Mem Inst Oswaldo Cruz, Rio de Janeiro, 99(4) June 2004
Original Article

Detection of Lymnaea columella Infection by Fasciola hepatica through Multiplex-PCR

Kelly Grace Magalhães, Liana Konovaloff Jannotti Passos, Omar dos Santos Carvalho

Centro de Pesquisas René Rachou-Fiocruz, Av. Augusto de Lima 1715, 30190-002 Belo Horizonte, MG, Brasil

Page: 421-424
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From complete mitochondrial DNA sequence ofu00a0Fasciola hepaticau00a0available in Genbank, specific primers were designed for a conserved and repetitive region of this trematode. A pair of primers was used for diagnosis of infectedu00a0Lymnaea columellau00a0byu00a0F. hepaticau00a0during the pre-patent period simultaneously with another pair of primers which amplified the internal transcribed spacer (ITS) region of rDNA fromu00a0L. columellau00a0in a single Multiplex-PCR. The amplification generated a ladder band profile specific foru00a0F. hepatica. This profile was observed in positive molluscs at different times of infection, including adult worms from the trematode. The Multiplex-PCR technique showed to be a fast and safe tool for fascioliasis diagnosis, enabling the detection ofu00a0F. hepaticau00a0miracidia inu00a0L. columellau00a0during the pre-patent period and identification of transmission areas.

Fascioliasis is regarded to be one of the most important parasitic diseases in domestic ruminants of economical importance and its aethiological agent, Fasciola hepatica (Linnaeus, 1758), is a cosmopolitan trematode in areas of bovine, caprine, ovine, and buffaloes breading (Lessa et al. 2000). Great economical losses are believed to be caused by such parasitism, leading to the decrease of meat and milk production as well as to high mortality rates in several countries in the world (Saleha 1991). Human cases have been reported in the five continents (Mas-Coma et al. 2001), but man participates as an accidental host in the parasite life cycle.

F. hepatica intermediate host snails belong to the genus Lymnaea (Saleha 1991). Lymnaeidae are widely distributed fresh water hermaphrodite pulmonata with a dextral shell and without operculum. There are over 20 species of the genus and many of them are F. hepatica and F. gigantica transmitters. F. hepatica Brazilian intermediate hosts are: L. columella (Say, 1817) and L. viatrix (Orbigny, 1835). The distribution of L. columellain South America has been reported in Venezuela, Colombia, and Equator, to the Andes east (Paraense 1982a). Prepelitchi et al. (2003) reported the first evidence of natural infection of L. columella with F. hepatica in Argentina. In Brazil, the geographical distribution of L. columella is quite broad and it has been reported to be present in the following states: Rio Grande do Sul, Santa Catarina, Paraná, São Paulo, Rio de Janeiro, Minas Gerais, Goiás, Distrito Federal, Mato Grosso, Mato Grosso do Sul, Amazonas, and Bahia (Paraense 1982a, b, 1983, 1986). In Minas Gerais, in the municipality of Itajubá, Silva et al. (1995) remarked the first finding of L. columella naturally infected by F. hepatica.

Traditional methods of F. hepatica detection in L. columella are usually performed by exposing the snails to light and/or posterior dissection, through which cercariae may be observed (Souza et al. 2002). Such method does not allow the infection diagnosis during the pre-patent period, compromising field trials.

Molecular techniques have been extensively used as diagnosis tools. Up to the present, most of the works have reported the use of radioactive probes which hybridise with a ribosomal RNA (ribonucleic acid) region from F.hepatica (Shubkin et al. 1992, Heussler et al. 1993, Rognlie et al. 1994, Kaplan et al. 1995), but such methods are time-consuming besides demanding manipulation of radioactive materials.

The Multiplex-PCR technique consists of using specific primers, simultaneously, in a single reaction, under high stringency conditions. It has been widely used for detection and identification of a wide range of organisms, including: yeast strains; measles virus from clinical samples; isomorphic species from the complex Anopheles dirus; free-living Amoeba in the environment; and snails from the group Bulinus africans (Fujita et al. 1993, Mosqueira et al. 2002, Pelandakis & Pernen 2002, Stohard et al. 2002).

The present work was aimed at designing a pair of primers for the conserved and repetitive region of mitochondrial DNA from F. hepatica that could be used to detect infections by this trematode. Those primers were simultaneously used in a Multiplex-PCR with another pair of primers that amplified the internal transcribed spacer (ITS) region of rDNA of the trematode and molluscs which worked as an internal control.



Parasites, snails, and experimental infections - Adult worms and eggs from F. hepatica (Uruguai strain) were obtained from experimentally infected rabbit gall bladder. The eggs were incubated at 27°C in distilled water in the dark for 12-14 days and then exposed to artificial light for approximately 2 h, at 28 ± 1ºC, for miracidia eclosion. Miracidia were collected with a micropipette under a stereomicroscope and then transferred to cell culture plate with 24 wells of the 2.5 ml liquid capacity. Into each well one L. columella snail was introduced together with five new-ecloded miracidia and the total volume was completed with distilled water. The dishes were covered with a lid for approximately 2 h in order to ensure that the snails were totally immersed in distilled water. The snails were killed at different times of infections (1, 5, 7, 10, 15, 20, and 34 days). The specimens of L. columella, measuring approximately 3-5 mm length, were reared and kept at the Snail Rearing of Centro de Pesquisas René Rachou-Fiocruz, according to Souza and Magalhães (2000). F. hepatica adult worms were frozen and kept at _70°C. The species Cercaria macrogranulosa and Cercaria caratinguensis, obtained from snails collected at Barreiro de Cima, city of Belo Horizonte, state of Minas Gerais, were also included in this study.

DNA extraction - Total DNA was extracted from the snails body, a cercariae pool, and adult worms, using the Genomic DNA Purification Kit Wizard (Promega). Briefly, the material was mechanically disrupted in 200 µl of nucleic lysis solution and incubated overnight with 50 µg/ml proteinase K. Thereafter, 80 µl of protein precipitation solution was added to the initial mix. The mixture was placed in vortex for 10-20 s and centrifuged at 14,000 rpm for 5 min. The supernatant was transferred to a microcentrifuge tube containing 300 µl of isopropanol at room temperature precipitation. The mixture was gently mixed by inversion for 20 min and centrifuged at 14,000 rpm for 5 min. The DNA pellet was washed with 300 µl of 70 % ethanol and centrifuged for 10 min. The pellet was treated with 50 µl of DNA rehydration solution for 30 min at 65°C and stored at _20°C.

Primers design - From the complete mitochondrial DNA sequence of F. hepatica (Le et al. 2001), available in NCBI (National Center for Biotechnology Information) Genbank, specific primers were designed for the conserved and repetitive region of mitochondrial DNA of the trematode, which consisted of identical tandem repeats of 85 nucleotides, rich in G and C. The designed primer pair was: FASCR (5' CCA AAT AAA TAG ATC AGC CC 3') and FASCF (5' ATA TTA AGA GTT GTG CCC C 3'). Anneal-ing temperature was set to be 56°C.

Multiplex-PCR and PCR product analysis - Multiplex-PCR consisted of using simultaneously 2 pairs of primers in a single reaction, under high stringency conditions. The primers FASCR and FASCF flanked a region of mtDNA from F. hepatica, while the other pair,ETTS1 and ETTS2 (Kane & Rollison 1994), flanked the ITS region of the trematode and the mollusc, which worked as an internal control. PCR amplification was carried out in a final volume of 10 µl, with 1ng target DNA, 5 pmol of each primer, 200 µM each desoxyribonucleotide triphosphate (dNTP- PROMEGA), 0.8 units TaqDNA polymerase (Cenbiot RS) in a buffer containing 10 mM Tris - HCl, pH 8.5, 50 mM KCl, 1.5 mM MgCl2. Afterwards, each reaction tube was covered with 20 µl of mineral oil in order to avoid evaporation during PCR cycles. The samples were amplified in thermocycler M J Research, Inc; model PTC-100 (Programmable Thermal Controller). The program used involved 26 cycles: denaturation step at 95°C for 3 min, annealing at 56°C for 1 min and extension at 72°C for 1 min, followed by 25 cycles with denaturation step at 95°C for 45 s, annealing at 56°C for 1 min and extension at 72°C for 1 min, and the last cycle extension period time was changed to 5 min. A negative control (no DNA) was included in all the experiments. PCR products were visualized on 6% silver stained polyacrylamide gels. The gels were photographed using a digital camera.

Reaction sensitivity to detect F. hepatica in L. columella - To establish the reaction sensitivity for F. hepaticadetection in L. columella, amplification of mtDNA was performed. DNA was extracted from 1 to 10 miracidia ofF. hepatica and 1 negative L. columella. DNA from such organisms was quantified by spectrophotometry. DNA from a variable number of miracidia was mixed to a constant amount of total DNA from negative snails, in order to simulate the infection. Thus, DNA from negative snails (7 ng/µl) was mixed with DNA extracted 10 miracidia from F. hepatica (9.8 ng/µl), 5 miracidia (4.8 ng/µl), 3 miracidia (3.1 ng/µl), and 1 miracidia (0.8 ng/µml). Afterwards, PCR was performed as described above. PCR products were visualised on 6% silver stained polyacrylamide gels.



Parasitemia - Evolution of parasitemia is shown in Fig. 1 for the several groups. Comparing the three strains, the Y strain determined the highest and earliest peak of parasitemia by the 10th day of infection; the 21SF strain determined the lowest peak at the 12th day; the Colombian strain showed intermediate parasitemic peak by the 14th day of infection.

Histopathological lesions

Y strain (Biodeme Type I): heart C. callosus infected with the Y strain showed an evolutive parasitism of the myocardium, from the 7th day of infection, increasing rapidly until the 10th day, with an intense parasitism of myocells. An intense myocarditis was present, with diffuse and focal mononuclear infiltrates, and focal necrosis of cardiac cells. From 15 to 20 days (Fig2A) of infection, regression of inflammation and parasitism were seen, with focal mononuclear infiltrates and mild deposits in the connective tissue. Lesions in the myocardium subsided by the 40th day post infection (Fig. 2B).

Skeletal muscles - The sections exhibited normal histology. No inflammation, fibrosis or parasitism was present, and the Picro Sirius staining did not reveal abnormal collagen deposits in the skeletal muscles.

21 SF strain (Biodeme Type II): heart - Lesions in the myocardium were absent until 10 days of infection. From 15 to 30 days of infection, scarce parasites were present with necrosis of cardiac cells as well as mild to moderate focal and diffuse inflammatory infiltration with macrophages and fibroblasts (Fig. 2C). From 35 to 60 days, mild focal inflammatory infiltrates were seen in the myocardium, in the absence of parasites (Fig. 2D); the interstitial matrix was scarce, with thin strands of collagen seen by Picro Sirius staining, in 6/15 cases.

Skeletal muscles - Small perivascular mononuclear infiltrates were seen by the 10th day of infection. No parasites were identified in the examined sections. From 15 to 20 days mild focal infiltrates, scarce parasites and focal necrosis of muscle cells were seen. Up to 35 days after infection, perivascular infiltrates of mononuclear cells were present, as well as focal necrosis of muscle cells, without parasites. Fibrotic alterations were absent. Total regression, of the histopathological alterations was registered from 40 to 60 days post infection.

Colombian strain (Biodeme Type III): heart - In the myocardium, mild to moderate diffuse mononuclear infiltration as well as focal infiltration around necrotic cardiac cells were seen from 15 to 20 days, with presence of macrophages and fibroblasts (Figs 3A, B). From 25 to 30 days of infection moderate to intense, diffuse and focal inflammatory infiltrate, with predominance of macrophages and lymphocytes, was present as well as proliferation of fibroblasts and interstitial matrix deposits ( Figs 3C, D). Picro-Sirius staining showed collagen deposits as slender bundles and peri-vascular thickening. Parasites were scarce in the heart. At 40th day, small foci of destroyed cardiac cells, with focal mononuclear inflammation were present. Picro-Sirius staining for collagen showed fragmentation and a progressive decrease of collagen until the 60th to the 70th day with residual foci of inflammation.

Skeletal muscles - Form 15 to 20 days, lesions of the skeletal muscles were mild and focal, with the presence of intracellular amastigotes, without inflammation, with focal destruction of parasitized muscle cells (Fig. 3E) The lesions became more intense in the skeletal muscle from 25 to 30 days of infection, with focal necrosis of parasitized myocells and the presence of large collections of amastigotes. Intense inflammatory process was present, with predominance of macrophages, lymphocytes and fibroblasts with parasitic debris and polymorphonuclear neutrophils (Fig. 3F,G). In the 35th day of infection, intense interstitial infiltrates were present (Fig. 3H). Strands of collagens were seen in the inflammatory foci (Fig. 3H) and confirmed in the sections stained with Picro Sirius. At the 40th day of infection, the inflammatory lesions became less intense. From 60 to 70 days they were limited to focal areas. Fine strands of collagen appeared in sections stained with Picro Sirius.

Normal controls - Sections of the heart showed normal cardiac structure, with scarce interstitial matrix. Skeletal muscle sections showed characteristic histological structure with a distinct and fine perimisial matrix deposit seen with the Picro Sirius red staining.

Morphometric evaluation of the inflammation

Myocardium - Results of quantitative morphometric evaluation of the number of inflammatory cells in five areas of inflammation of 12 mm2 in the myocardium are shown in Fig. 4A, revealing significant increasing of the number of inflammatory cells from the 25th to the 30th day of infection, followed by a significant decrease from the 40th to 60th day post infection (p < 0.01).

Skeletal muscle - Fig. 4B shows the quantitative evaluation of the inflammatory infiltrates in the skeletal muscle, with significant increase of the number of cells from 25 to 30 days of infection and decrease from 40 to 60 days (p < 0.01).

Morphometric evaluation of the fibrosis

Myocardium, Fig. 5A - The morphometric analysis of fibrosis showed slight increase in collagen deposits, as measured in 5 areas of 12 mm2 with decrease to normal levels by the 60th day. Statistical analysis did not show significance when compared with normal controls.

Skeletal muscle, Fig. 5B -Significant increase of collagen deposits was detected by the 30th to 40th days of infection, and show significant decrease in the 60th day as compared to normal controls (p < 0.01).



Findings of the present study indicate clear differences in C. callosus response to infection by strains of T. cruzifrom different biodemes, regarding the degree of the inflammatory process, parasite histotropism, the intensity of parasitism and the evolution of fibrogenesis. Furthermore, the Y and 21SF strains did not induce a significant increase of the interstitial matrix components. In contrast, the Colombian strain induced an intense inflammatory process in the heart and skeletal muscles. Significant fibrosis, in the skeletal muscle, that spontaneously subsided during the subacute phase of infection (40 to 60 days) has also been detected. The Colombian strain (Type III) is more pathogenic for the C. callosus than types I (Y) and II (21SF) strains, which determined a mild infection, with an early tendency towards the regression of the lesions, and null animal mortality. Histotropism for the myocardium of the three biodemes was maintained in C. callosus, as compared with mice. Presence of amastigotes of T. cruzi in cardiac myocells was detected, although with a low degree of parasitism with the Y and 21SF strain. However the macrophagotropism that is a hallmark of the infection with Type I strains in mice, was scarce in the spleen of C. callosus (data not shown). On the contrary, the tropism for skeletal muscles, characteristic of the Colombian strain, was very intense in the C. callosus, maintaining the same aspects as seen in mice. The C. callosus apparently controls the infection with the Types I and II strains, with inhibition of parasite multiplication and early regression of inflammation, as well as absence of significant fibrogenesis.

The Colombian strain, differently from the others, showed an exacerbation of virulence, expressed by a precocious increasing of the parasitemia differing from the murine model that shows late peaks, from 25 to 30 days. Also, an increased pathogenicity was detected in C. callosus as compared with the murine model. Extensive muscle necrosis and intense inflammatory and fibrotic lesions, were present in C. callosus, confirming the importance of the interaction host/parasite in the patterns of the lesions.

Apparently, several factors, dependent on the parasite strain and on the host response, are responsible for the characteristic lesions in this model. The Colombian strain shows a peculiar behavior in respect to several parameters. The antigenic analysis (Andrade et al. 1981) of the three types of strains showed clear cut differences in their antigenic profiles. Cross reacting antigens were present in the three strains, but Colombian strain has in addition, its own antigenic determinants and this may correspond to differences in its immunogenicity.

The specific neuraminidase activity (Pereira & Hoff 1986), can influence the predominant tropism for the skeletal myocells (Libby et al. 1986). Pereira and Hoff (1986), studying the neuraminidase activity of several strains, have shown higher activity of neuraminidase in the Colombian strain as compared with Y and 12 SF strains, which could be associated with the myotropism of the Colombian strain. The infective trypomastigote forms of T. cruziexhibit neuroaminidase activity and can desialylate cardiac cells in culture. These observations are in accordance with those of Shenkmann et al. (1991, 1992), who identified the transialidase that transfer the sialic acid from the surface of host cells to the surface of the parasite, and participates in the penetration of trypomastigotes into the host cells. These aspects are important but do not exclude the participation of the host response. The genetic types of collagen investigated in a previous study (Magalhães-Santos et al. 2002) do not influence the regression of the collagen deposits. Differing from C. callosus, evolution of the fibrogenesis in the murine model is slow and progressive until the chronic phase of infection (150 to 200 days post-infection). Reversion of fibrosis in mice occurred after specific treatment in the chronic phase of infection, but it did not occur spontaneously (Andrade et al.1991).

C. callosus apparently controls the infection with strains of T. cruzi of BiodemesTypes I and II, being more susceptible to the strains of sylvatic origin represented by the Biodeme Type III (T. cruzi I).

The results confirm the importance of the different biodemes in the determination of tissue lesions and the peculiarities of response of C. callosus to infection with T. cruzi.



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+Corresponding author: Fax: +55-31-3295.3115.
Received 21 January 2004
Accepted 29 April 2004
Partial financial support: Fapemig

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