Integrative taxonomy: combining molecular and morphological characteristics to identify Lymnaea (Galba) cubensis, intermediate host of Fasciola hepatica / Taxonomia integrativa: combinando características moleculares e morfológicas para identificar Lymnaea (Galba) cubensis, hospedeiro intermediário de Fasciola hepatica

Abstract Despite the epidemiological importance of the Lymnaeidae family regarding transmission of Fasciola hepatica, knowledge about the diversity and distribution of these molluscs and the role of each species in the expansion of fasciolosis remains sparse. Classical morphological (n=10) identification was performed in lymneids from Lagoa Santa, a municipality in the state of Minas Gerais, Brazil, along with molecular and phylogenetic analysis (n=05) based on the partial nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I gene (COI mtDNA) and ribosomal internal transcribed spacer II (ITS-2 rDNA). The shell morphology made it possible to distinguish the lymneids of Lagoa Santa from Pseudosuccinea columella. Differences found in the penile complex and prostate shape allowed this species to be distinguished from Galba truncatula. However, the homogeneity of reproductive tract characteristics among Lymnaea (Galba) cubensis, L. viator and L. neotropica confirmed that these characteristics show low taxonomic reliability for identifying cryptic species. Genetic divergence analysis for the COI mtDNA gene and ITS-2 region of rDNA revealed greater similarity to Lymnaea (Galba) cubensis. Thus, correct species differentiation is important for monitoring the epidemiological risk of fasciolosis in the state of Minas Gerais, where cases of the disease have increased over recent years.

Resumo Apesar da importância epidemiológica da família Lymnaeidae na transmissão de Fasciola hepatica, o conhecimento sobre a diversidade e a distribuição desses moluscos e o papel de cada espécie, na expansão da fasciolose, ainda é escasso. Realizou-se a identificação morfológica clássica (n=10) em limneídeos de Lagoa Santa, município do estado de Minas Gerais, Brasil, juntamente com a análise molecular e filogenética (n=05), baseada nas sequências parciais de nucleotídeos do gene mitocondrial da subunidade I do citocromo c oxidase (COI mtDNA) e espaçador interno, transcrito do DNA ribossomal II (ITS-2 rDNA). A morfologia da concha possibilitou distinguir os limneídeos de Lagoa Santa de Pseudosuccinea columella. As diferenças encontradas no complexo peniano e na forma da próstata permitiram que essa espécie fosse distinta de Galba truncatula. No entanto, a homogeneidade das características do trato reprodutivo entre Lymnaea (Galba) cubensis, L. viator e L. neotropica confirmou que essas características apresentam baixa confiabilidade taxonômica para a identificação de espécies crípticas. A análise da divergência genética para o gene COI mtDNA e região ITS-2 do rDNA revelou maior similaridade entre os limneídeos de Lagoa Santa com Lymnaea (Galba) cubensis.

Monitoring of Fasciola Species Contamination in Water Dropwort by cox1 Mitochondrial and ITS-2 rDNA Sequencing Analysis

Fascioliasis, a food-borne trematode zoonosis, is a disease primarily in cattle and sheep and occasionally in humans. Water dropwort (Oenanthe javanica), an aquatic perennial herb, is a common second intermediate host of Fasciola, and the fresh stems and leaves are widely used as a seasoning in the Korean diet. However, no information regarding Fasciola species contamination in water dropwort is available. Here, we collected 500 samples of water dropwort in 3 areas in Korea during February and March 2015, and the water dropwort contamination of Fasciola species was monitored by DNA sequencing analysis of the Fasciola hepatica and Fasciola gigantica specific mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear ribosomal internal transcribed spacer 2 (ITS-2). Among the 500 samples assessed, the presence of F. hepatica cox1 and 1TS-2 markers were detected in 2 samples, and F. hepatica contamination was confirmed by sequencing analysis. The nucleotide sequences of cox1 PCR products from the 2 F. hepatica-contaminated samples were 96.5% identical to the F. hepatica cox1 sequences in GenBank, whereas F. gigantica cox1 sequences were 46.8% similar with the sequence detected from the cox1 positive samples. However, F. gigantica cox1 and ITS-2 markers were not detected by PCR in the 500 samples of water dropwort. Collectively, in this survey of the water dropwort contamination with Fasciola species, very low prevalence of F. hepatica contamination was detected in the samples.

Ectopic Human Fasciola hepatica Infection by an Adult Worm in the Mesocolon

We report here an ectopic case of Fasciola hepatica infection confirmed by recovery of an adult worm in the mesocolon. A 56-year-old female was admitted to our hospital with discomfort and pain in the left lower quadrant of the abdomen. Abdominal CT showed 3 abscesses in the left upper quadrant, mesentery, and pelvic cavity. On surgical exploration, abscess pockets were found in the mesocolon of the sigmoid colon and transverse colon. A leaf-like worm found in the abscess pocket of the mesocolon of the left colon was diagnosed as an adult fluke of F. hepatica. Histologically, numerous eggs of F. hepatica were noted with acute and chronic granulomatous inflammations in the subserosa and pericolic adipose tissues. Conclusively, a rare case of ectopic fascioliasis has been confirmed in this study by the adult worm recovery of F. hepatica in the mesocolon.

A Case of Fasciola hepatica Infection Mimicking Cholangiocarcinoma and ITS-1 Sequencing of the Worm

Fascioliasis is a zoonotic infection caused by Fasciola hepatica or Fasciola gigantica. We report an 87-year-old Korean male patient with postprandial abdominal pain and discomfort due to F. hepatica infection who was diagnosed and managed by endoscopic retrograde cholangiopancreatography (ERCP) with extraction of 2 worms. At his first visit to the hospital, a gallbladder stone was suspected. CT and magnetic retrograde cholangiopancreatography (MRCP) showed an intraductal mass in the common bile duct (CBD) without proximal duct dilatation. Based on radiological findings, the presumed diagnosis was intraductal cholangiocarcinoma. However, in ERCP which was performed for biliary decompression and tissue diagnosis, movable materials were detected in the CBD. Using a basket, 2 living leaf-like parasites were removed. The worms were morphologically compatible with F. hepatica. To rule out the possibility of the worms to be another morphologically close species, in particular F. gigantica, 1 specimen was processed for genetic analysis of its ITS-1 region. The results showed that the present worms were genetically identical (100%) with F. hepatica but different from F. gigantica.

Detection of genetic variabiltiy in non - human isolates of fasciola hepatica and Fasciola gigantica by the RAPD - PCR Technique

The present study showed the molecular characterization of Fasciola gigantica and F. hepatica isolates collected from cows and sheep, using the random amplified polymorphic DNA fragments-polymerase chain reaction [RAPDs-PCR] technique. Optimal standardization of amplification conditions and thermocyclation were made, using genetic markers. The methodology used compared the genetic patterns of the two species [interspecies] and inside each species [intra-species] between cow and sheep and the amplification fragments were between 135 and 741 base pairs of marker. The results showed genetic variations [polymorphisms] of Fasciola gigantica and F. hepatica with amplification fragment based on a 500-400 base pair [bp]. Inside each species, there were genetic variations in bovine and ovine and the amplification fragments were between 600 and 400 base pairs [bp]