Nanoemulsions with oleoresin of Copaifera reticulata (Leguminosae) improve anthelmintic efficacy in the control of monogenean parasites when compared to oleoresin without nanoformulation.

This study compared the in vitro anthelmintic activity of Copaifera reticulata oleoresin (200, 400, 600, 800 and 1,000 mg/L) and of nanoemulsions prepared with this oleoresin (50, 100, 150, 200 and 250 mg/L) against monogeneans on the gills of Colossoma macropomum. The major compounds present in the oleoresin of C. reticulata were γ-macrocarpene (14.2%), α-bergamotene (13.6%), ß-selinene (13.4%) and ß-caryophyllene (11.7%). All concentrations of the nanoemulsion and the oleoresin without nanoformulation showed anthelmintic efficacy against monogeneans, and higher concentrations led to more rapid parasite mortality. Structural damages to the tegument of the parasites exposed to C. reticulata oleoresin were observed with scanning electron microscopy. At two hours of exposure, fish showed 100% tolerance to all nanoemulsion concentrations used in the in vitro assays, whereas 100% mortality was shown in the fish exposed to the oleoresin without nanoformulation after one hour. The results of this study suggest that nanoemulsions with oleoresin of C. reticulata have advantages in the control and treatment of monogenean infections in C. macropomum when compared to the oleoresin without nanoformulation. In addition, since nanoemulsions with the C. reticulata oleoresin are safe to control monogeneans, the efficacy of these nanoformulations may be assayed in therapeutic baths to treat C. macropomum infected by monogeneans.

Screening of in vitro antitrematodal activities of compounds and secondary metabolites isolated from selected Pteridophytes.

Four ferns Blechnum orientale Linn. (BO), Dicranopteris linearis (Burm.f ) Underw. (DL), Marattia fraxinea Sm. (MF), and Microlepia speluncae (L.) Moore (MS) were extracted in varied combination of organic solvents followed by the preparation of eluates and isolation of secondary metabolites using chromatography on a glass column with silica gel as the fixed phase. The chemical components were identified using HPTLC and GC-MS analysis. The in vitro anti-trematodal activities of these eluates and compounds were evaluated against the sheep trematode worm Gastrothylax crumenifer (Plagiorrchiida: Gastrothylacidae) at increasing concentrations (1 to 5 mg/mL), to find the relative motility (RM) values for 0 to 60 min of incubation as reflection of paralysis and death of the worms. Hedon-Fleig salt solution was used as negative control and Oxyclozanide® 1% as standard control. In vitro incubation study showed DL and MS extracts had strong trematodicidal activity. BO extract (5 mg/mL) produced moderate trematodicidal activity and MF (5 mg/mL) showed the least trematodicidal activity. Phytochemicals analysis revealed that the ferns are a potential source of trematodicidal compounds such as phytol isomers fern-8-ene and fern-9(11)-ene (terpenoid derivative), quercetin 7,3 ́,4 ́-trimethoxy (flavonoid derivative), etc., which offer scope for a more elaborate study for exploitation of ferns for human welfare.

An in vitro confirmation of the ethonopharmacological use of Senna plants as anthelmintic against rumen fluke Paramphistomum gracile.

BACKGROUND: Paramphistomosis is a pathogenic disease of domesticated ruminants, causing great economic loss in dairy industry and meat production. It is considered as a neglected tropical disease with highest prevalence throughout tropical and subtropical regions, particularly in Africa, Asia, Europe, and Australia. There are few trematocidal drugs available in the market. Most are resistant and have elevated side effects. Therefore, alternative trematocidal drugs need to discover. This study was conducted to evaluate three plants leaf extracts (from Senna alata, S. alexandrina, and S. occidentalis) as effective remedies against gastrointestinal trematode parasite (Paramphistomum gracile) of ruminants. Live adult parasites were collected in 0.1 M phosphate-buffered saline (PBS) from fresh autopsied goat's rumen. Parasites were incubated in leaf extracts of S. alata, S. alexandrina and, S. occidentalis individually and in combination (1:1) ratio at 37 ± 1°C. Treatment media contain extracts at different concentrations (10, 20 and 40 mg/mL) in 10 mL of 0.1 M PBS with 1% dimethylsulphoxide (DMSO). Parasites in control group were incubated in PBS without extract. The efficacy of three Senna extracts was evaluated on the basis of dose-dependent motility and mortality of the trematode. Immediately after paralysis, all treated parasites were collected for histology, SEM and biochemical study. RESULTS: Dose-dependent efficacy was observed in terms of motility and time of mortality in all treated parasites after exposure in various concentrations of the Senna plant extracts. S. occidentalis and S. alexandrina showed better efficacy in combination than comparing with individual treatment groups. Histological study and scanning electron microscopic observations revealed conspicuous deformity of surface architecture in all treated parasites. Scanning electron micrographs also revealed shrinkage, vacuolization, infoldings and blebbings on the body surface of treated worms. Activities of tegumental enzymes were inhibited in all treatment groups compared to control. CONCLUSION: The overall findings from this study revealed that all three Senna leaf extracts individually and in combination showed potential antitrematocidal activity against Paramphistomum gracile by damaging body tegument and neural propagation. Thus, this study confirmed that all three Senna extracts can be considered as a potential drug-like candidate in indigenous system of traditional medicine against trematode infections in livestock.

The Anthelmintic Effects of Medicinal Plant Extracts Against Paramphistome Parasites, Carmyerius spatiosus.

INTRODUCTION: Paramphistomosis is a disease caused by the rumen flukes which cause an acute gastroenteritis and anemia with high mortality particularly in young ruminants. MATERIALS AND METHODS: In this study, we have investigated the anthelmintic effect of medicinal plant extracts from leaves and heartwoods of Cassia siamea L., roots of Plumbago zeylanica L. and Plumbago indica L., and leaves of Terminalia catappa L. against Carmyerius spatiosus. RESULTS: The highest anthelminthic effect on the flukes after 24 h of exposure was found in heartwood ethyl acetate extract of C. siamea (LC50 = 374.30; LC90 = 749.03 ppm), root n-butanol extract of P. zeylanica (LC50 = 1005.12; LC90 = 2411.55 ppm), root hexane, ethyl acetate, and n-butanol extract of P. indica (LC50 = 34.38, 211.34, 506.92; LC90 = 64.09, 496.05, 934.86 ppm), and leaf n-butanol and water extract of T. catappa (LC50 = 487.17, 470.28; LC90 = 913.27, 848.23 ppm). When observed by scanning electron microscopy, the tegument showed similar sequence of morphological changes after treatments with all plant extracts, comprising of swelling of ridges and folds, blebbing, rupturing of the blebs, erosion, lesion and disruption of the tegument. CONCLUSION: This study is the first report on the anthelmintic activity of plant extracts to C. spatiosus; therefore, these plant extracts are highly effective in the elimination of adult rumen flukes.

Increased parasite resistance of greater amberjack (Seriola dumerili Risso 1810) juveniles fed a cMOS supplemented diet is associated with upregulation of a discrete set of immune genes in mucosal tissues.

The main objective of this study was to determine the effect of two forms of mannan oligosaccharides (MOS: Bio-Mos® and cMOS: Actigen®, Alltech Inc, USA) and their combination on greater amberjack (Seriola dumerili) growth performance and feed efficiency, immune parameters and resistance against ectoparasite (Neobenedenia girellae) infection. Fish were fed for 90 days with 5 g kg-1 MOS, 2 g kg-1 cMOS or a combination of both prebiotics, in a Seriola commercial base diet (Skretting, Norway). At the end of the feeding period, no differences were found in growth performance or feed efficiency. Inclusion of MOS also had no effect on lysozyme activity in skin mucus and serum, but the supplementation of diets with cMOS induced a significant increase of serum bactericidal activity. Dietary cMOS also reduced significantly greater amberjack skin parasite levels, parasite total length and the number of parasites detected per unit of fish surface following a cohabitation challenge with N. girellae, whereas no effect of MOS was detected on these parameters. Of 17 immune genes studied cMOS dietary inclusion up-regulated hepcidin, defensin, Mx protein, interferon-γ (IFNγ), mucin-2 (MUC-2), interleukin-1ß (IL-1B), IL-10 and immunoglobulin-T (IgT) gene expression in gills and/or skin. MOS supplementation had a larger impact on spleen and head kidney gene expression, where piscidin, defensin, iNOS, Mx protein, interferons, IL-1ß, IL-10, IL-17 and IL-22 were all upregulated. In posterior gut dietary MOS and cMOS both induced IL-10, IgM and IgT, but with MOS also increasing piscidin, MUC-2, and IL-1ß whilst cMOS induced hepcidin, defensin and IFNγ. In general, the combination of MOS and cMOS resulted in fewer or lower increases in all tissues, possibly due to an overstimulation effect. The utilization of cMOS at the dose used here has clear benefits on parasite resistance in greater amberjack, linked to upregulation of a discrete set of immune genes in mucosal tissues.

Effects of a nanoemulsion with Copaifera officinalis oleoresin against monogenean parasites of Colossoma macropomum: A Neotropical Serrasalmidae.

Monogeneans are ectoparasites that may cause losses in production and productivity in the aquaculture of Colossoma macropomum. Chemotherapeutics used in aquaculture usually have major adverse effects on fish; hence, the use of essential oils has been considered advantageous, but these are not soluble in water. Thus, the use of nanostructures to enhance water solubility of compounds and improve bioactivity may be very promising. This study investigated the antiparasitic activity of nanoemulsion prepared with Copaifera officinalis oleoresin (50, 100, 150, 200 and 300 mg/L), against monogenean parasites from the gills of C. macropomum. The particle size distribution and zeta potential suggested that a potentially kinetic stable system was generated. The nanoemulsion from C. officinalis oleoresin achieved high efficacy (100%) at low concentrations (200 and 300 mg/L) after 15 min of exposure. This was the first time that a nanoemulsion was generated from C. officinalis oleoresin using a solvent-free, non-heating and low-energy method. Moreover, this was the first time that an antiparasitic against monogeneans on fish gills, based on nanoemulsion of C. officinalis oleoresin, was tested.

Combined effects of Chinese medicine feed and ginger extract bath on co-infection of Ichthyophthirius multifiliis and Dactylogyrus ctenopharyngodonid in grass carp.

Dactylogyrus ctenopharyngodonid and Ichthyophthirius multifiliis are two important ectoparasites of freshwater fish. Co-infection by the two parasites leads to high fish mortality and results in heavy economic losses. This study aimed to evaluate the efficacy of medicated feed and a ginger extract bath against D. ctenopharyngodonid and I. multifiliis on grass carp and investigate the hematological response of grass carp co-infected by the two parasites. These results demonstrated that red blood cell (RBC) and thrombocyte percentage among leucocytes significantly decreased after grass carp were co-infected by D. ctenopharyngodonid and I. multifiliis. The monocyte and neutrophil percentages significantly increased with the increment of parasite mean intensities, while the lymphocyte percentage decreased. The activities of serum acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LZM), and superoxide dismutase (SOD) significantly increased after co-infection. When grass carp treated with medicated feed containing 4% of Astragalus membranaceus, Allium sativum, Morus alba, and Glycyrrhiza uralensis, the activities of ACP, AKP, LZM, and SOD were significantly enhanced, and the mean intensities of D. ctenopharyngodonid and I. multifiliis were significantly decreased. When grass carp was treated with medicated feed and a 4-mg/L ginger extract bath, all parasites were eliminated during 28 days. The bath of ginger extract at a concentration of 4 mg/L kept a low mean intensity of I. multifiliis and D. ctenopharyngodonid, then the two parasites were eliminated by oral administration of the medicated feed with an immunostimulant (Chinese medicine compound).

Effects of anthelmintic treatment and feed supplementation on parasite infections and morbidity parameters in Cambodian cattle.

Helminth infections are the cause of morbidity in Cambodian cattle but other factors such as nutritional deficiencies and concurrent diseases may enhance the effects of parasites. The present study aimed to investigate the impact of anthelmintic treatment, feed supplementation, or both on gastrointestinal strongyle (GIS) and trematode infections as well as on morbidity parameters in Cambodian village cattle. At the beginning of the dry season, cattle populations in six villages were randomly assigned to a group: (A) receiving anthelmintic treatment (ivermectin+clorsulon) at week 0; (P) feed pellet supplementation during week 0-13 or both (AP). On five visits (week 0-29), faecal and blood samples were obtained for parasitological examination and haematocrit determination, respectively. Body condition (BCS), hind quarter fouling (HQFS), diarrhoea (DS), and conjunctiva colour (FAMACHA©) were scored and heart girth circumference was determined. To investigate the impact of treatment over time (week 0-29), a mixed model was used with treatment, time, and their interaction as fixed effects, and animal and village as random factors. At baseline, the proportion of GIS positive animals was high (67.9%), whereas trematode infections were low (Paramphistomum: 8.8%; Fasciola: 2.6%). Very thin to emaciated cattle (BCS 1-2) were more prevalent (11.4%) and FAMACHA© scores of ≤3 or below (65.8%) less prevalent than in an earlier study in the region. A Time ⨯ Treatment interaction was present for faecal egg counts (FEC) of GIS, GIS prevalence (both p<0.0001), PCV (p=0.0034), DS (p=0.0086) and HQFS (p=0.0241). For GIS FEC, treatment groups differed at a specific time point, with levels of treatment group P being higher than in A at week 6 (p=0.0054). For Paramphistomum prevalence as well as FAMACHA© scoring, heart girth and BCS, the interaction between treatment and time was not significant, yet, time in itself had a significant impact on all (p<0.0001). The beneficial effects of protein supplementation were unclear from the current study.

Antiparasitic activity, histopathology and physiology of Colossoma macropomum (tambaqui) exposed to the essential oil of Lippia sidoides (Verbenaceae).

In vivo and in vitro antiparasitic activity of the essential oil of Lippia sidoides and blood and histological alterations were assessed in Colossoma macropomum (tambaqui). Essential oil concentrations of 10, 20, 40, 80, 160 and 320mg/L were assayed in vitro against monogenoideans Anacanthorus spathulatus, Notozothecium janauachensis and Mymarothecium boegeri from fish gills. Lippia sidoides essential oil concentrations of 320 and 160mg/L were 100% effective against monogenoideans in 10min and 1h of exposure, respectively. However, the effectiveness of 100% concentrations of 80mg/L and 40mg/L occurred in 3 and 6h, respectively. In the in vivo tests, juvenile fish were submitted to 60min of baths with 10mg/L and 15min of baths with 20mg/L of the essential oil of L. sidoides. These therapeutic baths were not efficient against Ichthyophthirius multifiliis, and monogenoideans present in the gills of C. macropomum. In addition, 10 and 20mg/L of the essential oil of L. sidoides caused an anesthetic effect on the fish and did not influence total glucose and protein plasma levels; however, it decreased the number of total erythrocytes in fish exposed to the higher concentration of this essential oil. Severe alterations and irreversible damage were observed in the fish gills just after L. sidoides essential oil baths and after 24h of recovery. The most recurrent lesions found were hyperplasia and fusion of the lamellar epithelium, vasodilation, detachment of the gill epithelium and lamellar aneurism, epithelial breakdown with hemorrhage, congestion, edema and necrosis, proliferation of the mucous cells and chloride cells and lamellar hypertrophy. Therefore, since the essential oil of L. sidoides has in vitro antiparasitic activity and low concentrations of it have shown toxic effects, the bioactive potential of its main chemical components should be investigated, as well as more efficient forms of its administration in therapeutic baths in order to eliminate fish parasites.

Application of compound mixture of caprylic acid, iron and mannan oligosaccharide against Sparicotyle chrysophrii (Monogenea: Polyopisthocotylea) in gilthead sea bream, Sparus aurata.

We have evaluated the therapeutic effect of a compound mixture of caprylic acid (200 mg/kg fish), organic iron (0.2% of diet) and mannan oligosaccharide (0.4% of diet) in gilthead sea bream, Sparus aurata Linnaeus, infected with Sparicotyle chrysophrii Beneden et Hesse, 1863 in controlled conditions. One hundred and ten reared and S. chrysophrii-free fish (197 g) located in a cement tank were infected by the parasite two weeks following the addition of 150 S. chrysophrii-infected fish (70 g). Growth parameters and gill parasitic load were measured in treated against control fish after a ten-week-period. Differences in final weight, feed conversion ratio, specific growth rate and feed efficiency were not statistically significant between the experimental groups, suggesting no evident effect with respect to fish growth during the study period. Although the prevalence of S. chrysophrii was not affected by the mixture at the end of the experiment, the number of adults and larvae was significantly lower. The mean intensity encompassing the number of adults and larvae was 8.1 in treated vs 17.7 in control fish. Individual comparisons of gill arches showed that the preferred parasitism site for S. chrysophrii it the outermost or fourth gill arch, consistently apparent in fish fed the modified diet and in control fish. In conclusion, the combined application of caprylic acid, organic iron and mannan oligosaccharide can significantly affect the evolution of infection with S. chrysophrii in gilthead sea bream, being capable of reducing adult and larval stages of the monogenean. However, no difference in growth improvement was observed after the trial period, potentially leaving space for further optimisation of the added dietary compounds.

Seasonal changes in infection with trematode species utilizing jellyfish as hosts: evidence of transmission to definitive host fish via medusivory.

In the Seto Inland Sea of western Japan, metacercariae of three species of trematodes, Lepotrema clavatum Ozaki, 1932, Cephalolepidapedon saba Yamaguti, 1970, and Opechona olssoni (Yamaguti, 1934), were found in the mesoglea of the jellyfish Aurelia aurita s.l., Chrysaora pacifica, and Cyanea nozakii. Moreover, these jellyfish frequently harbored juveniles of the fish species Psenopsis anomala, Thamnaconus modestus, and Trachurus japonicus. The former two fish species are well-known medusivores. We investigated seasonal changes in the prevalence and intensity of these metacercariae in their host jellyfish from March 2010 to September 2012 and presumed that infection by the trematodes of the definitive host fish occurs through these associations. The mean intensity of metacercariae in A. aurita s.l. clearly showed seasonality, being consistently high in June of each year. The intensity of metacercariae in C. nozakii was highest among all jellyfish hosts and appeared to be enhanced by medusivory of this second intermediate, and/or paratenic host. Trophic interactions between jellyfish and associated fish were verified using both gut content and stable isotope analyses. The detection of trematodes and nematocysts in the guts of P. anomala and T. modestus juveniles, in addition to stable isotope analysis, suggests that transmission of the parasites occurs via prey-predator relationships. In addition, the stable isotope analysis also suggested that P. anomala is more nutritionally dependent on jellyfish than Th. modestus and Tr. japonicus.

The anthelmintic effects of the ethanol extract of Terminalia catappa L. leaves against the ruminant gut parasite, Fischoederius cobboldi.

Presently, no effective anthelmintic drugs have been used to treat and control paramphistomosis, a severe disease of ruminants. In this study, we have investigated the in vitro anthelmintic effect of the leaves of Terminalia catappa L. crude extract (TcCE) and albendazole (ABZ) on adult Fischoederius cobboldi after incubating the flukes in RPMI-1640 medium containing the TcCE at various doses and times. The TcCE-treated flukes at all dosages exhibited rapid decrease of motility, and the relative motility (RM) values were decreased sharply from start to 3 h. Worms were killed after 6 and 12 h of treatment with 1000, 1500 and 2000 µg mL(-1) as well as 500 µg mL(-1) of TcCE, respectively. By light microscopy examination, the flukes exhibited the earliest alteration in a limited area of the tegument. At scanning electron microscopy level, the flukes' tegument showed similar sequence of morphological alterations after treatment with ABZ and TcCE that consisted of swelling of ridges and folds, followed by blebbing and rupturing of the blebs, leading to the erosion, lesion and disruption of the tegument. Hence, in vivo studies should be performed to examine whether the TcCE may serve as a powerful anthelmintic drug for treatment of paramphistomosis.

The role of rice fields, fish ponds and water canals for transmission of fish-borne zoonotic trematodes in aquaculture ponds in Nam Dinh Province, Vietnam.

BACKGROUND: Fish-borne zoonotic trematodes (FZT), such as Clonorchis sinensis, Opistorchis viverini (Opisthorchiidae) and intestinal trematodes of the family Heterophyidae, constitute a public health hazard in Vietnam and infections with these trematodes has been linked to consumption of raw or undercooked fish from aquaculture. The FZT transmission pathways, however, are more complicated than just the presence of intermediate snail hosts in aquaculture ponds as ponds may exchange water with surrounding habitats such as rice fields and irrigation canals and thereby these surrounding habitats may be a source of snails and cercariae and contribute to FZT infection in cultured fish. METHODS: This is a longitudinal descriptive study on selected farms (n = 30) in Nam Dinh Province which is endemic for FZT. At each farm, we sampled one pond, a small irrigation canal used to supply the pond with water, and a nearby rice field. At each of these three sites, we estimated the density of the FZT intermediate snail hosts and determined their trematode infection status. Comparative analysis was performed for the prevalence and density of FZT infections in fish and snails. RESULTS: Species of the Thiaridae, and most notably Melanoides tuberculata, the most important host species for FZT belonging to the Heterophyidae, were particularly abundant in ponds and small canals, i.e. M. tuberculata was found in 27 ponds and 13 small canals. Bithynia fuchsiana, a potential host for both Heterophyidae and Opisthorchiidae, was rarely found in fish ponds but common in rice fields. A total of 12 types of cercariae were found in the snails and pleurolophocercous cercariae, primarily FZT, constituted about 40 % of all cercarial infections. The fish species cultured were mainly carp species and Haplorchis pumilio was the dominating trematode species infecting fish. Clonorchis spp. were not recorded in any of the ponds. FZT transmission to fish was intense during the summer period (May-June to November) but less intense during the winter months (December-January) partly because cercarial emergence ceases due to the low temperature. CONCLUSION: Our findings highlight the complexity of FZT transmission within aquaculture farm settings and suggest that efforts to control these infections must take a holistic approach using interventions against all stages of the transmission cycle.

Efficacy of commercially available products against Gyrodactylus turnbulli infections on guppies Poecilia reticulata.

The demand for ornamental fish has led to a steep rise in aquaculture for the hobbyist trade, promoting the emergence, persistence and spread of various infectious diseases. Complete control of disease outbreaks with antibiotics and chemical-based medicines is rare, but plant compounds may herald potential alternatives effective against a range of pathogens. Melafix® and Pimafix® are formulated with the essential oils cajuput (Melaleuca cajuputi) and West Indian bay (Pimenta racemosa) and are marketed against bacterial and fungal infections, respectively. Previous experiments showed high efficacy of emulsified cajuput oil against gyrodactylids; the current study tested Melafix® and Pimafix® and their individual compounds against Gyrodactylus turnbulli infecting the guppies Poecilia reticulata. In particular, a combination treatment of Melafix® and Pimafix® was highly effective at reducing in vitro survival of parasites from 15 to 2 h and eradicating 95% of gyrodactylids in vivo. The unexpected high efficacy of this combination treatment is likely explained by the high content of terpenes and phenol propanoids in the cajuput and West Indian bay oils, as well as the anti-helminthic properties of the emulsifier Crovol PK 70. Hence, Melafix® and Pimafix® effectively reduce gyrodactylid burdens on fish, increasing the chances of efficient disease control in ornamental fish.

Efficacy of ginger-based treatments against infection with Gyrodactylus turnbulli in the guppy (Poecilia reticulata (Peters)).

Monogenean infections of commercially farmed fishes are responsible for significant economic losses and existing chemical therapeutants, often stressful to the fish, pose associated risks. As part of a recent trend to move towards the use of alternative, plant-based remedies for commonly occurring aquaculture-related diseases, the efficiency of ginger (Zingiber officinale) was investigated against the monogenean parasite Gyrodactylus turnbulli in the guppy. In vitro trials revealed the clear anti-parasitic effects of ginger. Ethanolic and aqueous extracts, prepared from freeze dried ginger, were tested. An increase in extract concentration was associated with reduced time to parasite immobilisation, with ethanolic extract being more efficient; at 75 and 200ppt aqueous ginger extract parasites died at 65.6±2.8 and 1.8±0.2min, respectively, whereas at 5 and 40ppt ethanolic extract parasites died at 26.1±0.7 and 4.9±0.3min, respectively. Bathing G. turnbulli-infected fish in ethanolic ginger extract (i.e. 5 and 7.5ppt for 90 and 30min, respectively) significantly reduced infection prevalence and intensity when compared to the water and ethanol controls. The higher concentration (i.e. 7.5ppt) proved as equally effective as Praziquantel, the conventionally used chemical treatment for gyrodactylosis, with the fish appearing to be completely cleared of the infection in both cases. Oral treatments of G. turnbulli-infected guppies with diets supplemented with 10 and 20% ginger powder proved to be ineffective in decreasing parasite load. These findings demonstrate that immersion in ginger extract offers an effective, alternative treatment against monogenean infection in fish.

Total mercury and selenium concentrations in Sarpa salpa and Balistes capriscus and in their respective Digenean endoparasites Robphildollfusium fractum and Neoapocreadium chabaudi from Tunisia.

The present study reports the levels of mercury and selenium in Sarpa salpa and Balistes capriscus collected along the coast of Mahdia and Sfax (Tunisia). The systems constituted by S. salpa and Robphildollfusium fractum and by B. capriscus and Neoapocreadium chabaudi were tested as potential bioindicators to monitor environmental Hg pollution in marine ecosystems. Mercury and selenium concentrations were assessed in kidney, liver and muscle of 51 S. salpa and of 45 B. capriscus as well as in their respective endoparasites R. fractum and N. chabaudi. The Se:Hg molar ratios were evaluated for both species across the study areas. Surprisingly, the Se:Hg molar ratio in B. capriscus muscle from Mahdia is significantly lower than in Sfax. Our results indicate that some parasites may also be implicated in the amount of Se and Hg available in tissues and therefore contribute to oscillations of the Se:Hg molar ratios. In the model involving the carnivorous species (B. capriscus), the 5.1-times higher levels of mercury in N. chabaudi than in B. capriscus muscle in Sfax enable this fluke to be a sensitive biomonitoring tool for Hg pollution. The present results confirm that the habitual consumption of S. salpa should not suppose any potential health risk for Tunisian people. On the other hand, the consumption of B. capriscus may be of concern and further monitoring is advisable, since the Hg average concentration in Mahdia was above the maximum allowed Hg concentration in the edible portion of fish fixed by the European Union.

Efficacy of garlic based treatments against monogenean parasites infecting the guppy (Poecilia reticulata (Peters)).

Monogenean infections of commercially farmed fishes are responsible for significant economic losses. Garlic (Allium sativum) is a well-known spice which also possesses anti-microbial and anti-parasitical properties. The current work aimed to test the efficacy of garlic-based treatments against infection with monogenean sp. in the guppy (Poecilia reticulata). Clipped sections of tail fins of guppies heavily infected with Gyrodactylus turnbulli were exposed to aqueous garlic extract (7.5 to 30 mL L(-1)) and visually observed under a dissecting microscope. Results revealed that exposure to garlic caused detachment of parasite and cessation of movement indicating death. A positive correlation was seen between garlic concentration and time to detachment and death of parasites, which, at the highest concentration of 30 mL L(-1), occurred at 4.1 and 8.6 min, respectively. Bathing in aqueous garlic extract (7.5 and 12.5 mL L(-1)) was tested in guppies infected with G. turnbulli. Prior acute toxicity tests revealed the maximum tolerance levels of guppies to garlic extract to be 12.5 mL L(-1) for 1h. Bathing of infected fish in garlic extract (7.5 and 12.5 mL L(-1)) significantly (p<0.05) reduced infection prevalence and intensity as compared to the control. Oral treatments using dry garlic powder-supplemented diet were tested on guppies infected with G. turnbulli and Dactylogyrus sp. Fish were fed with food containing 10% and 20% dry garlic powder for 14 days. Groups fed with garlic supplemented diets showed significantly reduced (p<0.05) mean prevalence and mean intensity of parasites as compared to the control. Dietary application of garlic did not appear to affect palatability. Fresh crushed garlic was added at a level of 1 gL(-1) and applied as an indefinite bath for 14 days. This treatment was seen to significantly reduce (p<0.05) parasite prevalence and mean intensity as compared to the control. Histopathology revealed elevated muscular dystrophy in the 20% garlic-fed group, as compared to control. These findings demonstrate the potential of garlic as a natural alternative to currently used chemical treatments for monogenean sp. infection in the guppy.

In vitro and in vivo efficacy of garlic compounds against Gyrodactylus turnbulli infecting the guppy (Poecilia reticulata).

Traditional compounds used to treat fish diseases in aquaculture and the ornamental fish industry (such as formalin and malachite green) can be more toxic to the hosts than their parasites. With the reviviscence in the use of herbal products, various botanicals have been heralded as cures for particular pathogens, but the efficacy of these compounds for parasitic worms is questionable. Here, we tested a range of garlic (Allium sativum) products against a major aquarium pathogen, Gyrodactylus turnbulli, infecting the guppy (Poecilia reticulata). All garlic products significantly reduced parasite mean survival time in vitro, from 13 h to <1 h. In fully randomised trials, the number of parasites was also significantly reduced on infected fish exposed to garlic from different sources. Two garlic treatments (minced and granule forms) reduced worm burdens by 66% and 75% after three doses, whereas Chinese freeze-dried garlic and allyl disulphide were 95% effective after a single application. In fact, Chinese freeze dried garlic was equally effective as Levamisole, a licensed livestock dewormer that is highly effective against G. turnbulli but not routinely prescribed for use in fish; hence, garlic may be a potential alternative treatment for gyrodactylosis.

Anthelmintic efficacy of Santalum album (Santalaceae) against monogenean infections in goldfish.

Dactylogyrus spp. and Gyrodactylus spp. are helminth ectoparasites that are a significant threat to the aquaculture industry. Existing treatments could cause the threats of anthelmintic resistance, risk of residues, environmental contamination, and toxicity to fish. Importantly, there is no report on a treatment against these two parasites. This study explored the possibility of using the extracts of Santalum album to treat Dactylogyrus sp. and Gyrodactylus sp. infections in goldfish. Results showed that among the four extracts (chloroform, methanol, ethyl acetate, and water) of S. album, the chloroform extract is the most effective and 40 mg/L is a safe and the lowest effective dosage. In addition, we found that Gyrodactylus elegans is more sensitive than Dactylogyrus intermedius when exposed to the extract of the medicinal plant. Finally, it is substantiated that bath treatment with long duration and multiple administrations could eliminate a greater proportion of monogenean infections. These findings show the potential for the development of effective and safe therapy to treat Dactylogyrus sp. and Gyrodactylus sp. infections of fishes.

Bioassay-guided isolation and identification of active compounds from Semen pharbitidis against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus).

The present study was undertaken to isolate the active compounds responsible for the anthelmintic activity of methanol extract of Semen pharbitidis against Dactylogyrus intermedius in goldfish (Carassius auratus). The active methanol extract was fractionated on silica gel column chromatography in a bioassay-guided fractionation, eventually yielding two bioactive compounds: palmitic acid and pharnilatin A by comparing spectral data (NMR and ESI-MS) with literature values. According to in vivo anthelmintic assays, they were found to be 50% effective at the concentrations (EC(50)) of 5.3 and 1.4 mg L(-1), respectively. The promising palmitic acid and pharnilatin A from S. pharbitidis were also subjected to acute toxicity tests for the evaluation of their safety to the host (goldfish). After 48h exposure, the mortalities of goldfish were recorded, and the established LC(50) values were 2.45- and 5.29-fold higher than the corresponding EC(50), demonstrating that pharnilatins A may have better application potential than palmitic acid. The present results provide evidence that pharnilatins A might be potential source of new anti-parasitic drug for the control of Dactylogyrus.