Download - Naegleria lovaniensis tarasca New Subspecies, and the Purepecha Strain, a Morphological Variant of N. I. lovaniensis, Isolated from Natural Thermal Waters in Mexico

J. Protozool., 37(4), 1990, pp. 301-310 0 I990 by the Society of Protozoologists

Naegleria lovaniensis tarasca New Subspecies, and the Purepecha Strain, a Morphological Variant of N, 2, Zovaniensis,

Isolated from Natural Thermal Waters in Mexico FERMIN RIVERA,* LUBOR CERVA,** JULIO MARTINEZ,*** GEORG KELETI,**** FERNANDO LARES,*

ELIZABETH RAMIREZ,* PATRICIA BONILLA,* SCOTT R. GRANER,*** ASlSH K. SAHA***** and ROBERT H. GLEW***** *Project of Conservation and Improvement of Environment (P. CYMA). Unit of Interdisciplinary Research on Health and Education

Sciences (UIICSE). ENEP- Iztacala, UNAM. Av. de Los Barrios s/n, Los Reyes Izlacala, Tlalnepantla Edo. de Mgxico, Mkxico. **Postgraduate Medical and Pharmaceutical Institute. Research Laboratory of Tropical Medicine.

Rusk6 85. 10000 Pruha 10-Vinohrady. Czechoslovakia, ***Department of Pathology, University of Pittsburgh. School of Medicine,

****School of Public Health, University of Pittsburgh, and *****Department of Microbiology, Biochemistry, and Molecular Biology, University of Pittsburgh, School of Medicine.

Pittsburgh, Pennsylvania 15261. USA

ABSTRACT. Amoebae were isolated from a natural thermal water source in Michoacan, Mexico, in September 1986. Two 500-ml samples were taken from pools with water at 45" C and 46" C and concentrated at 2,000 g for I 5 min. The sediment was seeded on nonnutritive agar plates and incubated at 42" C. The isolates were axenized in bactocasitone-serum medium. The identification of the isolates was based on their morphology, total protein and isoenzyme patterns by agarose isoelectric focusing, serology, fine structure, agglutination with Concanavalin A, sensitivity to trimethoprim, capacity to kill mice, and their cytopathic effect in Vero cells. The results showed several morphophysiological, biochemical and serological differences between the isolates and the type strain Aq/9/ I / 45D of Naegleria lovaniensis. These remarkable differences provide sufficient evidence to consider one of the isolates a new subspecies, and the other one a morphological variant of N. 1. lovuniensis, which can be differentiated from other Naegleriae by their morphology, biochemistry, serology and physiology. The authors propose the name tarasca for the subspecies and purepecha for the morphological variant.

Key words. Atypical nucleolus, gymnamoebae, nonpathogenic, taxonomy, thermophilic.

AEGLERIAfowleri is the etiologic agent of primary amoe- N bic meningoencephalitis (PAM), a fatal disease found in man [I 11. During further investigations of the natural occur- rence and systematics of this species and other small free-living amoebae in Mexico, a culture examination of thermal water from the recreational center "Los Azufres" in the state of Mi- choacan was undertaken in September 1986. Two amoebic strains were isolated by culturing from two thermal water pools at 42" C. Several morphophysiological and biochemical characteristics of these isolates are so peculiar as to make them distinctly different from all known taxonomic entities of the genus Naegleria [a.

MATERIALS AND METHODS Description of the biotope. The natural thermal resort sur-

veyed is located at 19" 45' 46" N, and 100" 39' 20" W, at 2,920 meters above sea level. The climate of the area is of the type C (W,) (W), that is to say, temperate subhumid with summer rains [9]. The described strains have been isolated from man-made reservoirs receiving their water supply from one natural spring of thermal water. The temperature and pH of the pools, at the site of sampling, were 46" C and 45" C, and 6.7 and 6.5, respectively. Water in the reservoirs was very clear and without any striking odor. Green algae and other plant organisms oc- curred on the inner surfaces of the pools. Detailed chemical analysis of this thermal spring was not available.

Sampling. A water sample was collected from each pool into a sterile 500-ml glass bottle. Samples of encrusting material growing on the walls of the pool were also collected in the same manner.

Cultivation. Each 500-ml sample was thoroughly shaken and aliquots placed in two 50-ml tubes and centrifuged at 2,000 g for 15 min. The sediments were deposited on the surface of nonnutrient agar plates streaked with living Escherichia coli (NNE medium), sealed into polyethylene bags and incubated upside down at 42" C [15]. The plates were examined with a microscope daily. Both isolates, as well as the reference strains, were grown simultaneously in culture and subjected to similar nutritional regimes, both in monoxenic and axenic conditions.

Cloning. Each isolate was cloned from isolated cysts in NNE plates. Then, I-cm2 agar blocks were transferred to axenic conditions in bactocasitone-serum medium (BCSM) [2]. The isolates did not grow well in the modified serum-casein-glucose- yeast extract medium (SCGYEM) [16] that the authors commonly use to maintain the reference strains and the gymnamoebae collection of the laboratory.

Light microscopy. Morphological and biometrical data from 100 individuals of each strain were obtained by phase-contrast microscopy, at 10 x , 40 x , and 100 x , using living samples or preserved material stained with Giemsa and trichromic Go- mori. Mitotic figures were studied in slides stained with Giemsa- Robinow after acid hydrolysis.

Electron microscopy. The trophozoites and cysts of both strains from axenic cultures were fixed at room temperature in Karnovsky's fixative and post fixed for 1 h in 1% osmium te- troxide buffered in 0.1 M sodium cacodylate. They were centrifuged at a low speed for 20 min. Pellets were dehydrated in graded series of alcohol and propylene oxide and embedded in Epon resin. Sections 1 pm thick were cut in a LKB ultramicro- tome with a diamond knife and stained with toluidine blue. A general survey of the sections was made. Ultrathin sections were made from the chosen areas, stained with lead citrate and uranyl acetate and examined in a Philips 200 electron microscope.

Indirect immunofluorescence antibody technique (IFAT). Antisera produced in rabbits (2.5 kg) [4], against whole organisms of N . fowleri (strain KUL), and N . lovaniensis (strain Aq/ 91 1 /45D), were used to identify the strains studied. New antisera were prepared in rabbits against the new isolates, using whole amoebae, according to Stevens et al. [18]. For adsorption, the cross-reacting species were harvested, concentrated, and washed twice with phosphate buffer (pH 7.2). A sample of 2.4 ml antiserum (diluted 1%) was added to a pellet of 3 x lo6 amoebae, The adsorption was performed at 37" C with continuous agitation. After 2 h of incubation, the amoebae were removed from the antiserum by centrifugation at 2,000 g for 10 min. This adsorption procedure was repeated five times as suggested by Stevens et al. [ 181.

Double immunodiffusion and immunoelectrophoretic analysis

301

302 J. PROTOZOOL., VOL. 37, NO. 4, JULY-AUGUST 1990

Fig. 1-4. Light microscopy morphology and promitosis of the isolates. 1. Trophozoite of N . 1. taruscu. Nu- nucleolus; Ur-uroid; Ant. Lob.- anterior lobopode; I-insertion of the nucleolus to the nuclear membrane. x 800. 2. Prophase of promitosis. {Ch-chromosomal material; Nu- nucleolar material forming polar bodies; Nm-nuclear membrane. 3. Anaphase of promitosis. Ch. p.-chroinosonal plate; Pm-polar masses. 4.-Telophase of promitosis. Pm-polar mass; Ib-interzonal bodies; Ch-chromosomal material.

(DID and IEF). The method was according to Willaert & Le Ray [ 191. Precipitation reactions were done in 1% (w/v) agarose in pH 8.2 Verona1 buffer (ionic strength 0.1). The specific conditions used in these procedures have been described previously [20, 2 11. Water soluble extracts from axenically growing amoebae constituted the antigens and were prepared as suggested by Willaert [20].

Agarose isoelectric focusing (AIEF). The isoenzyme patterns for acid phosphatase (AP), propionyl esterase (PE) at pH 3-10, and leucine amino peptidase (LAP) at pH 4-6.5, of the strains from axenic cultures were studied following procedures described by De Jonckheere [6]. Total protein patterns at pH 3- 10 and 4-6.5 were also studied according to De Jonckheere et al. [7 ] .

Lectin studies (Concanavalin A). Concanavalin A concentrations of 62.5, 125,250 and 500 pdml were used as employed by Stevens et al. [ 181. After agitation of the samples for 30 min at room temperature, the percentage of agglutination was scored on a scale of 1 + to 4 +, with 4 + representing 100% agglutination of cells in 1 4 large clumps. The end-point titres given in the Results section represent the lowest concentration of the lectin that produced 50% agglutination of cells in clumps of 10-15 cells.

Animal pathogenicity test. A 0.02-ml aliquot of axenic medium containing from 1 O4 to 1 O6 amoebae was inoculated both intranasally (IN), and intracerebrally (IC), into white mice according to Rivera et al. [ 151.

Cytopathic effect. Vero cell (cultures (5 x lo4 amoebae per

RIVERA ET AL. - N . L. TARASCA GYMNAMOEBAE FROM THERMAL WATERS 303

Fig. 5-6. Light microscopy morphology and promitosis of the isolates. 5 . Metaphase of promitosis. Pm-polar mass; Ch. p.-chromo- sonal plate. 6. Cyst of N. 1. iarasca. Nu-nucleolus; P--pore. x800.

75 cm2 monolayer) were used with the isolates grown axenically

Sensitivity to trimethoprim. Using N. fowleri (KUL strain), and N . lovaniensis (Aq/9/1/45D strain) as controls, the test was performed on both isolates according to Cerva [3] in BCSM, using the trimethoprim at a concentration of 2, 4, 10, and 400

Amoeboflagellate transformation test. This test was done using distilled water in slides with living trophozoites according to Rivera et al. [14].

Histopathological study of mice inoculated with the isolates. Sagittal sections of the entire head of five mice intranasally inoculated with the strains studied were examined. The entire head was decalcified, sagittally cut, embedded in paraffin and sections were cut at 6-8 Wm in thickness. The sections were stained with H&E [ 121.

Reference strains. Preparations were obtained from the Lab- oratory of Protozoology of the Institute of Epidemiology and

151.

wg/ml.

Fig. 7. Trophic form of the purepecha strain. Nu-nucleolus; Ps- pseudopodium. x 800.

Hygiene of the Ministry of Public Health from Brussels, Bel- gium, as a kind donation from Dr. Johan F. De Jonckheere as follows: Naegleria .fowleri (30808), Naegleria lovaniensis type strain (Aq/9/ 1/45D), Naegleria australiensis australiensis (PP397), and Naegleria jadini (0400).

RESULTS AND DISCUSSION The strains isolated originated in different pools at slightly

different temperatures, 45" C and 46" C, respectively; thus, the isolates came from different but nearby sites nourished by the same spring. The methodology used and the results obtained clearly document that the observed differences in the organisms studied are stable characteristics and not ecophenotypic varia- tions, since the isolates have been kept in identical axenic culture conditions in BCSM and growth phase, showing always the same morphophysiological characteristics, for more than 3 yr in the laboratory.

Light microscopy of the isolates. Shape and movement of the trophic form. The body shape is typical of limax amoebae and varies constantly depending on the temperature. Between 37" C and 42" C, the trophozoites are usually elongated with one main hyaline anterior pseudopodium and a well developed uroid at the posterior end (Fig. 1, 7). At room temperature the trophozoites acquire a more rounded shape, forming usually lobous or finger-like pseudopodia with hyaline edges. In stained slides of fixed amoebae layers, the prevailing shape is oval with short pseudopodia.

Nucleus. The nucleus is spherical and usually slightly elongated. In phase contrast and in stained preparations the outline of the nucleus is very distinct. In interphasic nucleus the nucleolus has a spindle-like form (Fig. 1, 7). Both ends of the nucleolar spindle appear to be fixed in the nuclear membrane (Fig. 1). Occasionally filamentous formations are visible lying in the axis ofthe spindle (Fig. 1,7). In the prophase of promitosis the nucleolar material transforms and moves to form typical

304 J. PROTOZOOL., VOL. 37, NO. 4, JULY-AUGUST 1990

Table 1. Measurements of the strains studied in fixed preparations (w-4

Nucleolus (in rounded

Strain Length Ikeadth Nucleus projection)

N. I. tarasca Trophic form

Range 12.2-25.5 7.8-18.9 3.3-5.5 1.6-3.3 Mean 18.15 12.89 4.18 2.09

cyst Range 7.0-13.0 7.0-1 1.0 Mean 10.0 9.1

Purepecha Trophic form

Range 12.2-21.1 11.1-17.8 3.9-5.5 1.9-3.3 Mean 16.54 13.56 4.76 2.75

cyst Range 13.61 6.2 9.5-1 3.1 Mean 14.2 11.4

Fig. 8. Flagellate stage of the purepecha strain. uole; Nu-nucleolus. x 800.

Pv-pulsating vac-

polar masses (Fig. 2-5). Interzonal bodies are distinctly visible in anaphase and telophase (Fig. 4). The chromosomal plate is evident in metaphase and in anaphase (Fig. 3, 5). As in all amoebae with promitosis, locomotion continues during nuclear division.

Flagellated stage. When transferred from the agar plates to distilled water, biflagellated forms appear after several minutes. Their body shape is ovoid with one active pulsating vacuole (Fig. 8). However, approximately 50% of flagellates retained the amoebic body shape.

Cysts. Cysts are only spherical in one of the isolates (the proposed purepecha strain), and spherical or lens-like in the other (the proposed N . 1. tarasca subspecies). Their walls are smooth and appear to be double-layered in light microscopy. The cells are mononuclear and their general features closely resemble the cysts of other Naegleriae, including the aggregation of fine granules around the nuclear membrane. The typical spindle-like nucleolus can be observed in the cysts when a conve- nient position of the nucleus is found (Fig. 6). Unlike N . fowi'erz the described strains produce well formed cysts in axenic cultures. Table l shows the measurements of the strains studied in fixed preparations.

Electron microscopy of the isolates compared with the one described for the N. lovaniensis (Stevens, De Jonckheere and Willaert, 1980) type strain. The most remarkable ultrastructural, biochemical, serological, and physiological differential

characteristics found between N. lovaniensis type strain, the purepecha strain, and N. 1. tarasca are shown in Table 2. Other features such as cytoplasmic rough endoplasmic reticulum (RER), Golgi-like structures, free-ribosomes, and membrane-bound dense bodies, although present in the three strains, do not differ significantly between them or with other Naegleriae.

If the ultrastructural taxonomic criteria most commonly used at present for the gymnamoebae are considered [ 1, 8, 10, 13, 18, 191, the most important difl'erences between the purepecha strain and the type strain of A'. lovaniensis are the nucleolar shape and composition, and the attachment of the nucleolus to the nuclear membrane. These rnorphological differences-also shown by light microscopy-are sufficient to declare the purepecha strain as a morphological variant of N. lovaniensis, given that these features are not shown by any other known strain of the same species and even of the same genus. Likewise, the most important additional ultrastructural difference found between N . 1. tarasca and the type strain of N . lovaniensis-that hence- forth should be named Naegleria lovaniensis lovaniensis-is, the outer surface of the external layer of the nuclear membrane which is covered with ribosom'ss, a typical feature found previously only in N . fowleri Cartes, 1970, N . jadini Willaert & Le Ray, 1973 and N . gruberi Schardinger, 1899, but not in N . lovaniensis [ 1, 171. Of greater importance is the lack of a perinuclear layer of RER, shown by N. l. tarasca, provided that the presence of this character was considered species-specific when the description of N . lovaniensbs as a new species was done by Stevens et al. in 1980 [18]. Another constant morphological characteristic that appears in all specimens of N . 1. tarasca, no matter if the cultures are monoxenic or axenic, young or old, or if the amoebae are in trophic or in cystic stage, is the presence of a perinuclear layer of lipid globules. The authors have not

~ ~ ~~

3

Fig. 9-12. Ultrastructure of trophozoite and cyst of the isolates. 9. Trophozoite of the purepecha strain. 1. Nucleus contour; 2. Nucleolus; 3. Fibrous core of nucleolus; 4. Outer surface of external layer of the nuclear membrane, deprived of ribosomes; 5 . Pennuclear layer of RER 6. Lipid globules; 7. Oval and elongated mitochondria; 8. Cytoplasmic RER, 9. Smooth endoplasmic reticulum (SER); 10. Free-ribosomes and polysomes; 11. Golgi-like structures; 12. Mass of heterochromatic chromatin that may correspond to a chromocenter. 10. Trophic and cystic forms of the purepecha strain. 1 & 2. Pore with plug with high rim; 3. Ectocyst; 4. Endocyst; 5 . Annular mitochondria; 6 . Lipid globules; 7. Granular material of nucleolus; 8. Fibrous core of nucleolus; 9. Evagination of the nuclear membrane surrounded by a layer of RER, 10. Perinuclear

RIVERA ET AL-N. L. TARASCA GYMNAMOEBAE FROM THERMAL WATERS 305

layer of RER. 11. Trophic form of N. 1. tarasca. 1. Outer layer of nuclear membrane provided with ribosomes; 2. Spindle-shaped nucleolus; 3. Fibrous core of nucleolus; 4. Granular material of nucleolus; 5. Lack of a perinuclear layer of RER; 6. Perinuclear layer of lipid globules which are surrounded by ribosomes; 7. Oval, cup-shaped, and dumbell-shaped mitochondria; 8. Free ribosomes; 9. Polysomes. 12. Cyst of N. 1. taruscu. 1. Fibrous core of nucleolus; 2. Granular material of nucleolus; 3. Perinuclear layer of lipid globules; 4. Cytoplasmic lipid globules; 5 . Mitochondria; 6. Endocyst; 7. Ectocyst; 8. Pores with plugs with short rim; 9. Microtubules between the endocyst and the cell membrane.

306 J . PROTOZOOL., VOL. 37, NO. 4, JULY-AUGUST 1990

Table 2. Comparative ultrastructural, biochemical, serological and physiological characteristics found among N. lovaniensis type strain, the purepecha strain and N. lovaniensis tarasca.

N . lovaniensis Ultrastructure of trophozoite, Feature Strain Aq/9/1/45D purepecha strain N . 1. tarasca Figure

Nucleus contour Spherical or slightly elongated Slightly elongated irregular “wavy” outline, waves higher than in N. 1. tarasca

Spindle-shaped, oval or rounded depending on the plane of section

Present

Same as in purepecha with short waves

Same as in purepecha strain

9, 10, 11

9, 10, 1 1

9, 10, I 1

9, 10, 1 1

9, 10, 1 1

9, 10

1 1

9, 1 1

9, 11

10,12 10,12 10,12

10

10,12

12

10,12

10

13 14 15

16

17

Nucleolar shape Rounded

Fibrillar core across

Nucleolar composi- the nucleolus

tion

Insertion of nucleolus to nuclear membrane

Nuclear membrane

Present and bulkier than in purepecha strain

The fibrous core and the granular material have approximately the same mass

Present

Absent

Only granular material The bulky substance is granular, being the fibrous core, ‘13 to % of the nucleolar mass

Present Absent

Outer surface of external layer

Present

Absent

deprived of ribosomes Same as in N. lovaniensis type

strain Present

Absent

Outer surface cif external lyaer

Absent covered with ribosomes

Perinuclear layer of


Mitochondria

RER

lipid globules Present, surrounded by ribo-

Same as in purepecha strain somes

Oval, cup-shaped, digitiform ctistae

Dumbbell-, cup-shaped, annular or oval with laminar ctistae

Absent

Present, sparse, not always surrounded by ribosomes

Abundant and conspicuous

Ultrastructure of cyst Spherical 1 to 3, plugs with high rim Same as in N. lovaniensis type

strain

Rosette like struc-

Lipid globules in

SER

tures

cytoplasm

Absent

Not described

Present

Abundant, scattered, always surrounded by ribosomes

Scanty Not described

Spherical or lens-like 1 to 3, plugs with short rim Double layered, endocyst

thicker than ectocyst

Present, surrounded by tibo-

Absent

Oval and abundant

somes

Shape Pores Wall

Spherical 1 to 4, plugs with high rim Double layered, ecto- & endo-

cyst of approximately same thickness

Absent Perinuclear layer of


Mitochondria

lipid globules

RER

Absent

Present Present

Oval, with dense matrices Oval, abundant, surrounded

Absent by ribosomes

Present Microtubules between the endocyst and the cell membrane

Nucleating sites

Lipid globules in cytoplasm

Nucleolar shape

Absent

Present, related to microtubules

Abundant, scal tered, always surrounded by ribosomes

Same as in purepecha strain

Absent

Abundant, scattered, not always surrounded by ribosomes

Spindle-shaped, rounded or oval depending on the plane of section

Biochemistry

Absent

Not described

Rounded or oval

Isoenzyme and total protein patterns Same running patterns in the 3 strains Same running patterns in the 2 isolates, 3 bands of the isolates not present in the type strain Different running patterns between the isolates, several bands of the isolates not present in the type

Slight differences among the isolates and between the latter and the type strain

Remarkable differences among the isolates and the type strain

strain

Acid phosphatase Propionyl esterase Leucine amino pep-

Total proteins (pH

Total proteins (pH

tidase

3-10)

4-6.5) Serology

N. lovaniensis strain Aq/9/1/45D Purepecha strain N. I. tarasca Results show isolates are very similar to N. lovaniensis type strain

Test DID

RIVERA ET AL.-N. L. TARASCA GYMNAMOEBAE FROM THERMAL WATERS 307

Table 2. Continued.

N . lovaniensis Ultrastructure of trophozoite, Strain Aq/9/1/45D purepecha strain N. I. tarasca Figure Feature

Test N. lovaniensrs strain Aq/9/1/45D Purepecha strain Physiology

Agglutination with Positive

Inhibition with tri- Positive Con A

methoprim Maximal growth 45" c

Positive

Positive

N. I. tarasca

Negative

Negative

45" c 46" C temperature

mice Pathogenicity in Negative Negative Negative

Cytopathic effect Positive at 48 h Positive at 24 h Growth in modified Easy Difficult Difficult

Positive at 24 h

SCGYEM

observed this characteristic in the purepecha strain nor is it described for the strains of the species N. lovaniensis in the literature [ 1, 13, 181. The authors also consider that the unique presence of rosette-like structures in the cytoplasm of the trophozoites, of nucleating sites in the cytoplasm of cysts, and of microtubules between the endocyst and the cell membrane of N . I. tarasca-provided they are constant and not occasional characters of the specimens-can also be used to segregate this strain from N . 1. lovaniensis at the subspecies level.

Compared biochemical findings obtained by AIEF with the strains studied. The AP running patterns at pH 3-10 were identical in the three strains (Fig. 13). However, when PE was run at the same pH, the banding patterns of the isolates-although identical-were different from the one shown by the type strain (Fig. 14). Furthermore, the LAP patterns at pH 4-6.5 differed among the strains and the type strain (Fig. 15). There- fore, the AIEF of isoenzymes supported in this case that the isolates-although belonging to the species N. lovaniensis, since they share identical AP patterns with the type strain [8]-have their own and peculiar biochemical differences as evidenced by the PE and LAP runnings, that make them distinct from the known strains of the species N. lovaniensis. These peculiar differences among the isolates and the type strain were also evidenced by the AIEF of total proteins (Fig. l6), especially in the run done at pH 4-6.5 (Fig. 17). Thus, the total protein banding pattern-considered an important tool in the taxonomy of Nae- gleria [8]-suggests that the isolates are distinct among them and to N. lovaniensis type strain.

Compared serological findings obtained by IFAT, IEF & DID with the strains studied. The results of the IFAT test show a close relationship between N . lovaniensis type strain and the

purepecha strain, and a less remarkable relationship with N. I. tarasca. This supports the assignment of the isolates to the species N. lovaniensis, but at the same time emphasizes some important differences, especially between the type strain and N . 1. tarasca (Table 3 ) . On the other hand, the immunoelectrophoretic analysis corroborated the difference between the type strain and N. 1. tarasca, and the similarity between the type strain and the purepecha strain (Table 4). The results of the double immunodiffusion done revealed that the strains share antigenic structures with N. lovaniensis type strain. The serum ofthe latter recognizes the strains studied and N. fowleri. The serum of the isolates recognizes N. Iovaniensis type strain. Also, when adsorbed sera were used, the cross-reactions, including the homologous ones, disappeared. This fact supports the finding that the isolates are very similar to N. lovaniensis type strain (Table 2).

Compared physiological findings obtained with the strains studied by agglutination with Con A, growth inhibition with trimethoprim, maximal growth temperature, pathogenicity test in mice, cytopathic effect, and growth in modified SCGYEM. According to the criteria of Stevens et al. [ 181, the purepecha strain showed 50% ( 2 f ) agglutination with Con A at a concentration of 125 pg/ml, comparable to that shown by strain Ar- 9Mi of N. lovaniensis [ I 71, while N. t! tarasca showed very little agglutination (1 +) even at concentrations of 250 and 500 pg/ ml of the lectin. This result does not satisfy the 50% of agglutination criterion for a positive result. On the other hand, N. lovaniensis type strain showed 50% of agglutination even at a concentration of 62.5 pg/ml of Con A. N. jadini, used as a control, also was not agglutinated, even at a concentration of 500 wg/ml of Con A [ 191. Thus, the agglutination of the purepecha strain constitutes, in this case, additional evidence that

Table 3. Results obtained by immunofluorescence antibody technique.

Serum dilutions

Antisera Antigens 1:64 1:128 1.256 1:512 1:1,024 1 :2,048

Type strain Type strain 4 i 4+ 4+ 4+ 4+ 4+ Purepecha strain 4+ 4+ 4+ 3 + 2+ 1+ N. 1. tarasca 4+ 4+ 3 + 2+ 1+ 0

N. I, tarasca Type strain 2+ 2 + I + 1+ 0 0 Purepecha strain 3 + 3 + 2+ 1+ 0 0 N. I. tarasca 4+ 4 + 4+ 4 + 4+ 3 +

Purepecha strain Type strain 3 + 3 + 1 + I + 0 0 Purepecha strain 4+ 4+ 4+ 4+ 4+ 3+ N. I. tarasca 4 + 4+ 4+ 3 + 2 + 2 +

308

-

- -

L

AP pH 3-10

- A B C D E

13 TP pH 3-10

J. PROTOZOOL., VOL. 37, NO. 4, JULY-AUGUST 1990

PE pH 3-10 LAP pH 4-6,5

A

A B C D E

16

E

TP pH 4-6.5

A B

17

B C D E

15

C I1 E

Fig. 13-17. Isoenzyme and total protein patterns of the isolates and reference strains obtained by agarose isoelectric focusing. 13. Acid phosphatase running done at pH 3-10. A) N. fowleri (30808); B) N. lovaniensis type strain (Aq/9/1/45D); C) N. 1. tarasca; D) Purepecha strain; E) N. a. australiensis (PP397). 14. Propionyl esterase running done at pH 3-10. A) N. fowleri (30808); B) El. lovaniensis type strain (Aq/9/1/ 45D); C) N. 1. tarasca; D) Purepecha strain; E) N. a. australiensis (PP397). 15. Leucine amino peptidase running done at pH 46.5. A) N. fowleri (30808); B) N. lovaniensis type strain (Aq/9/1/45D); C) N. 1. tarasca; D) Purepecha strain; E) N. a. atktraliensis (PP397). 16. Total protein (Coomassie blue R250) running done at pH 3-10. A) N. fowleri (30808); B) N. lovaniensis type strain (Aq/9/1/4 SD); C) N. 1. tarasca; D) Purepecha strain; E) N. a. australiensis (PP397). 17. Total protein (Coomassie blue R250) running done at pH 4-6.5. A) N . fowleri (30808); B) N. lovaniensis type strain (Aq/9/1/45D); C) N. 1. tarasca; D) Purepecha strain; E) N . a. australiensis (PP397).

this isolate belongs to the N. lovaniensis species, and supports its denomination as a morphological variant of this species. Likewise, nonagglutination of N. 1. tarasca constitutes a strong biological difference that can warrant the taxonomic segregation of this strain at the subspecies level [ S , 7, 8, 18, 191.

Another physiological test that is useful in distinguishing crit- ical taxonomic characteristics is .the sensitivity to trimethoprim [3], which in the present case indeed helped to corroborate that the purepecha strain belongs to ithe species N. lovaniensis. The type strain of this species also W,BS inhibited at a concentration

309 RIVERA ET AL.-N. L. TARASCA GYMNAMOEBAE FROM THERMAL WATERS

Table 4. Results obtained by immunoelectrophoresis." ~ ~~ ~

Antiserum

Purepecha Strain Type strain strain N. I. Idrusca

Type strain 6 Not done 2 Purepecha strain 4 3 3 N . 1. tarasca 3 1 4

~~~ ~

Number of precipitation bands.

of 400 Kg/ml. On the other hand, N. 1. tarasca grew normally at the same concentration, showing once more a different physiological behavior when compared with the type strain and the purepecha strain. The authors consider that this biological difference can also be used to segregate N . 1. tarasca as a separate subspecies.

The maximal growth temperature is considered in the literature as a paramount character in the taxonomy of Naegleria andAcanthamoeba[5,7,8, 10, 13, 18, 19,21],andintheprcsent case proved to be a useful tool to ascertain on one hand, the similarity between the purepecha strain and the type strain of N . lovaniensis, and on the other hand, the difference between N . 1. tarasca and the same type strain (Table 2). Thus, the temperature tolerance shown by N . 1. tarasca constitutes another strong characteristic that validates its taxonomic segregation as a new subspecies, provided that known strains of the genus Naegleria cannot grow at 46" C, as N . 1. tarasca does.

The pathogenicity test using mice corroborated that the studied strains, as the described strains of N . lovaniensis, did not kill mice either IC or IN. Likewise, the cytopathic effect in Vero cells yielded the same results for the three strains (Table 2), with the only difference being that the effect was present earlier in the cells in contact with the isolates, than in those in contact with the type strain of N . lovaniensis.

Another physiological character used in the taxonomic segregation of strains of the genus Naegleria is the capacity of the strains to grow in SCGYEM [5, 7, 81, which has been modified by Rivera et al. [ 161. In this latter medium the type strain of N . Iovaniensis grew easily-as it grows in the original SCGYEM- whereas the two isolates grew in it with great difficulty the 1st three days and eventually stopped growing in the next two days (Table 2). This demonstrates again that the isolates, although belonging to the species N . lovaniensis, show peculiar characteristics, in this case nutritional, that make them somehow different from the type strain.

Histopathological study of the inoculated mice. Microscopic examination showed no signs of encephalitis. No amoebae were detected in the olfactory mucosa, olfactory neuroepithelium, or within the olfactory bulbs, frontal lobes, or within any structures of the CNS. Some of the animals, however, showed signs of acute rhinitis with some purulent exudate in the olfactory cavity, possibly related to the cytopathic character of both isolates.

To the authors, the distinction between the strains described and N . lovaniensis does not lie in the number of differences, but in their significance. Indeed, the morphological differences: shape and composition of nucleolus; attachment of the nucleolus to the nuclear membrane; presence or absence of a perinuclear layer of RER; of a perinuclear layer of lipid globules; of rosette- like structures; of nucleating sites in the cytoplasm of cysts; and of microtubules between the endocyst and the cell membrane; together with the biochemical differences in the runnings of PE, LAP and total proteins, the serological ones shown by IFAT and IEF, and the physiological differences elicited by agglutination with Con A, inhibition with trimethoprim, maximal

growth temperature, and growth in modified SCGYEM, are the most significant distinctions. All the latter distinctive characteristics, each with a specific taxonomic weight, helped in the decision to separate the isolates from N. I. lovaniensis, one sim- ply as a morphological variant, and the other one as a subspecies.

The name tarasca proposed for the subspecies has been chosen in honor of the ancient culture developed by the Indians of MichoacLn, and the name purepecha proposed for the morphological variant is related to the Purepechan Indians who 1st inhabited the area where the isolates were found.

ACKNOWLEDGMENTS This work was supported, in part, by CONACYT, The Uni-

versity of Michoach, and by the Pathology Education and Re- search Foundation (PERF) of the Department of Pathology, University of Pittsburgh, and by the United States Public Health Service, grant Al l 8945, from the National Institutes of Health. The authors thank Dr. Blanca L. Barrbn, QBP Hugo Guzman, Dr. Fausto Quezada and Irma Jimknez for their valuable assis- tance.

LITERATURE CITED I . Carosi, G., Scaglia, M., Filice, G. & Willaert, E. 1977. A com-

parative electron microscope study of axenically cultivated trophozoites of free-living amoebae of the genus Acanthamoeba and Naegleria with special reference to the species N. gruberi (Schardinger, 1899), N. fowleri (Carter, 1970), and N. jadini (Willaert & Le Ray, 1973). Arch. Protis- tenkd., 119:264-273.

2. Cerva, L. 1969. Amoebic meningoencephalitis: axenic culture of Naeglcria. Science, 163576.

3. Cerva, L. 1986. Naegleria fowleri and Naegleria lovaniensis: differences in sensitivity to trimethoprim and other antifolates. Z. Par- asitenk., 72: 5 8 5-590.

4. De Jonckheere, J. F. & Van de Voorde, H. 1977. Comparative study of six strains of Naegleria with special reference to nonpathogenic variants of N. fowleri. J. Protozool., 24:304-309.

5. De Jonckheere, J. F. 198 1. Naegleria australiensis sp. nov., another pathogenic Naegleria from water. Protistologica. 17:423-429.

6. De Jonckheere, J. F. 1982. Isoenzyme patterns of pathogenic and nonpathogenic Naegleria spp. using agarose isoelectric focusing. Ann. Microbiol. (Paris), 133A:3 19-342.

7. De Jonckheere, J. F., Pernin, P., Scaglia, M. & Michel, R. 1984. A comparative study of 14 strains of Naegleria australiensis demonstrates the existence of a highly virulent subspecies: N. australiensis italica n. ssp. J. Protozool., 31:324-331.

8. De Jonckheere, J. F. 1987. Taxonomy. In: Rondanelli, E. G. (ed.), Amphizoic Amoebae Human Pathology. Piccin Nuova Libraria, Padova, Italia, pp. 25-48.

9. Garcia-Amaro, E. 1983. Modificaciones al Sistema de Clasifi- caci6n Climatica de Koppen, la. ed., Publicaciones del lnstituto de Geografia de la Universidad Nacional Autbnoma de Mtxico, Mtxico.

10. Martinez, A. J. 1985. Protozoology, taxonomy, and nomencla- ture of free-living amebas. In: Martinez, A. J. (ed.), Free-living Amebas: Natural History, Prevention, Diagnosis, Pathology, and Treatment of Disease. CRC Press, Inc., Boca Raton, Florida, 3:21-42.

11. Martinez, A. J. 1985. Ecology, epidemiology, and environ- mental factors. In: Martinez, A. J. (ed.), Free-living Amebas: Natural History, Prevention, Diagnosis, Pathology, and Treatment of Disease. CRC Press, Inc., Boca Raton, Florida, 4:43-62.

12. Martinez, A. J . 1985. Animal models: PAM and GAE. In: Martinez, A. J. (ed.), Free-living Amebas: Natural History, Prevention, Diagnosis, Pathology, and Treatment of Disease. CRC Press, Inc., Boca Raton, Florida, 9: 127-1 30.

13. Page, F. C. 1988. Taxonomic introduction. In; Page, F. C. (ed.), A New Key to Freshwater and Soil Gymnamoebae, CCAP. Published by the Freshwater Biological Association, The Feny House, Ambleside, Cumbria, LA22 OLP, United Kingdom, pp. 9-15.

14. Rivera, F., Paz, M. E. & Lopez-Ochoterena, E. 1978. Trans- formaci6n ameboflagelar espontanea e inducida en especies del gCnero Naegleria Alexeieff 19 12 emend. Calkins 19 13, recolectadas en piscinas,

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grifos, y reservorios naturales de agua duke de la Cd. de Mexico. Arch. Mex. Anat., 15:9-19.

15. Rivera, F., Medina, F., Ramirez, P., Alcocer, J., Vilaclara, G. & Robles, E. 1984. Pathogenic and free-living protozoa cultured from the nasopharyngeal and oral regions of dental patients. Environ. Res., 33:428440.

16. Rivera, F., Roy-Ocotla, G., Rosas, I., Ramirez, E., Bonilla, P. & Lares, F. 1987. Amoebae isolated from the atmosphere of Mexico City and environs. Environ. Res., 42: 149-154.

17. Stevens, A. R., Gallup, E. & Willaert, E. 1978. Evaluation of membrane-bound black bodies in trophozoites and cysts of Naegleria spp. J. Invert. Pathol., 31~63-76.

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mophilic strains of a new species found in association with Nuegleriu fowleri. Int. J. Parasitol., 103 1-64

19. Willaert, E. & Le Ray, D. 1973. Caracteres morphologiques, biologiques et immunochemiques de Naegkria jadini sp. nov. (Amoe- bida, Vahlkampfiidae). Protistologica, 9:4 17426.

20. Willaert, E. 1976. Etude?, immunotaxonomiques des genres Naegleria et Acanthamoeba (Protozoa: Amoebida). Acta Zoologica et Pathologica Ant verpiensia, 59 : 1 -2:;9.

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Received 4-21 -89: accepted 3-20- 90

J. Prolozool., 37(4), 1990, pp. 310-318 0 I990 by the Society of Protozoologists

On the Cytology and Taxonomic Position of Nudispora biformis N. G., N. Sp. (Microspora, Thelohaniidae), a Microsporidian Parasite of the

Dragon Fly Coenagrion hastulatum in Sweden J. I. RONNY LARSSON

Department of Zoology, University of Lund, S-223 62 Lund, Sweden

ABSTRACT. The microsporidium Nudispora biformis n. g., n. sp., a parasite of a larva of the damsel fly Coenagrion hastulutum in Sweden, is described based on light microscopic and ultrastructural characteristics. Merogonial stages and sporonts are diplokaryotic. Sporogony comprises meiotic and mitotic divisions, and finally eight monokaryotic sporoblasts are relea!;ed from a lobed plasmodium. Sporophorous vesicles are not formed. The monokaryotic spores are oval, measuring 1.4-1.8 x 2.8-3.4 ym in living condition. The thick spore wall has a layered exospore, with a median double-layer. The polaroplast has two lamellar parts, with the closest packed lamellae anteriorly. The isofilar polar filament is arranged in 6 (to 7) coils in the posterior half of the spore. Laminar and tubular extracellular material of exospore construction is present in the proximity of sporogonial stages. In addition to normal spores teratological spores are produced. The microsporidium is compared to the microsporidia of the Odonata; its possible relations to the genus Pseu- dothelohania and to the Thelohania-like microsporidia are discussed. The new genus is provisionally included in the family Thelo- haniidae.

K e y words. Pseudothelohaniu, ultrastructure.

N the summer of 1988 a diseased larva of the damsel fly I Coenagrion hastulatum was given to m e by my colleague Dr. Ulf Norling, Lund. T h e agent was a microsporidium, which in fresh smears appeared t o have ungrouped spores. Further investigations made it clear that the species exhibited octosporo- blastic sporogony, and the cytology was basically of the The- Inhania-type. It differed only in one respect: sporophorous vesicles (pansporoblasts) were not formed. As the presence of persistent or subpersistent sporophorous vesicles is a crucial character for microsporidia of the family Thelohaniidae Hazard & Oldacre, 1975, the microsporidium could not be accommo- dated in an established genus of the family [2]. However, it might be related to the microsporidium for which the genus Pseudothelohania Codreanu & Codreanu-Balcescu, 1982 (J . Protozool., 29:301) was created. This genus was not placed in a family, and to m y knowledge it was never described in the manner required by the International Code of Zoological No- menclature [3].

The microsporidium, which is considered new t o science, is briefly described. The taxonomic position is discussed, and a new genus is created.

MATERIALS A N D METHODS A single infected larva was present in a sample of the damsel

fly Coenagrion hastulatum, collected in a bog at Bokeberg, in southern Sweden, by Dr. Ulf Norling, University of Lund, on August 2 , 1988.

Fresh squash preparations were made by the agar method of

HostounskL & Ziika ( J . Protozool., 26:4 1 A 4 2 A ) , and studied using phase contrast microscopy and dark field illumination.

Permanent squash preparations were lightly air-dried and fixed in Bouin-Duboscq-Brasil solution overnight. For paraffin sec- tioning a part of the body was iiixed in the same fixative overnight, washed and dehydrated in a n ascending series of ethanols, cleared in butanol, and embedded in paraplast. Sections were cut longitudinally a t 10 pm. Squash preparations and sections were stained using Giemsa scilution and Heidenhain's iron haematoxylin. For details on the histological techniques used see the manual by Romeis [ 131. All permanent preparations were mounted in DePeX. Measurements were made with an eye-piece micrometer a t x 1,000.

For transmission electron microscopy infected segments were excised and fixed in 2.5% (v/v) glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) a t 4" iC for 8 and 96 h. After washing in cacodylate buffer and post fixation in 2% (w/v) osmium te- troxide in cacodylate buffer for 1 h a t 4" C, the pieces were washed and dehydrated in an ascending series of buffer-acetone solutions t o absolute acetone, and embedded in epon. Sections were stained with uranyl acetate and lead citrate.

RESLJLTS Pathology. T h e infected larva was recognized from a n anom-

alous white colour of the entire body, including the head. The fat body was almost completely ,disintegrated (Fig. 1). The basal membrane of the lobes formed sacs, where parasite cells were floating among organelles of the host cells. In addition the hy-