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Revista do Instituto de Medicina Tropical de São Paulo

On-line version ISSN 1678-9946

Rev. Inst. Med. trop. S. Paulo vol.42 n.5 São Paulo Sept./Oct. 2000

http://dx.doi.org/10.1590/S0036-46652000000500010 

BRIEF COMMUNICATION

SUSCEPTIBILITY OF Biomphalaria tenagophila AND Biomphalaria straminea TO Schistosoma mansoni INFECTION DETECTED BY LOW STRINGENCY POLYMERASE CHAIN REACTION

 

Liana Konovaloff JANNOTTI-PASSOS & Cecilia Pereira de SOUZA

 

 


SUMMARY

In order to determine Schistosoma mansoni infection rates in Biomphalaria tenagophila and B. straminea, low stringency polymerase chain reaction (LS-PCR) technique was used as a complementary method to light exposure technique. LS-PCR has already been standardized in our laboratory to detect the trematode DNA in B. glabrata. Higher S. mansoni infection rates were detected using conventional method and LS-PCR. The parasite DNA profile was detected in both species after 7-day exposure to miracidia, using LS-PCR. This technique enables early detection of schistosomiasis transmission focuses, in endemic areas, before the beginning of cercariae shedding.

KEYWORDS: Schistosoma mansoni; Biomphalaria tenagophila; Biomphalaria straminea; Susceptibility; LS-PCR technique


 

 

INTRODUCTION

In Brazil, the three Biomphalaria species found naturally infected by Schistosoma mansoni are: Biomphalaria glabrata, B. straminea and B. tenagophila. Biomphalaria tenagophila plays an important role in the transmission of schistosomiasis through the south and southeastern regions of Brazil. Biomphalaria straminea is present from the north to the south of the country, being an important schistosomiasis vector in the northeastern region, in spite of the low natural infection rates in these snails8,9.

The current methods to detect S. mansoni infection in Biomphalaria are: exposition of the snails to light and squeezing them between two glass slides. The first method does not detect the infection during the prepatent period and it is also necessary snails manipulation during an indefinite time in the laboratory. In B. straminea and B. tenagophila species the prepatent period is longer, once it naturally occurs a delay on S. mansoni development. Thus, the conventional techniques are not suitable for detecting S. mansoni in these less susceptible species, due to the long time spent and the mortality of the snails10. Biomphalaria straminea and B. tenagophila infection rates, in experimental infections, according to several authors, are normally lower1,2,3,13,14,16, probably due to the snails mortality before eliminating cercariae. Facing the limitations of the traditional diagnosis methods, other faster and more efficient techniques have been developed based on the detection of the parasite DNA profile in B. glabrata5,6,7.

The aim of the current work was to verify whether the low stringency polymerase chain reaction (LS-PCR) technique, already standardized in our laboratory7 to detect S. mansoni DNA in B. glabrata, would also detect the parasite in other Biomphalaria species, once their profile are different from B. glabrata17. Another objective was to detect the total S. mansoni infection rate in B. tenagophila and B. straminea using light exposure and LS-PCR.

 

MATERIAL AND METHODS

A hundred specimens of B. straminea from Paracatu, MG, and a hundred of B. tenagophila from Pampulha lake Belo Horizonte, MG, reared in laboratory, were individually exposed to 50 S. mansoni miracidia of the LE strain, from Belo Horizonte. After 7 days of exposure, twenty snails of each species were killed and used for DNA extraction. After days 30, 37 and 42 post exposition, the snails were examined after light exposure and the ones shedding cercariae were separated. The DNA was extracted from snails after 7 days of exposure to miracidia, positive and negative snails, snails 42-day after exposure and control group. The DNA extraction was performed using Wizard genomic DNA purification kit (Promega). For polymerase chain reaction (PCR), one ng of template DNA was amplified using 0.8 units of taq polymerase (Cenbiot, RS, Brazil), 200 mM dNTP's, 1.5 mM MgCl2, 50 mM KCL, 10mM Tris-HCl, pH 8.5, and 5 pmol of each primer in a final volume of 10 ml. The pair of primers used in these reactions was designed to amplify across adjacent tandem minisatellite units from S. mansoni mtDNA11. DNA amplification was performed using a MJR thermocycler with the same program described in our previous work7.

 

RESULTS AND DISCUSSION

The infections rate using LS-PCR in 20 snails of each species, killed 7 days after miracidia exposition, were 15.0% for B. straminea and 50.0% for B. tenagophila.

The infection rates obtained by exposing 80 B. straminea and 80 B. tenagophila to light until 42 days after miracidia exposure were 20.0 and 45.0%, respectively (Table 1). When the LS-PCR technique was performed using the specimens, which have not been shedding cercariae until 42 days of exposure, the infection rates increased for 55.0 and 67.6%, respectively (Table 1). The Figs. 1A and B represent the DNA profiles of B. straminea and B. tenagophila infected with S. mansoni after 7 days of exposure to miracidia (lanes 5 to 7), snail shedding cercariae (lane 1), negative snail (lane 2), snails exposed to miracidia that were not infected (lanes 3 and 4), snails after a 42- day exposure that did not shed cercariae, but showed the presence of S. mansoni (lanes 8 to 10), and S. mansoni cercariae (lane 11). The obtained PCR gel electrophoresis profiles (Figs. 1A and B: lanes 1 and 5 to 11) allowed the detection of the parasite by the presence of bands far from each other approximately 62 bp, correspondent to S. mansoni mtDNA amplification (indicated by arrow). The characteristic band standard of the amplified S. mansoni mtDNA was not observed in B. straminea and B. tenagophila, exposed to miracidia and did not shed cercariae (Figs. 1A and B lane 3 and 4), and in negative snails (Figs. 1A and B lane 2). In these lanes we can observe the band complex correspondent to the random annealing of the primers due to the low stringency conditions of the LS-PCR reactions. Those low stringency conditions allow an intern control of the reactions avoiding the necessity of a second pair of primer, which enables the standardization of the technique.

 

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SOUZA et al.15 studied B. straminea and B. tenagophila exposed to miracidia and could observe inflammatory diffused reactions with encapsulated sporocysts by amebocytes. This cellular reaction suggests a low susceptibility and a delay on the parasite development into the snail, which increases the prepatent period making the diagnosis through light exposure very difficult. LS-PCR technique was able to detect S. mansoni infection in B. straminea and B. tenagophila in the prepatent period, 7 days after exposure to miracidia. When both detecting methods were used, it was possible to obtain a higher S. mansoni infection rate (Table 1). In susceptible B. glabrata specimens the infection rate was approximately 90.0% using light exposure technique to detect S. mansoni. However, in partially resistant B. glabrata, small sporocysts in ectopic regions12 were observed as well as a delay on S. mansoni development, with late elimination of the cercariae until seven months after miracidia exposition4, similar to B. tenagophila and B. straminea snails.

Therefore, LS-PCR technique can be used as a complementary tool to the light exposition method in field snail studies, for the three host species, once it detects S. mansoni DNA presence, enabling to distinguish the infected specimens from other trematode infections. The presence of a background parasite`s DNA derived bands are detectable clearly in the three species of infected Biomphalaria using LS-PCR after seven day exposure, despite the extensive interspecific genomic variation in these species17 .

This technique may also be used for early detection of schistosomiasis transmission focuses in the field before cercariae elimination, avoiding larvae contamination of the watercourses and the transmission to man, rodent and other hosts in endemic regions.

 

 

RESUMO

Suscetibilidade de Biomphalaria tenagophila e Biomphalaria straminea a infecção por Schistosoma mansoni detectada pela reação em cadeia da polimerase em baixa estringência

Para determinar a taxa de infecção pelo Schistosoma mansoni em Biomphalaria tenagophila e em B. straminea foi utilizada a reação em cadeia da polimerase em baixa estringência (LS-PCR), como técnica complementar ao método de exposição à luz. A LS-PCR já foi padronizada no nosso laboratório para detectar o DNA do trematódeo em B. glabrata. A taxa de infecção pelo S. mansoni foi maior quando utilizou-se a técnica convencional e a LS-PCR. O perfil do DNA do parasita foi detectado após 7 dias de exposição a miracídios em ambas as espécies, quando utilizou-se a LS-PCR. Esta técnica possibilita a detecção precoce de focos de transmissão, em áreas endêmicas, antes do início da eliminação de cercárias.

 

 

ACKNOWLEDGMENTS

To Dr Alvaro Romanha, Centro de Pesquisas René Rachou, for providing the reagents and equipment for this work.

 

REFERENCES

1. CARVALHO, O.S.; SOUZA, C.P. & FIGUEIREDO, P.Z. - Suscetibilidade de Biomphalaria straminea (Dunker, 1848) de Piripiri (Piaui, Brasil) a duas cepas de Schistosoma mansoni (Sambon, 1907). Rev. Saúde públ. (S. Paulo), 14: 224-229, 1980.         [ Links ]

2. FREITAS, J.R.; JUNQUEIRA, D.V. & GERKEN, S.E. - Habitats primitivos de hospedeiros do Schistosoma mansoni na região de Lagoa Santa, MG. Ciênc. e Cult., 24: 377, 1972.         [ Links ]

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5. HAMBURGER, J.; HE-NA; XIN, X.Y. et al. - A polymerase chain reaction assay for detecting snails infected with bilharzia parasites (Schistosoma mansoni) from very early prepatency. Amer. J. trop. Med. Hyg., 59: 872-876, 1998.         [ Links ]

6. HANELT, B.; ADEMA, C.M.; MANSOUR, M.H. & LOKER, E.S. - Detection of Schistosoma mansoni in Biomphalaria using nested PCR. J. Parasit., 83: 387-394, 1997.         [ Links ]

7. JANNOTTI PASSOS, L.K.; VIDIGAL, T.H.D.A.; DIAS NETO, E. et al. - PCR amplification of the mitochondrial DNA minisatellite region to detect Schistosoma mansoni infection in Biomphalaria glabrata snails. J. Parasit., 83: 395-399, 1997.         [ Links ]

8. LUCENA, D.T. - Epidemiologia do Schistosoma mansoni. An. Soc. Med. Pernambuco, 2: 11-27, 1950.         [ Links ]

9. LUCENA, D.T. - Planorbídeos transmissores da esquistossomose no nordeste do Brasil. Rev. bras. Malar., 15: 13-26, 1963.         [ Links ]

10. PARAENSE, W.L. & CORREA, L.R. - A potential vector of Schistosoma mansoni in Uruguay. Mem. Inst. Oswaldo Cruz, 84: 281-288, 1989.         [ Links ]

11. PENA, H.B.; SOUZA, C.P.; SIMPSON, A.J.G. & PENA, S.D.J. - Intracellular promiscuity in Schistosoma mansoni: nuclear transcribed DNA sequences are part of a mitochondrial minisatellite region. Proc. nat. Acad. Sci. (Wash.), 92: 915-919, 1995.         [ Links ]

12. RICHARDS, C.S.; KNIGHT, M. & LEWIS, F.R. - Genetics of Biomphalaria glabrata and its effect on the outcome of Schistosoma mansoni infection. Parasit. today, 8: 171-174, 1992.         [ Links ]

13. SOUZA, C.P. - Estudo de moluscos do gênero Biomphalaria de Minas Gerais, com relação a adaptação parasito hospedeiro e importância na epidemiologia da esquistossomose. Rev. Inst. Med. trop. S. Paulo, 28: 287-292, 1986.         [ Links ]

14. SOUZA, C.P.; ARAUJO, N.; MADEIRA, N.C. & CARVALHO, O.S. - Suscetibilidade de Biomphalaria tenagophila de Belo Horizonte e adjacências a infeção com três cepas de Schistosoma mansoni. Rev. Inst. Med. trop. S. Paulo, 25: 168-172, 1983.         [ Links ]

15. SOUZA, C.P.; BORGES, C.C.; SANTANA, A.G. & ANDRADE, Z.A. - Comparative histopathology of Biomphalaria glabrata, B. tenagophila and B. straminea with variable degrees of resistance to Schistosoma mansoni miracidia. Mem. Inst. Oswaldo Cruz, 92: 517-522, 1997.         [ Links ]

16. SOUZA, C.P.; RODRIGUES, M.S. & ARAUJO, N. - Suscetibilidade de Biomphalaria straminea (Dunker, 1848) de Belo Horizonte (MG) a infeção por cepas de Schistosoma mansoni. Rev. Inst. Med. trop. S. Paulo, 23: 188-193, 1981.         [ Links ]

17. VIDIGAL, T.H.D.A.; DIAS NETO, E.; SIMPSON, A.J.G. & CARVALHO, O.S. - A low-stringency-PCR approach to the identification of Biomphalaria glabrata and B. tenagophila, intermediate snail hosts of Schistosoma mansoni in Brazil. Mem. Inst. Oswaldo Cruz, 91: 739-744, 1996.         [ Links ]

 

Received: 19 April 2000
Accepted: 13 July 2000

 

 

Centro de Pesquisas René Rachou, FIOCRUZ, Laboratório de Malacologia, Belo Horizonte, MG, Brasil.

Correspondence to: Liana Konovaloff Jannotti Passos, Centro de Pesquisas René Rachou, Laboratório de Malacologia, FIOCRUZ, Av. Augusto de Lima 1715, 30190-002 Belo Horizonte, MG, Brasil.

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