Rodrigo Sanabria

Review and update of paramphistomosis

SummaryDespite records of ruminal paramphistomes in Argentina dating back to the beginning of the XX century, in the last decade cases have increased in number with evidence of spreading to new geographical areas. This fact led us to carry out some studies in the last few years in order to enhance the poor availability of reports in South America, some of which are actually performed in our group. This paper reviews the characteristics of the life cycles and some aspects of the disease both at world and local level, and updates the latest paramphistome reports in domestic ruminants of Argentina.

Identification of a field isolate of Fasciola hepatica resistant to albendazole and susceptible to triclabendazole

The experiments described here were designed to characterize the status of susceptibility/resistance to albendazole (ABZ) and triclabendazole (TCBZ) of a Fasciola hepatica isolate (named CEDIVE isolate) recovered from infected sheep (Gualeguay, Argentina) and maintained under laboratory conditions. Two separate clinical efficacy experiments were performed on sheep artificially infected with the CEDIVE isolate. Experiment 1: sheep were randomly distributed either in an untreated control group or in an ABZ (7.5 mg/kg) treated group (n=4 each). Additionally, the systemic exposure of ABZ metabolites was assessed in those ABZ-treated infected animals. In Experiment 2, an untreated control group and a TCBZ (10 mg/kg) treated group was included (n=4 each). The fluckicidal efficacy of ABZ and TCBZ was assessed by the comparison of the number of flukes recovered from untreated and treated sheep at 15 days post-treatment. The efficacy against the CEDIVE isolate of F. hepatica was 29% (ABZ) and 100% (TCBZ). The plasma drug exposure (expressed as AUC and C(max)) observed in the ABZ treated animals (Experiment 1) was in agreement with data obtained in the previous studies, which indicate that the low ABZ efficacy was not related to the quality of the pharmaceutical product and/or to a low systemic availability of the active drug/metabolite. The results reported here clearly show that the CEDIVE isolate of F. hepatica behaves as resistant to ABZ and susceptible to TCBZ.

Testing albendazole resistance in Fasciola hepatica : validation of an egg hatch test with isolates from South America and the United Kingdom

The main goal of the current work was to develop and validate an in vitro fluke egg hatch test, as a method for the detection of albendazole (ABZ) resistance in the liver fluke, Fasciola hepatica. Fluke eggs (200/ml, n= 5) from six different isolates were used in the current experimental work. They were obtained from different geographical locations and named Cullompton (UK), CEDIVE (Chascomus, Argentina), INTA-Bariloche (Bariloche, Argentina), Rubino (Uruguay), Cajamarca (Perú) and Río Chico (Catamarca, Argentina). The fluke eggs were incubated (25 °C) for a 12-h period in the presence of either ABZ or its sulphoxide metabolite (ABZ.SO) (5, 0.5 or 0.05 nmol/ml). Untreated eggs were incubated as a control. Incubated eggs (with or without drug present) were kept in darkness at 25 °C for 15 days. Afterwards, the trematode eggs were exposed to daylight over a 2-h period. Hatched and unhatched eggs were evaluated using an optical microscope, and the ovicidal activity was assessed for each fluke isolate. A very low ovicidal activity ( ≤ 13.4%) was observed in the ABZ-resistant CEDIVE isolate for both ABZ and ABZ.SO. Conversely, in the INTA-Bariloche and Río Chico isolates, which are suspected to be susceptible to ABZ, ovicidal activities ≥ 70.3% were observed after incubation with ABZ at the lowest concentration tested (0.05 nmol/ml). This finding correlates with that previously described for the ABZ-susceptible Cullompton. Finally, the Cajamarca and Rubino isolates behaved as ABZ resistant, since no ovicidal activity was observed after eggs were incubated with ABZ at 0.5 nmol/ml. Considering the specific results obtained for each isolate under assessment, the egg hatch test described here may be a suitable method for detection of ABZ resistance in F. hepatica.

Molecular characterization of the ITS-2 fragment of Paramphistomum leydeni (Trematoda: Paramphistomidae)

Paramphistomosis has shown an increased spread over the last years in Argentina, being in some regions an emerging parasitosis, which has motivated their study and identification. Even when morphological features were reported, molecular characterization appears as a complementary and reliable tool. Samples of Paramphistomum leydeni collected since 2002 from different origin, definitive hosts, natural or experimental infections and preservation method were subjected to PCR-RFLP for ITS2+ fragment, and compared to Fasciola hepatica and Notocotilidae cercarie. Frozen and ethanol fixed samples amplified fragments of 500 bp for P. leydeni and Notocotilidae samples, while F. hepatica eggs produced a 540 bp amplicon. Restriction fragments obtained from endonucleases HinfI, HhaI, BsuRI, TaqI, and TruI were identical for all P. leydeni samples, supporting the morphological classification previously performed. Four selected amplicons were sequenced and reported at GenBank, given a consensus sequence ITS-2+ of 441 bp. This first report of molecular characterization for P. leydeni, improves the current knowledge of the genus and establishes precedents for further specimen classifications.

Lymnaea neotropica and Lymnaea viatrix, potential intermediate hosts for Fascioloides magna

Experimental infections of two South American lymnaeid populations with Fascioloides magna were carried out to determine whether these snails may sustain larval development of this digenean and, if so, to quantify their potential for cercarial production. The reference group was a French population of Galba truncatula infected and raised according to the same protocol. According to the internal transcribed sequence (ITS)-1 segment of their genomic rDNA, these South American populations were identified as Lymnaea neotropica (origin, Argentina) and Lymnaea viatrix var. ventricosa (origin, Uruguay). In the snail groups followed for cercarial shedding, longer prepatent periods and lower numbers of shed cercariae were noted in South American lymnaeids. In other snails dissected at day 65 post-exposure, the redial and cercarial burdens of F. magna found in the bodies of L. neotropica and L. v. ventricosa were significantly lower than those noted in G. truncatula. Compared to the total cercarial production noted in the dissected snails, the percentage of cercariae that exited from snails was 51.3% for G. truncatula, 32.2% for L. neotropica and 46.8% for L. v. ventricosa. The two South American species of snails can thus be considered as potential intermediate hosts of F. magna.

Intermediate snail hosts of French Fasciola hepatica: Lymnaea neotropica and Lymnaea viatrix are better hosts than local Galba truncatula.

Allopatric and sympatric infections of Lymnaea neotropica and Lymnaea viatrix var. ventricosa with Argentinean and French isolates of Fasciola hepatica were carried out to determine the capacity of these snails to produce metacercariae and to verify if this capacity changed with snail generation. The same process was also made with a French population of Galba truncatula known to be highly susceptible to French isolates of the parasite. In each lymnaeid species separately considered, the survival rate at day 30 post-exposure and prevalence of F. hepatica infection in the group infected with Argentinean miracidia were significantly greater than those recorded in the corresponding French one. Compared to infected G. truncatula, both South American lymnaeids had longer patent periods and produced a higher number of metacercariae. The highest infections were noted with L. v. ventricosa. In the three snail species, metacercarial production was more important with the Argentinean isolate of miracidia than with the French one. If three successive generations of L. v. ventricosa are exposed to the same French isolate of miracidia, cercarial production significantly increased from parents to the F2 generation, while the other characteristics of infection only showed insignificant variations. L. neotropica and L. v. ventricosa are better intermediate hosts for French F. hepatica than local G. truncatula. The numerical increase of shed cercariae in the F1 and F2 generations of L. v. ventricosa demonstrates a rapid adaptation of this species to the French isolate of the parasite.

First report of Paramphistomum leydeni Näsmark, 1937 (Trematoda: Paramphistomidae) in Argentina, and re-examination of Cotylophoron cotylophorum sensu Racioppi et al. (1994)

SummaryCotylophoron cotylophorum and Balanorchis anastrophus are the only amphistomes of ruminants that have been described so far in Argentina. Recent observations suggest that C. cotylophorum could have been misclassified. To confirm this, we examined specimens, collected in Corrientes Province and classified by Racioppi et al. (1994) as Cotylophoron cotylophorum, and re-classified these as Paramphistomum leydeni Näsmark, 1937. Flukes collected from Zárate (Buenos Aires) were also studied and compared against the former, resulting in the same species. The present study reports the existence of P. leydeni in Argentina, modifies the previous classification of C. cotylophorum and suggests that, based on the evidence of the amphistomes described so far, P. leydeni would be the most prevalent species in cattle of Argentina.

Efficacy of oxyclozanide against adult Paramphistomum leydeni in naturally infected sheep

The aim of the current study was to assess oxyclozanide (OCZ) efficacy against Paramphistomum leydeni in naturally infected adult sheep. OCZ concentrations in blood stream and gastrointestinal fluids collected from treated animals were also measured. Fifteen P. leydeni naturally infected sheep were randomly divided into two groups: untreated control (n=5) and treated (n=10). The treated group was orally drenched with OCZ (20mg/kg, day 0). A second dose was administered 72 h later. Faecal samples were taken at days 0, +3 and +5. Five sheep from both groups were slaughtered at day +5. At necropsies, rumen, abomasum and small intestine were examined for adult and immature flukes. All recovered flukes were counted and the treatment efficacy was estimated. Additionally, serum and gastrointestinal fluid content (ruminal, abomasal and small intestine) samples, obtained from five treated animals at day +5, were analyzed by HPLC to measure OCZ concentrations. OCZ showed high efficacy (99%) against mature P. leydeni. The post-treatment egg reduction was also high after the first dose with values ranging from 98.4% (day +3) to 99.5% (day +5). The highest OCZ concentrations were measured in serum (20.7 ± 11.5 μg/mL) followed by the small intestinal fluid (6.00 ± 4.50 μg/mL). Very low OCZ concentrations (ranging between 0.05 and 0.02 μg/mL) were measured in ruminal and abomasal fluids. OCZ administered to sheep twice (20mg/kg) by the oral route was highly efficacious against mature stages of P. leydeni in naturally infected sheep. Despite a high drug concentration at the intestinal fluid, OCZ efficacy against immature stages could not be assessed. OCZ efficacy and assessment of its concentration profiles in different tissues are considered a contribution to the scarce information available on this ruminant fluke.

Metaphylactic effect of Diclazuril 0.25% in suckling beef calves, during a coccidiosis outbreak in extensive farming.

The weight gain performance and oocysts reduction, as response to the metaphylactic treatment with Diclazuril 0.25% at the start of a coccidiosis outbreak, was studied by a cases-controls transverse study. Fifty-eight suckling calves of approximately 90 days old were randomly selected from an infected beef herd on extensive farming. The calves were weighted and individual faecal samples were taken for oocyst per gram count (OPG). Out of those, 29 were drenched with 1 mg kg(-1) of Diclazuril in one oral dose (group T), since the other 29 remained as control group (group C). Samples for OPG and weights were measured again at days 7 and 21 after treatment, respectively. Later, the groups were divided (by the median) in higher or lower OPG counts, and were compared in the same way, in order to remove those without apparent infection (lower OPG counts). The faecal oocysts reduction reached 99.5% (p<0.0001), for the treated group. Along the three weeks of study, an increment of 2.65 kg in 21 days (125 g day(-1), p=0.036), was seen in treated group respect to controls, but this difference increased to 3.94 kg in 21 days (p<0.0001), when only the calves with higher OPG were taken into account. These results highlight the magnitude of the subclinical impact of coccidiosis, biased by the individual susceptibility to infection, which leads to get heavier infections and express higher oocysts output.

Fasciola hepatica: variations in redial development and cercarial production in relation to the geographic origin of the parasite

Two hundred Galba truncatula, originating from a French population, were subjected to single-miracidium infections with an allopatric isolate (origin, Argentina) of Fasciola hepatica. The control group was constituted by 100 snails coming from the same population and exposed to sympatric miracidia of F. hepatica according to the same protocol. Snail samples were collected bimonthly from both groups between day 14 and day 112 p.e. (at 20 °C) and snail dissections were performed to count free rediae, intraredial morulae and free cercariae. Third and fourth generation rediae were significantly more numerous in the allopatric group, while the number of first generation rediae was significantly lower. In the sympatric group, the decrease in the number of intraredial morulae in the first, second and third redial generations was significantly faster. Free cercariae within the snail body were significantly more numerous in the sympatric than in the allopatric groups, whatever the date of snail dissection. The changes in redial development and cercarial production noted in the Argentinean group might be due to the evolution of South American flukes in a divergent way after the introduction of foreign infected ruminants in this continent from the 15th century.