Parasitol ires (1992) 78:479-481
Parasitnlngy Research 9 Springer-Verlag1992
Appearance in Europe of Naegleria fowleri displaying the Australian type of restriction-fragment-length polymorphism* Pierre Pernin 1 and Johan F. D e Jonckheere 2
1 Facult6 de Pharmacie, Laboratoire de Biologic Cellulaire, 3, rue du Professeur Laguesse, F-59045 Lille Cedex, France 2 Protozoology Laboratory, Department of Microbiology, Institute of Hygiene and Epidemiology, 14, Juliette Wytsmanstraat, B-1050 Brussels, Belgium Accepted March 15, 1992
Abstract. We report for the first time the isolation in
Europe of Naegleria fowleri showing a type of restriction-fragment-length polymorphism ( R F L P ) usually found in Australia. The presence o f this type as welt as the European type fluctuated with time in the cooling waters o f the nuclear power station investigated. Two possible explanations for the appearance o f the Australian N. fowleri type in Europe are presented.
The ameboflagellate Naegleriafowleri has been the subject o f much research since its description in 1970 (Carter 1970) as an agent of primary amoebic meningoencephalitis. Strains of this pathogen have been identified worldwide. Although this species is the most homogeneous within the genus Naegleria, restriction-fi'agment-length polymorphism ( R F L P ) of multicopy D N A has been detected among strains of N. fowleri (De Jonckheere 1987; McLaughlin et al. 1988). The multicopy D N A consists mainly of m t D N A and plasmid r D N A (Clark 1990). Although different R F L P types of N. fowleri have been found in the United States, only one type has been isolated in Europe. In Australia, one type has been detected, together with a subtype, that is also the only one present in New Zealand (De Jonkcheere 1988). It has recently been shown that N. fowleri in Japan is closely related to the Australian type (De Jonckheere et al., submitted for publication). The present report discusses the sudden appearance in Europe of N. fowleri exhibiting the Australian R F L P type.
Materials and methods
During a systematic biological water surveillance conducted between 1987 and 1991, samples were taken along the course of the * This work was supported by a grant from Electricit6 de France, D@artment Etudes et Recherches Correspondence to: P. Pernin
Moselle river (France) and inside a nuclear power plant discharging its cooling water into the river. Naegleriafowleri was isolated according to established procedures (De Jonckheere and van de Voorde 1977) but was quantified by most-probable-number methods. Monoxenic Naegleria strains were identified to the species level by isoenzyme analysis using polyacrylamide isoelectric focusing (Pernin and Grelaud 1989). Isolates identified as N. fowleri by isoenzyme analysis were cultured axenically for the isolation of total-cell DNA as described elsewhere (De Jonckheere 1987)~ The RFLP was visualized following ethidium bromide staining of DNA separated by agarose gel electrophoresis after digestion with BamH I, Alu I, Eco RI, Ava I, Bgl II, Hind III, and Pst I. In each run, restriction-enzyme-digested DNA from reference strains was included.
Results and discussion
The origin and date of isolation of the Naegleriafowleri strains studied are listed in Table 1. Two strains of N. fowleri that had been isolated at 3-year intervals from non-thermally stressed water in the Moselle river at a site located 50 km upstream from the nuclear power plant showed the R F L P previously reported for N. fowleri isolates from Europe (De Jonckheere 1987). However, eight strains that had been isolated over a 1-year period (1987-1988) from the thermally stressed water inside the nuclear power station on the same river displayed R F L P patterns (Fig. 1) identical to those of N. fowleri strains from Australia (De Jonckheere 1988). In August and September of 1991, N. fowleri were once more screened for RFLP. This time, only one strain exhibited the Australian pattern, whereas the other three strains showed the pattern usually found in Europe. No N. fowleri strain was recovered during a sampling campaign conducted in March 1991. About 10 years previously, N.fowleri had been isolated from the same river, but at another power plant (Dive et al. 1981), but all of these strains showed the European R F L P type (De Jonckheere 1987). Therefore, the present study is the first to identify the Australian R F L P pattern in strains isolated in Europe.
480 Table 1. Origin and RFLP type of Naegleriafowleri isolates Place
Isolate
Date of isolation
RFLP type
Upstream
Moj200a B120blbR2 Moj32b Moj31c Na420c Na1083c Na1104c Nal 167c Na2195b Na2199c H4B1 H5B4 J2B2 J2A3b
July 1987 June 1990 July 1987 July 1987 July 1988 August 1 9 8 8 August 1 9 8 8 August 1 9 8 8 October 1 9 8 8 October 1 9 8 8 August 1 9 9 1 August 1 9 9 1 September 1991 September 1991
European European Australian Australian Australian Australian Australian Australian Australian Australian European European European Australian
In nuclear power station
The populations of N. fowleri appeared to be unstable. Although N. fowleri seemed to have disappeared in March 1991, in subsequent months the site was recoIonized and the predominant R F L P pattern appeared to be the European type. Since the latter type has occurred in Europe for a long time (De Jonckheere 1987), its prevalence must be higher than that of the recently appearing Australian type and it has a better chance of recolonizing. The observation that populations of N. fowleri are transient has previously been reported in a fish farm using artificially heated water near a nuclear power station in Belgium (De Jonckheere et al. 1983). Although N. fowleri displaying several different R F L P patterns have been isolated in the United States (De Jonckheere 1987, 1988), no change in R F L P type over time has been detected in a power-plant cooling reservoir in Illinois (Huizinga et al. 1990). It is most likely that the Australian type has accidentally been introduced into Europe by human intervention. Investigation of the R F L P type of N. fowleri in screening for this pathogen may provide a better understanding of its dispersal and evolution. It would appear that populations of N.fowleri can travel over large distances, and it could be that we are witnessing the global dispersal of an evolutionary, recently established pathogenic species. Another explanation for the discovery of the Australian R F L P type in Europe might be that mutations leading to the Australian R F L P type have occurred several times on different continents. Although this might seem implausible, identical mutations in different geographic locations have been reported in human genes (Pagnier et al. 1984).
Fig. 1. DNA gel electrophoresis of Naegleriafowleri isolates after digestion with Barn HI, Alu I, and Eco RI as compared with the reference strains of different species. ~, represents the size markers (2 DNA Hind/III+q~X-174 RF DNA/Hae III). Arrows indicate the difference between the European and the Australian profiles
481
References Carter RF (1970) Description of a Naegleria sp. isolated from two cases of primary amoebic meningoencephalitis, and the experimental pathological changes induced by it. J Pathol 100:217244 Clark CG (1990) Genome structure and evolution of Naegleria and its relatives. J Protozool 37:2S-(~S De Jonckheere JF (1987) Characterisation of Naegleria species by restriction endonuclease digestion of whole-cell DNA. Mol Biochem Parasitol 24:55 66 De Jonckheere JF (1988) Geographic origin and spread of pathogenic Naegleriafowleri deduced from restriction enzyme patterns of repeated DNA. Biosystems 21 : 269-275 De Jonckheere JF, Voorde H van de (1977) The distribution of Naegleriafowleri in man-made thermal waters. Am J Yrop Med Hyg 26 : 10-15 De Jonckheere JF, M61ard C, Phillipart JC (1983) Appearance of pathogenic Naegleria fowleri (Amoebida, Vahlkampfiidae) in artificially heated water of a fish farm. Aquaculture 35:73-78
Dive DG, Leclerc H, De Jonckheere J, Delattre JM (1981) Isolation of Naegleriafowleri from the cooling pond of an electric power plant in France. Ann Inst Pasteur MicrobioI 132 [A] :97-105 Huizinga HW, McLaughlin GL (1990) Thermal ecology of Naegleriafowleri from a power plant cooling reservoir. Appl Environ Microbiol 56 : 2200-2205 McLaughlin GL, Brandt FH, Visversvara GS (1988) Restriction fragment length polymorphism of the DNA of selected Naegleria and Acanthamoeba amoebae. J Clin Microbiol 26:1655-1658 Pagnier J, Mears JG, Dunda-Belkodja O, Schaefer-Rego KE, Beldjord C, Nagel RL, Labie D (1984) Evidence for multicentric origin of the sickle cell hemoglobin gene in Africa. Proc Natl Acad Sci USA 81:1771-1775 Pernin P, Grelaud G (1989) Application of isoenzymatic typing of the identification of monoaxenic strains of Naegleria (Protozoa, Rhizopoda). Parasitol Res 75:595-598
Parasitnlngy Research 9 Springer-Verlag1992
Appearance in Europe of Naegleria fowleri displaying the Australian type of restriction-fragment-length polymorphism* Pierre Pernin 1 and Johan F. D e Jonckheere 2
1 Facult6 de Pharmacie, Laboratoire de Biologic Cellulaire, 3, rue du Professeur Laguesse, F-59045 Lille Cedex, France 2 Protozoology Laboratory, Department of Microbiology, Institute of Hygiene and Epidemiology, 14, Juliette Wytsmanstraat, B-1050 Brussels, Belgium Accepted March 15, 1992
Abstract. We report for the first time the isolation in
Europe of Naegleria fowleri showing a type of restriction-fragment-length polymorphism ( R F L P ) usually found in Australia. The presence o f this type as welt as the European type fluctuated with time in the cooling waters o f the nuclear power station investigated. Two possible explanations for the appearance o f the Australian N. fowleri type in Europe are presented.
The ameboflagellate Naegleriafowleri has been the subject o f much research since its description in 1970 (Carter 1970) as an agent of primary amoebic meningoencephalitis. Strains of this pathogen have been identified worldwide. Although this species is the most homogeneous within the genus Naegleria, restriction-fi'agment-length polymorphism ( R F L P ) of multicopy D N A has been detected among strains of N. fowleri (De Jonckheere 1987; McLaughlin et al. 1988). The multicopy D N A consists mainly of m t D N A and plasmid r D N A (Clark 1990). Although different R F L P types of N. fowleri have been found in the United States, only one type has been isolated in Europe. In Australia, one type has been detected, together with a subtype, that is also the only one present in New Zealand (De Jonkcheere 1988). It has recently been shown that N. fowleri in Japan is closely related to the Australian type (De Jonckheere et al., submitted for publication). The present report discusses the sudden appearance in Europe of N. fowleri exhibiting the Australian R F L P type.
Materials and methods
During a systematic biological water surveillance conducted between 1987 and 1991, samples were taken along the course of the * This work was supported by a grant from Electricit6 de France, D@artment Etudes et Recherches Correspondence to: P. Pernin
Moselle river (France) and inside a nuclear power plant discharging its cooling water into the river. Naegleriafowleri was isolated according to established procedures (De Jonckheere and van de Voorde 1977) but was quantified by most-probable-number methods. Monoxenic Naegleria strains were identified to the species level by isoenzyme analysis using polyacrylamide isoelectric focusing (Pernin and Grelaud 1989). Isolates identified as N. fowleri by isoenzyme analysis were cultured axenically for the isolation of total-cell DNA as described elsewhere (De Jonckheere 1987)~ The RFLP was visualized following ethidium bromide staining of DNA separated by agarose gel electrophoresis after digestion with BamH I, Alu I, Eco RI, Ava I, Bgl II, Hind III, and Pst I. In each run, restriction-enzyme-digested DNA from reference strains was included.
Results and discussion
The origin and date of isolation of the Naegleriafowleri strains studied are listed in Table 1. Two strains of N. fowleri that had been isolated at 3-year intervals from non-thermally stressed water in the Moselle river at a site located 50 km upstream from the nuclear power plant showed the R F L P previously reported for N. fowleri isolates from Europe (De Jonckheere 1987). However, eight strains that had been isolated over a 1-year period (1987-1988) from the thermally stressed water inside the nuclear power station on the same river displayed R F L P patterns (Fig. 1) identical to those of N. fowleri strains from Australia (De Jonckheere 1988). In August and September of 1991, N. fowleri were once more screened for RFLP. This time, only one strain exhibited the Australian pattern, whereas the other three strains showed the pattern usually found in Europe. No N. fowleri strain was recovered during a sampling campaign conducted in March 1991. About 10 years previously, N.fowleri had been isolated from the same river, but at another power plant (Dive et al. 1981), but all of these strains showed the European R F L P type (De Jonckheere 1987). Therefore, the present study is the first to identify the Australian R F L P pattern in strains isolated in Europe.
480 Table 1. Origin and RFLP type of Naegleriafowleri isolates Place
Isolate
Date of isolation
RFLP type
Upstream
Moj200a B120blbR2 Moj32b Moj31c Na420c Na1083c Na1104c Nal 167c Na2195b Na2199c H4B1 H5B4 J2B2 J2A3b
July 1987 June 1990 July 1987 July 1987 July 1988 August 1 9 8 8 August 1 9 8 8 August 1 9 8 8 October 1 9 8 8 October 1 9 8 8 August 1 9 9 1 August 1 9 9 1 September 1991 September 1991
European European Australian Australian Australian Australian Australian Australian Australian Australian European European European Australian
In nuclear power station
The populations of N. fowleri appeared to be unstable. Although N. fowleri seemed to have disappeared in March 1991, in subsequent months the site was recoIonized and the predominant R F L P pattern appeared to be the European type. Since the latter type has occurred in Europe for a long time (De Jonckheere 1987), its prevalence must be higher than that of the recently appearing Australian type and it has a better chance of recolonizing. The observation that populations of N. fowleri are transient has previously been reported in a fish farm using artificially heated water near a nuclear power station in Belgium (De Jonckheere et al. 1983). Although N. fowleri displaying several different R F L P patterns have been isolated in the United States (De Jonckheere 1987, 1988), no change in R F L P type over time has been detected in a power-plant cooling reservoir in Illinois (Huizinga et al. 1990). It is most likely that the Australian type has accidentally been introduced into Europe by human intervention. Investigation of the R F L P type of N. fowleri in screening for this pathogen may provide a better understanding of its dispersal and evolution. It would appear that populations of N.fowleri can travel over large distances, and it could be that we are witnessing the global dispersal of an evolutionary, recently established pathogenic species. Another explanation for the discovery of the Australian R F L P type in Europe might be that mutations leading to the Australian R F L P type have occurred several times on different continents. Although this might seem implausible, identical mutations in different geographic locations have been reported in human genes (Pagnier et al. 1984).
Fig. 1. DNA gel electrophoresis of Naegleriafowleri isolates after digestion with Barn HI, Alu I, and Eco RI as compared with the reference strains of different species. ~, represents the size markers (2 DNA Hind/III+q~X-174 RF DNA/Hae III). Arrows indicate the difference between the European and the Australian profiles
481
References Carter RF (1970) Description of a Naegleria sp. isolated from two cases of primary amoebic meningoencephalitis, and the experimental pathological changes induced by it. J Pathol 100:217244 Clark CG (1990) Genome structure and evolution of Naegleria and its relatives. J Protozool 37:2S-(~S De Jonckheere JF (1987) Characterisation of Naegleria species by restriction endonuclease digestion of whole-cell DNA. Mol Biochem Parasitol 24:55 66 De Jonckheere JF (1988) Geographic origin and spread of pathogenic Naegleriafowleri deduced from restriction enzyme patterns of repeated DNA. Biosystems 21 : 269-275 De Jonckheere JF, Voorde H van de (1977) The distribution of Naegleriafowleri in man-made thermal waters. Am J Yrop Med Hyg 26 : 10-15 De Jonckheere JF, M61ard C, Phillipart JC (1983) Appearance of pathogenic Naegleria fowleri (Amoebida, Vahlkampfiidae) in artificially heated water of a fish farm. Aquaculture 35:73-78
Dive DG, Leclerc H, De Jonckheere J, Delattre JM (1981) Isolation of Naegleriafowleri from the cooling pond of an electric power plant in France. Ann Inst Pasteur MicrobioI 132 [A] :97-105 Huizinga HW, McLaughlin GL (1990) Thermal ecology of Naegleriafowleri from a power plant cooling reservoir. Appl Environ Microbiol 56 : 2200-2205 McLaughlin GL, Brandt FH, Visversvara GS (1988) Restriction fragment length polymorphism of the DNA of selected Naegleria and Acanthamoeba amoebae. J Clin Microbiol 26:1655-1658 Pagnier J, Mears JG, Dunda-Belkodja O, Schaefer-Rego KE, Beldjord C, Nagel RL, Labie D (1984) Evidence for multicentric origin of the sickle cell hemoglobin gene in Africa. Proc Natl Acad Sci USA 81:1771-1775 Pernin P, Grelaud G (1989) Application of isoenzymatic typing of the identification of monoaxenic strains of Naegleria (Protozoa, Rhizopoda). Parasitol Res 75:595-598
