Differentiation of Naegleria fowleri from other species of Naegleria using monoclonal antibodies and the polymerase chain reaction

Sparagano, Olivier; Drouet, Emmanuel; Denoyel, G. A.; Pernin, Pierre; Ruchaud-Sparagano, Marie-Hélène

doi:10.1016/0035-9203(94)90532-0

Cited by 10 publications

(9 citation statements)

References 13 publications

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“…This inoculated buffer was incubated 60 min at 55ЊC to release DNA, held for 10 min at 100ЊC to deactivate the proteinase, and then held at Ϫ20ЊC until later use for PCR. Aliquots of the DNA isolates (25 L each) were used for amplification reactions with primers specific for P. piscicida (Bowers et al 2000), N. fowleri (Sparagano et al 1994), and Acanthamoeba spp. (Vodkin et al 1992;Lai et al 1994).…”

Section: Methodsmentioning

confidence: 99%

Amoebae Associated with Ulcerative Lesions of Fish from Tidal Freshwater of the James River, Virginia

Webb

Garman

McIninch

et al. 2002

Journal of Aquatic Animal Health

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While surveying ichthyofaunal diversity in the tidal freshwater James River (near Richmond, Virginia) during March and September 1997, we observed that 40-50% of live fish collected had ulcerative lesions. Concomitant sampling upstream in nontidal reaches produced few fish with lesions (Յ2%). These observations were unprecedented based on similar surveys conducted annually since 1989 and subsequently through 2000. Affected taxa included cyprinids, catostomids, ictalurids, centrarchids, and moronids, representing both resident and migratory species. During this episode of tissue ulceration, free-living pathogenic amoebae, predominantly Acanthamoeba and Naegleria, were the primary organisms associated with lesions across all degrees of severity and across all host species. Based on the focal nature of the lesions, the lack of physical injury to surrounding tissues, the low numbers of fungi and pathogenic bacteria, the absence of Pfiesteria and related organisms, and the large numbers of free-living pathogenic amoebae isolated from all of the lesions, we suggest the epizootic event was an instance of ulcerative amebiasis. The 1997 event in the tidal James River was the first documented case of a fish epizootic from these amoebae in North America in over 20 years and is the first such event described for a mid-Atlantic coastal tributary.

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Section: Methodsmentioning

confidence: 99%

Amoebae Associated with Ulcerative Lesions of Fish from Tidal Freshwater of the James River, Virginia

Webb

Garman

McIninch

et al. 2002

Journal of Aquatic Animal Health

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“…The identification of Naegleria species has been carried out by an isoenzymatic procedure which allowed simultaneous detection of the three main thermophilic Naegleria species. Other immunological and molecular techniques are now available to specifically detect N. fowleri in environmental sites (14,25,26). Recently, ribosomal internal transcribed spacers (ITS) were reported to be useful markers for Naegleria (10,20), and species-specific primers were defined for N. fowleri (20).…”

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confidence: 99%

Use of Multiplex PCR and PCR Restriction Enzyme Analysis for Detection and Exploration of the Variability in the Free-Living AmoebaNaegleriain the Environment

Pélandakis

Pernin

2002

Appl Environ Microbiol

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A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment. Multiplex PCR was also capable of identifying N. fowleri isolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites.The free-living amoebae belonging to the genus Naegleria occur worldwide and inhabit soil and aquatic environments. One member of this genus, Naegleria fowleri, causes primary amoebic meningoencephalitis (PAM) in humans. Although PAM is rare (more than 190 cases reported worldwide [15]), this central nervous system disease is lethal within 1 week in most cases (3). The majority of the fatal infections due to N. fowleri occur in young people exposed to warm water in ponds, swimming pools, and lakes. N. fowleri is thermophilic and generally found in natural and artificially heated water, in particular in the cooling ponds of power plants, in which this species can proliferate intensively (4,11,12,27,28). Two other thermophilic Naegleria species are currently found in these sites: Naegleria lovaniensis, which is harmless, and Naegleria australiensis, which is pathogenic in mice. The thermophilic species Naegleria italica was also reported to be pathogenic in mice but is rarely encountered (7). These two species could be potentially dangerous for humans.In a preventive measure, water monitoring was performed regularly in the nuclear power plants of France in order to check for the proliferation of N. fowleri. The identification of Naegleria species has been carried out by an isoenzymatic procedure which allowed simultaneous detection of the three main thermophilic Naegleria species. Other immunological and molecular techniques are now available to specifically detect N. fowleri in environmental sites (14,25,26). Recently, ribosomal internal transcribed spacers (ITS) were reported to be useful markers for Naegleria (10,20), and species-specific primers were defined for N. fowleri (20). In addition, variations in the sequence and size of the ITS were found within this humanpathogenic species, with five different variants which were mostly detected in France. However, the geographic dispersal and the prevalence of these variants were not well established, since too few samples were examined at different sites.In this study, we applied a simplified ITS PCR procedure to the environmental Naegleria isolates for several reasons: (i) to rapidly and easily analyze a large number of isolates, (ii) to detect the presence of N. fowleri in the potential sites and to further explore the genetic diversity of this species, and (iii) to identify the other thermophilic Naegleria isolates at the sites by using PCR restriction fragment length polymorphism (RFLP) analysis.Amoeba isolation from the environment. Five hundred environmental strains were isolated from water of the cooling ponds and downstream of five different nuclear power plants ...

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“…Samples were initially subjected to PCR using primer sequences specific for Acanthamoeba spp. (Vodkin et al 1992;Lai et al 1994), Acanthamoeba polyphaga (GenBank accession numbers AF407668 and AF407669), Naegleria fowleri (Sparagano et al 1994), P. piscicida (Bowers et al 2000), and Kudoa clupeidae (Moran et al 1999a; KudoaL: 5¢-TGGTTCACTATCGGAATGAACGT-3¢ and KudoaR: 5¢-GCATTAGTTAGTCTTTGATGGGTCTA-AG-3¢) as well as specific primers described below.…”

Section: Molecular Diagnosticsmentioning

confidence: 99%

Etiology of ulcerative lesions of Atlantic menhaden (Brevoortia tyrannus) from James River, Virginia

et al. 2005

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We observed ulcerative lesions on live Atlantic menhaden, Brevoortia tyrannus, during ichthyofaunal sampling in the tidal James River in October 1999 (near Jamestown, VA, USA). Other synoptically collected fishes exhibited no signs of lesions or pre-ulcerative tissues. Live fish were classified as unremarkable (no dermal anomalies), pre-ulcerative (integument intact with boil-like swelling), and ulcerative (severe focal lesions). Specimens were analyzed for bacteria, fungi, and pathogenic protozoans including amphizoic amoebae, Pfiesteria piscicida, and Kudoa sp. No Pfiesteria were detected in any tissue specimen. All B. tyrannus examined, including tissues from unremarkable fish, tested positive for presence of the known fish parasite Kudoa. Only ulcerative lesions were also colonized by bacteria, fungi, and amphizoic amoebae. The absence of bacteria, fungi, and protozoans from unremarkable and pre-ulcerative fish suggests that association of other potential pathogens with B. tyrannus ulcers was due to secondary colonization following lesion formation as a result of Kudoa infection.

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Differentiation of Naegleria fowleri from other species of Naegleria using monoclonal antibodies and the polymerase chain reaction

Cited by 10 publications

References 13 publications

Amoebae Associated with Ulcerative Lesions of Fish from Tidal Freshwater of the James River, Virginia

Amoebae Associated with Ulcerative Lesions of Fish from Tidal Freshwater of the James River, Virginia

Use of Multiplex PCR and PCR Restriction Enzyme Analysis for Detection and Exploration of the Variability in the Free-Living AmoebaNaegleriain the Environment

Etiology of ulcerative lesions of Atlantic menhaden (Brevoortia tyrannus) from James River, Virginia

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