A new baseline for fascioliasis in Venezuela: lymnaeid vectors ascertained by DNA sequencing and analysis of their relationships with human and animal infection

Abstract

Background: Human and animal fascioliasis poses serious public health problems in South America. In Venezuela, livestock infection represents an important veterinary problem whereas there appear to be few human cases reported, most of which are passively detected in health centres. However, results of recent surveys suggest that the situation may be underestimated in particular areas. To obtain a baseline for future fascioliasis assessment, studies were undertaken by means of rDNA ITS-2 and ITS-1 and mtDNA cox1 sequencing to clarify the specific status of Venezuelan lymnaeids, their geographical distribution and fascioliasis transmission capacity, by comparison with other American countries and other continents.

Results: Results obtained completely change the lymnaeid scenario known so far. The relatively rich lymnaeid fauna of Venezuela has been proven to include (i) Lymnaea meridensis and L. neotropica as the only native members, (ii) L. cubensis and Pseudosuccinea columella introduced from the Caribbean area, and (iii) Galba truncatula and L. schirazensis introduced from the Old World. The absence of representatives of the stagnicoline and Radix groups is remarkable. Four species are fascioliasis vectors: G. truncatula, L. cubensis and L. neotropica, which have the capacity to give rise to human endemic areas, and P. columella, which is a source of animal infection and is responsible for the spread of disease. Vector capacity in the apparently highland endemic L. meridensis is to be confimed, although may be expected given its phylogenetic relationships. Similarly as elsewhere, the non-transmitting L. schirazensis has been confused with L. cubensis, also with G. truncatula and possibly with L. neotropica.

Conclusions: The new scenario leads to the re-opening of many disease aspects. In Venezuela, altitude appears to be the main factor influencing fascioliasis distribution. Human infection shows an altitude pattern similar to other Andean countries, although a differing highland/lowland impact on animal infection does not appear evident. The overlap of G. truncatula, L. cubensis and probably also L. neotropica in temperate and cold zones suggests a higher risk for human infection in mid and high altitude areas. A lymnaeid species mapping by means of DNA markers becomes a priority to determine human and animal fascioliasis distribution in Venezuela, owing to the importance of lymnaeid vectors in defining transmission and epidemiological patterns.

Figure 1

Geographical distribution of fascioliasis in Venezuela. Map of Venezuela showing localities where liver fluke infection in humans, livestock and lymnaeid snails has been reported.

Figure 2

Nucleotide variable positions and microsatellites found in the ITS-2 sequence of the L. cubensis populations and haplotypes studied. Position = numbers (to be read in vertical) refer to variable positions obtained in the alignment made with MEGA 4.0. Identical = .; Indel = -. *EMBL: AM412223; ** EMBL: FN182200; *** EMBL: FN182201.

Figure 3

Nucleotide and amino acid differences found in the mtDNA cox1 sequence of the Lymnaea neotropica populations studied from Venezuela. Position = numbers (to be read in vertical) refer to variable positions obtained in the alignment made with MEGA 4.0. Nt = nucleotides; Aa = amino acids; Identical = .; Indel = -. Haplotype codes only provisional due to incomplete sequences of the gene. *EMBL: AM494008; **EMBL: FN356741.

Figure 4

Nucleotide differences found in the mtDNA cox1 gene sequence of the Galba truncatula populations studied from Venezuela. Position = numbers (to be read in vertical) refer to variable positions obtained in the alignment made with MEGA 4.0. Nt = nucleotides; Identical = .; Indel = -. Haplotype codes only provisional due to incomplete sequences of the gene. *EMBL: AM494011; **GenBank: EU818799 (incomplete sequence: no haplotype available).

Figure 5

Nucleotide and amino acid differences found in the mtDNA cox1 gene sequence of the Lymnaea schirazensis populations studied from Venezuela. Position = numbers (to be read in vertical) refer to variable positions obtained in the alignment made with MEGA 4.0. Nt = nucleotides; Aa = amino acids; Identical = .; Indel = -. Haplotype codes only provisional due to incomplete sequences of the gene. *GenBank: JF272607, Iran, Spain, Egypt, Mexico, Peru, Dominican Republic; **GenBank: JF272608; ***GenBank: JF272609; ****GenBank: JF272610.

Figure 6

Nucleotide and amino acid differences found in the mtDNA cox1 gene sequence of Lymnaea meridensis from Venezuela and L. cousini haplotypes. Position = numbers (to be read in vertical) refer to variable positions obtained in the alignment made with MEGA 4.0. Nt = nucleotides; Aa = amino acids; Identical = .; Indel = -. Haplotype codes only provisional due to incomplete sequences of the gene. *EMBL: FN598161; **EMBL:FN598162; ***EMBL: FN598163.

Figure 7

Phylogenetic analysis of lymnaeid species from Venezuela. Phylogenetic tree of lymnaeid species studied, obtained using the planorbid Biomphalaria pfeifferi as outgroup, based on maximum-likelihood (ML) estimates. Scale bar indicates the number of substitutions per sequence position. Support for nodes a/b/c: a: bootstrap with NJ reconstruction using PAUP with ML distance and 1000 replicates; b: bootstrap with ML reconstruction using PAUP with 1000 heuristic replicates; c: Bayesian posterior probability with ML model using MrBayes.