facultad de Farmacia, Universidad Complutense,
28040 Madrid, Spain; *Deparmento de Farmacia y Tecnologı´a Farmace´utica, Facultad de Farmacia, Universidad Complutense, 28040 Madrid,Spain. e-mail: [email protected]
The antiparasitic activity of racemic albendazole-sulphox-
macokinetic behavior of ABZSO enantiomers have been documented
ide (Ricobendazole ϭ racRBZ) and its (ϩ) and (Ϫ) enantiomers was
in sheep (Capece et al., 2000). Furthermore, the selective uptake of
tested in an ex vivo murine model for Trichinella spiralis infection.
ABZSO enantiomers and their selective binding to cytosolic proteins
Larvae were isolated from the muscle of infected mice and exposed to
isolated from different helminth parasites has also been demonstrated
concentrations between 0.01 and 1 g/ml of the racemic mixture or to
(Alvarez et al., 2000; Solana et al., 2002). Thus, these factors must be
each of its enantiomers. The activity of each compound was then as-
taken into account because they may contribute significantly to the phar-
sayed by measuring the ability of the treated larvae to infect naive mice
macological properties of this chiral molecule.
(larval viability). At a concentration of 0.5 g/ml, all 3 compounds were
However, despite the interest in these agents, the antiparasitic activity
highly effective in reducing the viability of the larvae, achieving re-
of the (ϩ)ABZSO and (Ϫ)ABZSO enantiomers has never been directly
ductions of 91.26% (racRBZ), 96.7% (ϩ), and 89.2% (Ϫ), when com-
tested. Using optimized chiral high-performance liquid chromatography
pared with untreated controls. At lower concentrations (0.1 g/ml), only
(HPLC), we have been able to obtain very pure samples of (ϩ)ABZSO
treatment with (ϩ)RBZ rendered a significant reduction in larval via-
and (Ϫ)ABZSO. This has enabled us to test the anthelmintic activity of
bility in comparison with controls (84.3%; P Ͻ 0.01), whereas at 0.01
these enantiomers in an ex vivo model system, using the well-estab-
g/ml, none of the compounds altered larval viability (P Ͼ 0.05).
lished murine model of Trichinella spiralis infection.
To purify ricobendazole enantiomers from a racemic ABZSO mixture
Albendazole (ABZ), methyl (5-(propyl-thio)-1-H-benzimidazole-2yl)
(Ricobendazole ϭ RBZ), a modular liquid chromatography apparatus
carbamate, is a broad-spectrum drug that acts against the most important
was used, equipped with: a Jasco PU-1580 isocratic pump (Tokyo, Ja-
animal and human helminth parasite species (Horton, 2000). After ad-
pan); an automatic sampler (Gilson 231 XL, Villers le Bels, France)
ministration, ABZ is rapidly transformed by 2 distinct hepatic micro-
fitted to a 100-l sampler loop (Rheodyne, Rohnert Park, California);
somal enzyme systems: the flavin-containing monooxygenase system
a variable wavelength detector (UV-1575, Jasco); and a PC integrator
(Lanusse et al., 1993) and the cytochrome P-450 chain (Souhaili-El
(Borwin 1.5, JMBS developments [Jasco]). Before injection, the sam-
Amri et al., 1987). These modifications produce albendazole-sulphoxide
ples were filtered through a polyvinylidene fluoride Durapore 0.45-m
(ABZSO) and albendazole-sulphone, the main metabolites that can be
filter (Millipore Co., Boston, Massachusetts). Subsequently, the racemic
recovered from the plasma of sheep (Lanusse et al., 1995), cattle (San-
ricobendazole (racRBZ) sample mix was injected into the HPLC system
chez et al., 1997), mice (Rueda-Polo et al., 1998), and humans (Pro-
at a concentration of 0.1 mg/ml, and chiral HPLC separation was per-
chanzkova et al., 2000). Because of their affinity for the parasite’s beta-
formed according to a modified version of the method described pre-
tubulin, both ABZ and ABZSO exhibit antiparasitic activity (Lacey,
viously by Delatour et al. (1990) (Garcia et al., 1999). Briefly, a chiral–
1988). However, ABZ can be further metabolized and is thus only found
alpha 1-acid glycoprotein column (100 ϫ 4 mm, 5 m) and a mobile
at low levels in plasma. Hence, the ABZSO product is thought to be
phase containing sodium phosphate buffer (8 mM, pH 7.0), at a flow
the more active of the 2 in combating tissue-dwelling parasites (Mar-
rate of 0.9 ml/min, were used. The samples were analyzed at 290 nm,
riner and Bogan, 1980). The chiral core in the sulfur atom of ABZSO
and under these conditions, the retention times for (Ϫ) and (ϩ)RBZ
permits 2 enantiomers to be generated, (ϩ)ABZSO and (Ϫ)ABZSO.
were 3.1 and 10.5 min, respectively. The liquid samples were collected
The pharmacokinetic disposition of ABZSO enantiomers after oral ad-
in accordance with the retention times of the enantiomers. The samples
ministration of ABZ in distinct animal species and humans (Delatour,
were concentrated to dryness under vacuum at 70 C using a Savant
Benoit et al., 1991; Delatour, Garnier et al., 1991; Garcia et al., 1999)
Speedvac concentrator (Holbrook, New York). The samples recovered
reveals differences that possibly reflect the selective metabolism of ABZ
were quantified by the same HPLC method described above but in-
and ABZSO enantiomers (Marques et al., 1999; Solana et al., 2000;
cluded 2-propanol (1 ml/L) as an additive in the mobile phase. Under
Virkel et al., 2002). Indeed, even sex-related differences in the phar-
these conditions, the retention times after injection were 2.6 min for
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Prova no processo do trabalho Prof. J. A. Dallegrave Neto 1. Introdução: Princípio da concentração dos atos processuais Alegar e não provar mesmo que não alegar. O tema dentro dos Princípios da Primazia da realidade e do In dubio pro operario ; 2. Objeto de prova. Fatos, não o direito (art. 334, refere-se a fatos). - Pertinentes (à litiscontestatio: inicial x defes