A multiplex PCR for the detection of Fasciola hepatica in the intermediate snail host Galba cubensis

Alba, Annia; Vázquez, Antonio A.; Hernández, Hilda; Sánchez, Jorge; Marcet, Ricardo; Figueredo, Mabel; Sarracent, Jorge; Fraga, Jorge

doi:10.1016/j.vetpar.2015.05.012

Cited by 18 publications

(14 citation statements)

References 28 publications

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“…Table 2 summarises the assessment of the infection status of all field-collected G. cubensis by snail dissection, FasciMol-ELISA and multiplex PCR methods. Noteworthy, the band of 450 bp corresponding to G. cubensis 18S rDNA, which acts as a positive control of the amplification reaction per sample assayed in the multiplex PCR [ 17 ], was absent after the evaluation of 12 samples, most likely due to PCR inhibition. Other studies in which field-collected lymnaeid snails has been screened for infection with F. hepatica have also reported PCR inhibition [ 11 , 28 ] and therefore, these samples were eliminated from the investigation.…”

Section: Resultsmentioning

confidence: 99%

“…The difference in the number of positive samples between the multiplex PCR and the FasciMol-ELISA is likely related with the time points at which the parasite can be detected by each method . The limit for detecting DNA of F. hepatica in the multiplex PCR is lower than the DNA amount of a single miracidium enabling the detection of the parasite during all periods of the infection in snails [ 17 ]. Instead, the detection time frame of the parasite by the sandwich ELISA is extended only up to 80 % of the duration of the prepatent infection of F. hepatica in G. cubensis , i.e.…”

Section: Discussionmentioning

confidence: 99%

“…The remaining portion of each snail was preserved in 95 % ethanol for further DNA extraction and evaluation via multiplex PCR [ 17 ]. This method was considered as a reference technique to check the infection status of G. cubensis with respect to F. hepatica .…”

Section: Methodsmentioning

confidence: 99%

“…The aim of the present study is to assess the performance of the FasciMol-ELISA in monitoring G. cubensis populations occurring in sites at risk for fasciolosis in western Cuba, where high prevalence of infected livestock and several human disease outbreaks have been reported [ 4 ]. A multiplex PCR developed to detect F. hepatica in G. cubensis [ 17 ] was used as a reference method for classification of the samples. This DNA-based assay amplifies a specific segment of the second internal transcribed spacer of the parasite rRNA (ITS2) while amplifying simultaneously a conserved region of the 18S gene of the snail host G. cubensis.…”

Section: Introductionmentioning

confidence: 99%

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Assessment of the FasciMol-ELISA in the detection of the trematode Fasciola hepatica in field-collected Galba cubensis: a novel tool for the malacological survey of fasciolosis transmission

et al. 2016

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BackgroundFasciolosis is one of the food-borne neglected trematodioses that has reemerged as a human disease while its effects on domestic animal health remains of significant economic consideration. Being snail-borne disease, the accurate and time-saving epidemiological surveillance of the transmission foci where infected lymnaeid snails occur could be essential to effectively focus or redirect control strategies. For this purpose, the first monoclonal antibody-based immunoenzymatic assay to detect Fasciola hepatica-infected snails (FasciMol-ELISA) was recently developed and showed a high sensitivity and specificity when tested in an experimental F. hepatica – Galba cubensis system.MethodsHere, we surveyed populations of G. cubensis occurring in western Cuba for the assessment of the FasciMol-ELISA in determining natural F. hepatica infection in this intermediate host. A multiplex PCR, previously developed to detect F. hepatica in G. cubensis, was used for sample classification. Snail dissection method was also employed as screening technique. A Χ2 test and a Kappa index were calculated to evaluate the positivity and the level of agreement between the FasciMol-ELISA and the snail dissection methods with the multiplex PCR, respectively.ResultsGalba cubensis was found in nine out of 12 sampled localities of which four were positive for F. hepatica infection as detected by both immunoenzymatic and PCR-based assays. The overall prevalence was higher than the natural infection rates previously reported for Cuban G. cubensis (range from 4.1 to 7.42 % depending on the screening method). No significant differences were found between FasciMol-ELISA and multiplex PCR when determining parasite positivity (Χ2 = 6.283; P = 0.0981) whereas an excellent agreement was also noted (Kappa = 0.8224).ConclusionsOur results demonstrate the importance of malacological surveys in assessing parasite transmission risk and constitute an alert on the need of accurate measures to control fasciolosis in western Cuba. The sensitivity and specificity of the FasciMol-ELISA as well as its time-saving capacity and the easy of performing the determination of a large number of samples, point at this assay as a novel tool suitable for large-scale monitoring of natural snails populations. To our knowledge, this is the first study that explores natural infection by F. hepatica in field-occurring lymnaeid snails using an immunoenzymatic assay.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Assessment of the FasciMol-ELISA in the detection of the trematode Fasciola hepatica in field-collected Galba cubensis: a novel tool for the malacological survey of fasciolosis transmission

et al. 2016

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“…sperm from cross-fertilization). The extraction of DNA was performed using Chelex methodology following 26 and slightly modified for 96-well plates. In brief, a small portion of tissue was added to a mixture of 100 µL of 5% Chelex®100 (Bio-Rad) and 5 µL of proteinase K (50 mg/mL; Promega) in each well.…”

Section: Methodsmentioning

confidence: 99%

Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba

Alba

Vázquez

Sánchez

et al. 2019

Sci Rep

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Pseudosuccinea columella snails transmit the trematode Fasciola hepatica, but in Cuba, six naturally occurring populations successfully resist parasite infection. Here, we present an updated distribution of P. columella in Cuba; 68 positive sites with the earliest records more abundant in west-central Cuba and with east-central populations generally corresponding to the newest samples. No records were found farther east. The IPA site reported 10.5% prevalence of F. hepatica-infected snails. Population genetics, studied through microsatellites, showed low allelic and multilocus genotypic richness (MLGT), mainly in susceptible populations, strong deviations from panmixia and high self-fertilization rates. Susceptible individuals were grouped in one major cluster containing the majority of MLGT, and two independent clusters grouped the MLGT of resistant individuals from western and central populations, respectively. From these, we propose that several introductions of P. columella occurred in Cuba, primarily in the west, with the early arrivals deriving on the resistant populations. A more recent introduction of susceptible P. columella carrying MLGT T and Y may have occurred, where the latter spread quickly through the island and possibly increase the risk of parasite transmission in Cuba since all snails naturally infected with F. hepatica were carriers of the MLGT Y. Interestingly, even though resistant populations are highly diverse and are likely the oldest within Cuba, they are only found in six localities characterized by soft (total hardness, TH = 6.3 ± 1.03°d) and slightly acidic (pH = 6.2 ± 0.12) waters with low richness in snail species (3.2 ± 1.02). This tendency was also observed in a two-year follow-up ecological study that was conducted on a farm where both phenotypes occurred in sympatry; colonization events by resistant over susceptible snails coincided with a reduction in the pH and TH of the water. A comparison of life traits in susceptible and resistant isolates reared at two different pH/TH conditions (5.9/4°d or 7.8/14°d) showed that low pH/TH negatively affects P. columella, irrespective of the phenotype. However, evidence of higher tolerance (higher survival, life expectancy, egg viability) to such conditions was observed in resistant isolates. Finally, we speculate that the limited distribution of resistant populations might be related to a better exploitation of sites that are less suitable to snails (thus, with lower competition), rather than to a differential ecological restriction to specific environmental conditions from susceptible P. columella.

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A Brief History of the Lymnaeid Research

Vinarski,

Pointier,

Rondelaud

2023

The Lymnaeidae

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A multiplex PCR for the detection of Fasciola hepatica in the intermediate snail host Galba cubensis

Cited by 18 publications

References 28 publications

Assessment of the FasciMol-ELISA in the detection of the trematode Fasciola hepatica in field-collected Galba cubensis: a novel tool for the malacological survey of fasciolosis transmission

Assessment of the FasciMol-ELISA in the detection of the trematode Fasciola hepatica in field-collected Galba cubensis: a novel tool for the malacological survey of fasciolosis transmission

Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba

A Brief History of the Lymnaeid Research

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