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Evidence for geographic clusters: Molecular genetic differences among strains of Pythium insidiosum from Asia, Australia and the Americas are explored

     Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, R3T 2N2 Canada

Leonel Mendoza

     Medical Technology Program, Department of Microbiology, Michigan State University, East Lansing, Michigan 48824-1031

Arthur W. A. M. de Cock

     Centraalbureau voor Schimmelcultures, P.O. Box 85167, 3508 AD Utrecht, Netherlands

Glen R. Klassen ¹

     Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, R3T 2N2 Canada

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Twenty-eight isolates of Pythium insidiosum and P. destruens from Asia, Australia and the Americas were compared on the basis of restriction fragment-length polymorphisms of the amplified ribosomal intergenic spacer. Comparison of band profiles yielded three distinct clusters and an isolate that did not fall into any of the clusters. Cluster I consisted of 16 isolates, all from the Americas (Costa Rica, Brazil, Haiti, United States). Cluster II consisted of seven isolates from Asia (India, Thailand, Japan, Papua New Guinea) and Australia, including the two isolates of P. destruens. This cluster also included a United States isolate from a human who might have contracted an infection of P. insidiosum by contact with food from the Middle East. Cluster III was most distantly related to the other two clusters and consisted of two isolates from Thailand and one from the United States. The isolate excluded from all three clusters was from a spectacled bear in a zoo in the United States. These results indicate that all the isolates are more closely related to each other than to any other Pythium species and thus indeed might be one species, but they also point to geographical variants. Cluster III and Isolate M18 are so distant from the others that they might prove to be separate species. Knowledge of intraspecific variability in P. insidiosum might be important for the management of pythiosis in mammals.

Key words: intergenic spacer (IGS), Oomycetes, pythiosis, ribosomal DNA (rDNA)

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Pythium is a genus of the class Oomycetes that includes about 120 described species (Dick 1990). Pythium insidiosum (de Cock et al 1987) and P. destruens (Shipton 1987) are the only species capable of infecting mammalian hosts, while the other species are saprophytic or pathogenic to plants (Van der Plaats-Niterink 1981). The two species are widely considered to be synonymous (Mendoza and Marin 1989). Pythium insidiosum produces filamentous, noninflated sporangia, which give rise to biflagellate, motile zoospores that appear to be chemotactically attracted to skin, equine and human hair, and leaves of water lilies and grass (Mendoza et al 1993). It is suggested that tissue penetration by invading hyphae might be assisted by secreted proteinases that sufficiently reduce tissue strength (Ravishankar et al 2001). Pythium insidiosum is the etiological agent of pythiosis, a granulomatous disease that occurs in temperate, tropical and subtropical areas of the world. The disease has been reported in horses, dogs, cats, cattle and a captive spectacled bear with symptoms including cutaneous and subcutaneous infections (O'Neill-Foil et al 1984, Miller et al 1985, Mendoza and Alfaro 1986, Bissonnette et al 1991, Chaffin et al 1992, Hnilica 1998, Santurio et al 1998, Thomas and Lewis 1998, Dykstra et al 1999), bone lesions (Mendoza et al 1988, Alfaro and Mendoza 1990), esophagitis (Patton et al 1996), gastrointestinal disease (Miller et al 1983a, Miller 1985, Brown and Roberts 1988, Bentinck-Smith et al 1989, Allison and Gillis 1990, Fischer et al 1994, Purcell et al 1994, Helman and Oliver 1999, Buergelt 2000), and pulmonary infections (Goad 1984). Pythiosis also occurs in humans, causing arteritis (Sathapatayavongs et al 1989, Pracharktam et al 1991, Wanachiwanawin et al 1993, Imwidthaya 1994, Thitithanyanont et al 1998), keratitis (Virgile et al 1993), and cutaneous or subcutaneous infections (Shenep et al 1998).

Identification and classification of Pythium species is often based on morphological characteristics, but complications might arise due to the absence of sexual structures and the failure to induce zoosporogenesis in culture. In addition, environmental factors, such as temperature, media type and age of the culture, can affect morphological and physiological characters and thus hinder the identification process (Hendrix and Papa 1974). Pythium insidiosum is difficult to identify because it usually develops only hyphae, which even might be septate as in true fungi (de Cock et al 1987). The ability to distinguish P. insidiosum from other Pythium species and from other organisms that might cause similar symptoms in the host is crucial for treatment of the disease. Several serological tests have been developed to diagnose pythiosis and identify the etiological agent; these tests include complement fixation tests (Miller and Campbell 1982), fluorescent antibodies (Mendoza et al 1987), immunoperoxidase stain (Brown et al 1988), immunoblot analysis (Mendoza et al 1992a), enzyme-linked immunosorbent assay (ELISA) (Mendoza et al 1997), and immunodiffusion (ID) tests (Mendoza et al 1986, Imwidthaya and Srimuang 1989, Pracharktam et al 1991).

Despite the ability of P. insidiosum to cause potentially fatal infections in animals, very little molecular work has been reported on the identification and phylogeny of the species. In a phylogenetic analysis of Pythium species based on the mitochondrially encoded cytochrome oxidase II gene, two isolates of P. insidiosum formed a clade within a clade of species with filamentous to lobate sporangia but did not form a distinct lineage separate from the Pythium plant pathogens (Martin 2000). This suggested that P. insidiosum might be an opportunistic pathogen on the verge of true animal pathogenicity. The objectives of this study are to establish the applicability of restriction fragment length polymorphism (RFLP) analysis of the intergenic spacer (IGS) between the large subunit and small subunit ribosomal RNA (rRNA) genes for identification of P. insidiosum and for the examination of relationships among isolates from a variety of animal hosts and geographic origins. This simple approach to identification will allow rapid screening of new clinical isolates and will guide phylogenetic analysis of the species.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Culture conditions and DNA isolation – A collection of 31 Pythium isolates was used in this study (Table I). Strains were cultivated in 250 mL flasks containing 100 mL Sabouraud broth. Samples were incubated at 37 C while being rotated at 150 rpm for 5 d and their hyphae harvested by filtration. DNA was isolated according to the method of Möller et al (1992) with modifications as described previously (Klassen et al 1996).

Restriction endonuclease digestion – Restriction digestions of 10 µL amplified DNA were carried out with 2.5 U each of AluI, HaeIII, HincII, HinfI, MboI, RsaI, and TaqI, according to manufacturers' specifications (Invitrogen, Carlsbad, California). All restriction-digestion reactions were stopped after 2 h with the addition of 3.0 µL gel loading buffer (40% (w/v) sucrose, 0.25% bromophenol blue, 20 mM EDTA) to each reaction tube. Digested DNA fragments were separated on a 1.5% agarose gel in 1x TBE buffer, stained with ethidium bromide, and photographed under UV light.

Restriction-fragment data analysis – The length of each restriction fragment was estimated with the 1 kb Plus DNA Ladder (Invitrogen, Carlsbad, California). Only fragments ranging in size from 200–2000 bp were used for analysis. The presence or absence of each restriction fragment from each digestion was scored as "1" or "0" respectively in a binary matrix for the 31 isolates. Genetic distances were calculated for all pairwise comparisons of the isolates and used for the construction of a distance matrix, according to Nei and Li (1979). This was used to produce phenograms with the unweighted pair-groups method with averages (UPGMA) (Sneath and Sokal 1973) and neighbor-joining methods (Saitou and Nei 1987) with the Genetic Data Environment (GDE) (Smith et al 1994) through the Biological Research Computer Hierarchy (BIRCH) at the University of Manitoba (Fristensky 1999).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Isolates of P. insidiosum were selected from a variety of animal hosts representing a wide geographic distribution (Table I). No significant morphological differences among the isolates were observed. Amplification of the IGS between the large subunit and small subunit rRNA genes produced a single PCR product for each of the 28 isolates of P. insidiosum and P. destruens, ranging in size from 4.8 to 5.2 kb. Restriction digestions of the amplicons with seven different restriction endonucleases produced restriction-fragment patterns that were used to examine the intraspecific relationships (HaeIII and HinfI digestions shown in Fig. 1). The 28 isolates of P. insidiosum and P. destruens produced one of four restriction-fragment profiles. The unrooted UPGMA and neighbor-joining phenograms were produced by combining data from all seven restriction endonucleases. Three major clusters (I, II, and III) and one excluded isolate (M18) were observed in the UPGMA phenogram, and within the major clusters, smaller groupings could be noted (Fig. 2). The average genetic-distance values between isolates within each cluster were 0.0196, 0.0200 and 0.0215 for clusters I, II and III respectively. Average values for pairwise comparisons among isolates from different clusters and outgroups were significantly higher (Table II).

View larger version (122K): [in this window] [in a new window]    FIG. 1. (A) HaeIII and (B) HinfI restriction digestions of PCR products of the ribosomal intergenic spacer (IGS) for isolates of P. insidiosum and P. destruens. Lane numbers correspond to isolate designations in Table I. L stands for the 1 kb Plus DNA Ladder and fragment sizes (base pairs) are indicated

Cluster II contained eight isolates that formed two smaller groups. Pythium insidiosum isolates 296 and 393, from a mosquito larva and human respectively in India, were very similar and clustered with human Isolate M21 from Pennsylvania and equine Isolate 297 from Japan. The type culture for P. destruens (M23) clustered with the other P. destruens Isolate M24, both from horses in Australia, and also with P. insidiosum M15 from a horse in Papua, New Guinea. An additional P. insidiosum isolate, M25 from a human in Thailand, was the most divergent member of this cluster.

Cluster III contained three human P. insidiosum isolates (291, M7, and M19). Restriction-fragment profiles revealed prominent differences among these isolates and the profiles of those in clusters I and II. Two isolates, 291 and M7, came from patients in Thailand and were very closely related, while Isolate M19, from a patient in San Antonio, Texas, was located on its own branch.

An additional P. insidiosum isolate, M18, was isolated from a bony lesion on the paw of a spectacled bear (Tremarctos ornatus) in a zoo in Columbia, South Carolina. This isolate failed to group within clusters I, II, or III. Average genetic-distance values within each cluster (about 0.02) were significantly lower than average distances among clusters (Table II). Clusters I and II, with an average genetic distance of 0.0553, are more closely related. The single divergent isolate, M18, is more closely related to clusters I and II than are the outgroups (P. aphanidermatum, P. deliense, and P. grandisporangium), but Cluster III is not significantly closer to any of the other clusters than are the outgroups.

Cluster analysis also was performed with the neighbor-joining method. The resulting phenogram (not shown) essentially was identical to the one generated with UPGMA, with two minor exceptions. Pythium insidiosum 394 clustered with isolates M4 and M16 in Cluster I, and Isolate M25 clustered closer to the India and Japan isolates in Cluster II when compared to the UPGMA phenogram.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Pythium insidiosum is a cosmopolitan organism and the etiological agent of pythiosis in humans and animals. Identification of this organism based on morphology alone can be difficult due to the restricted morphological differentiation in culture. The use of RFLPs of the IGS between the large subunit and small subunit rRNA genes provides a molecular tool to identify isolates of P. insidiosum and contribute information on their relationships and geographic origins. In the description of the species (de Cock et al 1987), isolates from infected animals and humans from various locations were used for morphological and physiological analysis, and no significant differences were reported. Fluorescent antibody and immunodiffusion precipitin tests (Mendoza et al 1987) and immunoblotting analysis (Mendoza et al 1992a) revealed no differences among P. insidiosum isolates, regardless of their geographic origin or host, but distinguished them from other Pythium species. However, at the DNA level, there are significant differences among isolates in the IGS of the rDNA repeat unit that tend to correspond to their geographic origins.

The type cultures of P. aphanidermatum, P. deliense, and P. grandisporangium were used as outgroup species in this analysis. Pythium aphanidermatum and P. deliense are very similar morphologically to P. insidiosum, and the former shares the extraordinarily high optimum growth temperature of 37 C with P. insidiosum, while most other Pythium species grow best at 25–30 C (Van der Plaats-Niterink 1981). Phylogenetic analysis of the mitochondrially encoded cytochrome oxidase II gene also indicated that P. aphanidermatum, P. deliense, and P. insidiosum are closely related (Martin 2000). Sequence analysis of the internal transcribed spacer (ITS) of the rDNA repeats unit indicated that, although P. aphanidermatum and P. deliense are closely related to P. insidiosum, P. grandisporangium is even closer (C.A. Lévesque, pers comm).

Examination of restriction-fragment patterns revealed four general profiles for the 28 isolates of P. insidiosum and P. destruens in this study (Fig. 1). Restriction-fragment profiles of the outgroups (not shown) were significantly different from those of P. insidiosum and P. destruens, as well as from each other (see Table II). Frequently, the sum of the fragment sizes was greater than the size of the original PCR product. This might suggest that multiple variants of the IGS are present within a single isolate, a feature common in other Pythium species (Buchko and Klassen 1990, Klassen and Buchko 1990, Martin 1990).

Cluster analysis of restriction-fragment profiles produced a phenogram revealing three clusters, each comprising isolates from specific geographic locations, and a separate branch for Isolate M18 (Fig. 2). Cluster I consisted of isolates from North, Central and South America, predominantly in tropical and subtropical regions. The smaller groups of isolates within Cluster I corresponded with more proximate geographic origins. The seven isolates from Guanacaste, Costa Rica, formed a tight cluster, and the remaining isolates formed three additional clusters, one of which consisted of isolates from the United States only, while the isolates in the other two clusters were more diverse geographically. No correspondence could be demonstrated between host specificity and clustering patterns.

View larger version (21K): [in this window] [in a new window]    FIG. 2. Unrooted UPGMA phenogram showing relationships among isolates of P. insidiosum. Isolate numbers are in bold and correspond to those presented in Table I. The host and country of origin are indicated in parentheses (Host/Country) with the following abbreviations: Host—Ca, canine; Cq, Culex quinquefasciatus larva; Eq, equine; Hu, human; To, Tremarctos ornatus (Spectacled bear); Country—AU, Australia; BR, Brazil; CR, Costa Rica; HA, Haiti; IN, India; JA, Japan; NG, New Guinea; TH, Thailand; US, United States

The second grouping within Cluster II consisted of P. destruens and P. insidiosum isolates from Australia and New Guinea respectively. Pythium destruens was described by Shipton (1987) based on isolates from horses in Australia. Results from fluorescent antibody and immunodiffusion tests (Mendoza et al 1987, Mendoza and Marin 1989) concluded that the two Pythium species are antigenically and morphologically similar and that P. insidiosum and P. destruens are conspecific. In this report, isolates of P. destruens were present in Cluster II with P. insidiosum isolates from India, United States and Japan (isolate 297, which was originally identified by Ichitani and Amemiya (1980) as P. gracile) and were most closely related to P. insidiosum M15 from a horse in New Guinea. The fact that isolates of P. destruens were present in this cluster provides further evidence that P. destruens and P. insidiosum might be conspecific, or that all isolates in Cluster II should be considered to be P. destruens because the type culture for P. destruens is present. However, since Cluster III and Isolate M18 are genetically more distant from Cluster I than is Cluster II, such a resolution would necessitate the erection of new species names for Cluster III and possibly for isolate M18. Further phylogenetic work is needed to resolve this issue.

Pythium insidiosum M25 came from a patient in Thailand and grouped with the other Asian isolates in Cluster II. However, two other human isolates from Thailand are found in Cluster III. This might indicate that there are two different populations of P. insidiosum in Thailand that infect humans, one of which is very similar to human and animal isolates in Cluster II. Although it was believed that the patient who was the source of Isolate M25 was infected in Thailand, the possibility of infection from neighboring regions by way of visitors or imported foods cannot be excluded.

Cluster III consisted of three human isolates, two from Thailand and one from the United States. With only three isolates, it is difficult to draw conclusions about the genetic relationship among Cluster III and the other isolates. The average genetic distances between Cluster III and clusters I and II were relatively large and in the same range as the values for the outgroups (Table II), suggesting that Cluster III might represent a subspecies of P. insidiosum or a different species altogether. Additional isolates representative of Cluster III are needed to arrive at firm conclusions.

Isolate M18, from the spectacled bear, was divergent from clusters I, II and III, and average genetic distances between M18 and the clusters were large, yet slightly smaller than the values for the outgroups (Table II). This isolate therefore might represent a variant of P. insidiosum or a different species.

Our analysis thus points to the existence of at least three clusters with a high degree of geographical isolation, with two of them (clusters I and II) showing the greatest affinity (Fig. 2). Genetic diversity in P. insidiosum was demonstrated by McMeekin and Mendoza (2000), who showed that streptomycin had varying effects on the growth of P. insidiosum cultures. Growth of isolates from Florida, Costa Rica and Tennessee was inhibited or not significantly affected, while the growth of a human strain from Thailand was stimulated by streptomycin. These contrasting responses are reflected by the variation in the IGS restriction-fragment data presented here.

It is important to identify P. insidiosum accurately because infections caused by this pathogen often mimic the symptoms caused by other organisms, such as species in the genera Basidiobolus and Conidiobolus (Miller 1983). The ID test (Mendoza et al 1986, Imwidthaya and Srimuang 1989, Pracharktam et al 1991) has been used widely to detect pythiosis, but it sometimes fails to detect pythiosis in dogs and humans (Chetchotisakd et al 1992, Wanachiwanawin et al 1993, Mendoza et al 1997, Thitithanyanont et al 1998). Many clinical laboratories are not properly equipped to identify P. insidiosum quickly and this causes delays in treatment. RFLP analysis of the IGS of the rDNA repeat unit provides an additional technique for identifying the causative agent in pythiosis once the organism has been isolated from the host. This simple and inexpensive technique to identify P. insidiosum can produce results very quickly and provide valuable information on the geographic origin of an isolate and source of infection.

Treatment of pythiosis with antifungal agents, such as amphotericin B, which target cell membrane sterols, often is unsuccessful because ergosterol is lacking in P. insidiosum. However, the combination of itraconazole and terbinafine recently was tested successfully in a boy with pythiosis (Shenep et al 1998). In spite of this, surgical removal of the infected and surrounding tissue is often the only solution, although treatment with vaccines derived from P. insidiosum antigens has been shown to have curative properties when the vaccine is injected into horses (Miller 1981, Miller et al 1983b, Mendoza and Alfaro 1986, Mendoza et al 1988, 1992b) and humans (Thitithanyanont et al 1998). Results from these studies showed that some cases of pythiosis in the early stages could be successfully treated with immunotherapy. The unresponsiveness of an infected host to the P. insidiosum vaccine could depend not only on the immunological state of the host, as suggested by Mendoza et al (1992b), but by the strain of P. insidiosum causing the infection. Early identification would allow timely treatment or less invasive surgery.

The IGS of the rDNA repeat unit appears to be a useful tool for resolving intraspecific relationships within a species. In the case of P. insidiosum, variations in restriction-fragment patterns reflected the geographic origin of the isolates. There was, however, no apparent relationship between the host and clustering patterns. Each cluster of isolates represented a genetically distinct population. Results indicated that different populations of P. insidiosum might be endemic to various regions of the world, or that P. insidiosum consists of more than one species and might represent a recently diverging lineage of Pythium species with the ability to infect mammals. RFLP analysis of the IGS of the rDNA repeat unit provides a method for screening isolates rapidly and for inferring their relationships with other isolates and species. These studies will guide future molecular phylogenetic studies of P. insidiosum.

	ACKNOWLEDGMENTS

 
This work was supported by a research grant to GK by the Natural Sciences and Engineering Research Council of Canada and in part by a grant from the Center for Animal Production and Enhancement, Michigan State University to LM. We also thank James Bedard, Shikha Mittoo, Ans de Cock and Roger Herr for research assistance.

	FOOTNOTES

 
¹ Corresponding author. Glen_Klassen{at}umanitoba.ca

Accepted for publication July 10, 2002.

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