Studies of Laboratory Subcutaneous Infection of Naegleria Fowleri Carter, 1970 in Guinea Pigs| BCG Vaccination and Delayed Hypersensitivity

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1974

Studies of laboratory subcutaneous infection of Naegleria fowleri Carter, 1970 in guinea pigs| BCG vaccination and delayed hypersensitivity

Peter Diffley The University of Montana

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Recommended Citation Diffley, Peter, "Studies of laboratory subcutaneous infection of Naegleria fowleri Carter, 1970 in guinea pigs| BCG vaccination and delayed hypersensitivity" (1974). Graduate Student Theses, Dissertations, & Professional Papers. 3697. https://scholarworks.umt.edu/etd/3697

This Thesis is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. STUDIES OP LABORATORY SUBCUTANEOUS

INFECTION OF Naegleria fowleri CARTER, 1970

IN GUINEA PIGS:BOG VACCINATION

AND DELAYED HYPERSENSITIVITY

Peter Diffley

B.S., Tulane University, 1968

Presented in partial fulfillment of

the requirements for the degree of

Master of Science

University of Montana

1974

Approved by: UMI Number: EP33846

INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted.

In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. UMT Dissertation Publishing

ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ACKNOt^LEDGEMENTS

I would like to express rny sincere appreciation to the graduate

students and faculties of both the departments of Zoology and Micro

biology for their time, advice, and use of materials. Special thanks are extended to: Dr. S.H. Chang of the Environmental Protection Agency,

Cincinnati; who provided me with Naegleria fcwleri cultures and stim ulated much thought in my research. The staff of Stella Duncan Memorial

Institute, and especially Dr. Carl L. Larson provided BOG vaccine, various other materials, and advice throughout this study. Dr. Richard M. Ushijima provided cell cultures. Messrs. William Morrelles and

William Cowen were helpful in providing materials frcm the stock room and animal quarters respectively. Fellow graduate students, Michael Skeels, John Mitchell, and Sen Chi Lu donated time, thought, and energy throughout this study. Drs. Raymond P. Canham, E.W. Pfeiffer, and John F. Tibbs were very helpful as members of my graduate committee. I would especially like to express my gratitude to Dr. Franklin

Sogandares-Bernal, my conmittee chairman, without whose support and guidance this project would have never been undertaken.

And finally to Wanda, who made it all worthwhile.

ii TABLE OF CONTENTS

page ACKNOWLEDGEMENTS i i LIST OF TABLES iv LIST OF FIGURES v

CHAPTERS I INTRODUCTION 1 The Etiological Agent Primary Amoebic Meningoencephalitis Visceral Naegleriosis Host Immune Response Statement of Problem II MATERIALS AND METHODS 22 III RESULTS 28 IV DISCUSSION 32 V SUMMARY 36

LITERATURE CITED 56 LIST OF TABLES

1. Human cases of N. fcwleri-caused primary amoebic meningoencephalitis 37

2. Possible human cases of N. fcwleri-caused primary amoebic meningoencephalitis 39

3. Group I (Control) data: BCG irnmunization, skin tests, and weight change 40 4. Group II data: BCG inmunization, skin tests, initial weights, weight change, and days of infection 41 5. Sensitivity to soluble N. fcwleri derived antigen in Groups II and III 42 6. Comparison of sensitivity to BCG and N. fcwleri 43

iv LIST OF FIGURES

1. N. favleri, amodooid form 44

2. N. favleri, flagellate form. 45

3. N. fcwleri, cystic form 45

4. N. favleri nucleus, prophase 47

5. N. favleri nucleus, metaphase 47 6. N. favleri nucleus, anaphase 47

7. N. favleri, telophase 47 8. N. favleri, photanicrograph of the amoeboid form 49 9. N. fowleri, photanicrograph of the cyst form 49 10. Necrotic lesion of guinea pig at site of infection by N. favleri. 51

11. Comparison of organs frcm a Naegleria- infected and uninfected control guinea pigs 51 12. N. favleri in guinea pig lung tissue 51 13. Nodular lesions in lung tissue recovered from an infected guinea pig- 51 14. Lesions in liver tissue recovered frcm an infected guinea pig 53 15. Liver lesion under higher magnification 53

16. N. favleri shaving distinctive nucleus in liver tissue 53 17. Numerous N. favleri found in liver tissue surrounding liver lesion 53

18. Infiltration at skin test lesion elicited by antigen derived frcm N. fowleri. 55

19. Cell types found at lesion site 55

v CHAPTER 1 INTRODUCTION

Naegleria fowleri Carter, 1970, a member of the order Amoebida (Protozoa:Rhizopodia), has been found to be an etiological agent for primary amoebic meningoencephalitis. Defined by Duma (1972),

"primary amoebic meningoencephalitis is a disease of humans essentially confined to the central nervous system and produced by small, free- living amoebae." Secondary amoebic meningoencephalitis is the term reserved for infections caused by the normally enteric Entamoeba histolytica Schaudinn, 1903 which sometimes occurs in ectopic sites.

Naegleria fowleri was first isolated from a patient in Australia

(Fowler and Carter, 1965). Since then, over sixty cases of primary amoebic meningoencephalitis have been assigned to this etiological agent (Tables 1 and 2). N. fowleri exhibits a world-wide distribution.

Evidence points to a water-borne mode of infection. N. fowleri infects healthy, active, and young humans of both sexes. Human infections of N. fowleri are believed to be fatal within six days of symptomatic onset. Naeglerial-caused meningoencephalitis has been demonstrated in a variety of laboratory animals. The guinea pig (Cavia porcellus

(Linn.)) has been shown to be susceptible to subcutaneous infection of N. fowleri (Culbertson et al., 1972). In this mode of infection, the amoebae caused death to guinea pigs by general visceral invasion

1 2

of the viscera, apparently without involvement of the central nervous system. No human cases of visceral naegleriosis have been reported. Culbertson et al. (1972), however, warned that puncture wounds could be another route of infection by N. fowleri in humans.

The Etiological Agent

Taxonomy

At the supra-familial level, the classification system of Honigberg et al. (1964) was used. This system was the result of ten years of research and international discussion conducted by the Committee on Taxonomy and Taxoncmic Problems established by the Int ernational Society of Protozoologists. Below the supra-familial level, Singh's (1952) classification will be followed. This system is based on distinctive mitotic features and reflects possible phylogenetic relationships.

Carter (1970) named the pathogenic Naegleria sp., N. fowleri.

It was differentiated from the non-pathogenic N. gruberi when no interzonal bodies were observed during mitosis. Since then, Carter

(1972) has admitted possible error in the criteria for naming his new species. Culbertson et al. (1968), Butt et al.(1968), Singh and

Das (1970), and Chang (1971) have all observed interzonal bodies in

Naegleria sp. isolated from human infections in the United States.

Two months after Carter's article, Singh and Das (1970) desig nated Culbertson's HB-1 Naegleria isolate as N. aerobia. Using different taxonomic criteria, Chang (1971) named Culbertson"s 3

HB-1, HB-2, and HB-3 Naegleria isolates, N. invades. Thus, by reason of date priority as prescribed by Article 23 of the International Code of Zoological Nomenclature, N. fowleri is the correct name for the pathogenic Naegleria sp. To determine if more than one Naegleria sp. was responsible for primary amoebic meningoencephalitis, Willaert et al. (1973) sero- tested isolates taken from human patients from Belgium, Czechoslovakia, and the United States. Utilizing imnunoelectrophoresis they established that the four Naegleria strains were serologically identical. One Australian isolate, tested by the same technique, was also identical (Carter, 1973; personal communication). Classification of N. fowleri followed in this study is listed below:

PHYLUM: Protozoa Goldfuss, 1818 emend von Siebold, 1845. SUB-PHYLUM: Sarcomastigophora Honigberg and Balamuth, 1963.

SUPERCLASS: Sarcodina Hertwig and Lesser, 1874.

CLASS: Rhizopodia von Siebold, 1845. SUBCLASS: Lobosia Carpenter, 1861. ORDER: Amoebida Kent, 1880. FAMILY: Schizopyrenidae Singh, 1952. GENUS: Naegleria Alexeieff, 1912 emend Singh, 1952. SPECIES: N. fowleri Carter, 1970.

Synonyms: N. aerobia Singh, 1970; N. invades Chang, 1971.

Diagnosis: Schizopyrenidae. Polar masses and interzonal bodies present during mitosis; division taking place only in the amoeboid 4

phase. Trophic amoeboid phase dominant; locomotion via anteriad

pseudopodia. Limax (slug-like) in shape (22 by 7 microns). Contractile and food vacuoles present in well defined indoplasm. Biflagellate

phase present; achloroplastic; rigid, oval shape (15 by 8 microns);

prominent posteriad contractile vacuole; basal bodies present. Cystic phase is single walled (7 to 10 microns); spherical; with no pores in wall. Excystment via cyst wall disruption. All phases generally uninucleate; centrally placed nucleolus surrounded by a circular clear zone (3 microns in diameter). Pathogenic and serologically distinct. Maximum temperature tolerance of 45-46 C.

Morphology N. fowleri is best studied by use of the hanging drop method utilizing phase microscopy. Carter (1970) and Martinez et al. (1971) have also completed preliminary electron microscopic studies of the amoeboid phase of N. fowleri. Both Naegleria spp. have three phases; amoeboid, flagellate, and cystic. Only the amoeboid form has been observed in the bodies of laboratory-induced or naturally occurring human infections. Amoeboid phase. The trophic form of N. fowleri is amoeboid. The fully extended form is limax (slug-like) in shape with broad anterior and narrow posterior ends. Measurements vary in active forms from

15-30 microns (avg. 22 microns) by 6-9 microns (avg. 7 microns). The diameters of the rounded inactive form ranges from 10-15 microns. Locomotion is accomplished generally by a single pseudopod of clear hyaline ectoplasm. Carter (1970) stated: "pseudopod formation 5 can in no way be described as explosive, except when the amoeba is in an environment of high molarity." Contractile vacuoles, attaining a maximum diameter of 3 microns, vary from 1 to 6 in number.

N. fowleri is usually uninucleate but may rarely be found with 2-4 nuclei (Carter, 1970; Singh and Das, 1970). The resting nucleus consists of a circular, centrally located nucleolus, 1 micron in diameter; surrounded by a clear zone, 2 microns in diameter. A fine nuclear membrane can be observed. The only other structures observed by phase microscopy are numerous granules (0.25-1.00 microns) randomly distributed in the endoplasm.

N. fowleri is generally smaller than N. gruberi under similiar conditions (Singh and Das, 1970). Other than size, there are no differ ences noted between the amoeboid phases of the two species with light or phase microscopy. Both Carter (1970) and Martinez et al. (1971) however, reported many "dumbbell" shaped mitochondria in electron microscope studies of N. fowleri. Cup-shaped and oval mitochondria typical of Schuster's (1963) electron microscope study of N. gruberi were occasionally seen. Schuster (1963) noticed elongated mitochondria in the cystic phase of N. gruberi. The plasmalemma of N. fowleri is composed of a double-unit membrane.

The cytoplasm contains many non-membrane bound fat globules and a poorly organized rough endoplasmic reticulum. No Golgi apparatus was observed. Food vacuoles are bound by unit membranes. The contractile vacuole is a discrete membrane-bound vesicle. The mitochondria, bound by a double unit membrane have thick and prominent cristae irregularly arranged. Endoplasm and ectoplasm were considered 6

indistinct by Carter (1970) and distinct by Martinez et al. (1971). The nucleus of N. fowleri features pores in its double unit membrane. The mucleolus consists of electron dense granules.

Neither Carter (1970) nor Martinez et al. (1971) found viral inclusions noted by Schuster and Svihla (1969) and Dunnebache and Schuster (1971).

Flagellate phase. N. fowleri, taken from 24 hour-old cultures, can

easily be induced to form the flagellate phase. The cultures are flooded with distilled water and maintained at 37 C (Singh and Das, 1970). Chang (1971) noted lower . frequencies of natural flagellate transformation in cultures of N. fowleri than in N. gruberi under similiar conditions. The flagellate phases of both Naegleria spp. are morphologically indistinct except for size (Singh and Das, 1970) utilizing phase or light microscopy. Schuster (1963) has studied the

ultrastructure of N^ gruberi. No electron microscope studies have been undertaken for the flagellate form of N. fowleri.

The generally biflagellate N. fowleri has been observed to bear 3-4 flagellae. Basal bodies are present and centrioles lacking (Fulton, 1972). Flagellae are formed at the broad anterior end.

N. fowleri assumes a rigid oval configuration, 15-8 microns with a single posteriad contractile vacuole. The nucleus lies in the anterior one-half of the body. Its morphology is identical to that of the amoeboid form. Cystic phase. Presumably, in response to dessication, lack of food, or other unfavorable environmental conditions, the Naegleria spp. 7

form cysts. Excystation can be induced to the amoeboid phase within 3-4 hours in a distilled water medium. The cysts of N. gruberi are larger, having a double wall construction containing pores through which the trophozoite excysts. The cysts of N. fowleri have a single wall with no pores having been observed. Cyst wall destruction must be accomplished for excystment (Carter, 1970; Singh and Das, 1970). The cysts of N. fowleri are circular, 7-10 microns in diameter, with no nucleus visible by phase microscopy. Stained preparations show a nucleus typical of the amoeboid and flagellate stages. Carter (1970) reported in the hyaline cytoplasm of the cyst of N. fowleri, 1-3 sausage shaped inclusions (2.0-by 0.5 microns), 1-2 spherical bodies (2 microns in diameter) and about 6 very fine granules. He also reported two types of non-viable cysts constituting a majority of cystic types in his culture. Electron microscope studies of cysts of N. fowleri have not been undertaken. Schuster (1963) observed in N. gruberi encystations an increase in cytoplasmic density, elongated mitochondria, and contractile and food vacuoles.

Mitotic morphology Naegleria spp. reproduce by binary fission. The nuclear morph ology during mitosis is used as a taxonomic criteria in distinguishing

Schizopyrenidae and from Hartmannellidae. The text and illustrations for N. aerobia (=N. fowleri) mitotic phases are taken from Singh and

Das (1970). Prophase (fig. 4). The amoeba does not round off during division. 8

At the beginning of nuclear division, the nucleus swells and the

nucleolus elongates. The chromatin granules lie beside the nucleolus.

They the® begin to fuse and the nucleolus assumes a dumbell shape and divides into two halves, the 'polar masses'.

Metaphase (fig.5). After polar masses are formed, a spindle connection

can be seen, though there are no distinct spindle fibers. A solid mass of chromatin material, without distinguishable chromosomes, then occupies the position of the equatorial plane. Anaphase (fig. 6). The band of chromatin material divides into two halves and each moves towards its pole. When the chromatin material has reached its pole, granular material probably derived from the nucleolus during its division into polar masses (Chang, 1958), and shown to be non-chromatic by Kafalko (1947) in N. gruberi, is seen lying half-way between the polar masses. This 'interzonal beefy' increases in size and divides into two. The nuclear membrane persists during division; it becomes elongated and constricts, giving rise to two daughter nuclei. Telophase (fig. 7). After the nucleus has divided into two, the amoeba becomes elongated and constricts to give rise to two daughter individuals. In the process the thread-like structure joining the two interzonal bodies is ruptured, and each interzonal body fuses with its polar mass to form the nucleolus. In a few amoebae, two nuclei divide at the sane tine.

Polar caps, present in mitotic division of N. gruberi, appear not to be present in N. fowleri (Singh and Das, 1970). 9

Phylogeny

Schuster (1963), utilizing the theories of Corliss (1959), spec ulated on the ancestor of Naegleria. Amoebae are assumed to have arisen frcm flagellate stock. Chryscmonads and cryptcmonads most closely resembles the primitive stock giving rise to animal flagellates.

A chryscmonad with loss of pigment and trophic flagellate, plus develop ment of amoeboid tendencies and phagocytosis, was proposed as a hypothetical ancestor.

Singh (1952) also considered the mode of mitotic division in

Naegleria as primitive. The nuclear membrane and nucleolus remain intact during mitosis. Interzonal bodies and polar masses are formed.

In the course of evolution, the ability to form interzonal bodies and to produce a flagellate phase are lost. The nuclear membrane and nucleolus disappear during mitotic division.

As for the evolution of the pathogenic Naegleria sp., Carter (1972) believed N. fowleri to be a mutant of N. gruberi. This mutation may not have been a recent phenomenon. Because the symptoms of primary amoebic meningoencephalitis are like bacterial meningoencephalitis, the etiological agent might have been overlooked. Retrospective studies by Callicot et al.(1968), Dos Santos (1970), and Symmers (1970) shew possible cases dating frcm 1909 (Table 2).

Physiology

In vivo, N. fcwleri is apparently free-living and only accident ally infects humans. The amoeboid forms of the Naegleria spp. 10

capture bacteria by phagocytosis and digest them in food vacuoles.

The flagellate and cystic phases, have not been observed to feed. Excretion is the function of the contractile vacuoles in the flagellate and amoeboid forms. The contractile vacuole is lost in the cystic phase a-fter the cytoplasm has become condensed through dehy dration.

In mammalian cell cultures and within humans, N. fowleri has been thought to secrete a cytotoxin which breaks dcwn cells for digestion by the amoeba (Chang, 1971, 1974). Martinez et al.(1971) observed erythrocytes and leucocytes within N. fowleri. N. fowleri is aerobic. For this reason, Singh and Das (1970) named the pathogenic Naegleria sp., N. aerobia to distinguish it frcm anaerobic amoebic infections. N. fowleri has a maximum temperature tolerance of 45-46 C (Griffin, 1972) and an optimum temperature for growth at 35-37 C (Chang, 1972).

N. fowleri encysts when dessicated or short of food. The amoeba becomes rounded in appearance, cytoplasm becomes dense due to dehydration, and the cyst wall is secreted. The cytoplasm, filling the cyst, slowly decreases in observable activity. Excystation, when environmental conditions: " improve" begins with the cytoplasm disasso ciating from the cyst wall. In N. fowleri, cyst wall disruption is necessary for excystment. Locomotion in the amoeboid state is accomplished by pseudcpodial formation and cytcplamic streaming. Lastovica and Dingle (1971) isolated an actonyosin complex from N. gruberi. They proposed 11

that it functioned in a non-muscular contractile phenomenon associated with cytoplasmic streaming.

Flagellate transformation in N. gruberi has been studied by Chang (1958), Schuster (1963), and Yuyuma (1971). Factors affecting flagellate transformation were also reported by Willmer (1960). The flagellae are formed de novo by filamentous extension of an endoplasmic protrusion. Basal bodies are present at the base of the protrusion. Flagellate transformation is inhibited by temperature (Chang, 1958)? metabolic protein synthesis inhibitors (Yuyuma, 1971); and various salts and ions (Willmer, 1960). Frcm these results, it is hypothesized that flagellate transformation of the Naegleria spp. occurs with a flooding or dilution of its environment. It entails HSfA and protein synthesis, and utilizes enzymatic reactions with an cptimmi efficiency frcm 10-26 C. Acetylcholine is a metabolic requirement for transformation. Transformation to the amoeboid phase occurs usually within 4-6 hours. The flagellae are either cast-off or reabsorbed. The cystic phase of N. fcwleri appears not to be infectious

(Carter, 1970). Flagellate forms have not been tested for infectivity. Only the amoeboid form has been observed din necropsies of experimentally induced or natural human infections.

Routes of infection

In human infections it has been concluded by researchers that N. fcwleri gained entrance via the nasal passages, penetrated the nasal mucosa, passed through the cribriform plate, and thence to the brain. 12

In autopsies, the tissues surrounding the cribriform plate and olfactory lobes are very highly hemorrhagic and necrotic. Amoebae

have been found only once in organs other than the brains of human infections (Duma et al. 1969), and in the blood circulation twice (Carter, 1968; Duma et al., 1969). There is no other evidence that implicates other sites of entry into the human host (Duma, 1972).

Primary Amoebic Meningoencephalitis

According to Chang (1974), two species have been irrplicated as etiological agents for primary amoebic meningoencephalitis. Culbertson et al. (1958,1959) isolated a pathogenic Acanthamoeba sp. which was later reclassified by Singh and Das (1970) as Hartmannella culbertsoni and by Chang (1971) as Acanthamoeba rhysodes. It has been implicated in human infections of meningoencephalitis according to Chang (1974) in those cases reported by Patras and Andujar (1966) ; Roarke et al.

(1971); and Jagger and Stramm (1972). These infections were not acquired while swinming and involved individuals debilitated by chronic liver ailment, alcoholism, or undergoing immunosuppressive treatment. The route of entry in these cases is believed to have been the blood circulatory system (Chang, 1974). This pathogenic

Acanthamoeba sp. has also been irrplicated in respiratory ailments by

Wang and Feldman (1967) and Armstrong and Pereira (1967).

The first human cases of primary amoebic meningoencephalitis in which N. fowleri was isolated were located in Australia (Fowler and Carter, 1965). 13

Symptoriatology

The clinical course of primary amoebic meningoencephalitis caused by N. favleri has been explicitly described by Butt (1966), Callicot et al. (1968), Carter (1968), and Duma et al. (1971). The incubation period was determined by Serva et al. (1968) to be frcm 3-7 days.: ; The onset of symptoms in humans begins abruptly with a persistent and increasing frontal headache. Within three days it is accompanied by nuchal rigidity, fever (101-106!F), nausea, and vcmiting. A period of disorientation progresses to coma and death through cardio respiratory failure. Experimental intranasal infections of mice and guinea pigs by

N. fowleri have been described as running a similiar clinical course to that observed in humans (Carter, 1970; Cerva, 1971b).

Gross pathology

Human pathological findings have been summarized by Cerva et al.

(1968), Duma (1972), and Carter (1972). Generally limited to the central nervous system, human autopsies show a sanguinopurulent meningitis invilving the olfactory, frontal, and temporal lobes and the cerebellum. Only one human case has demonstrated amoebic invasion of organs outside of the central nervous system. Duma et al.(1969) isolated N. fowleri from the lung, liver, and spleen, of a fifteen year old girl.

Carter (1970), studying intracerebral infections of mice, found the pattern of invasion of N. fowleri identical to that observed 14

in man. Autopsies of mice revealed necrotic, hemmorrhagic olfactory lcbes.and brain swelling. Necrosis ceased at the proximal end of the olfactory lobes. Amoebic invasion proceded as far as the lateral

ventricles. Purulent meningoencephalitis was usually inconspicuous. Kidneys, lungs, spleen, and liver all contained N. fcwleri.

Myocarditis in humans has been reported by Carter (1968) and Duma et al.(1969). This was believed to be induced by a myotoxin produced by N. fcwleri in the brain.

Histqpathology

An electron microscope study of cerebral tissue of laboratory mice infected with N. fowleri was undertaken by Martinez et al. (1971). They observed numerous N. fcwleri "in close contact with neurons and

glial cells. Sane astrocytes showed marked degenerative changes.

Axons in the vicinity of the amoebae were swollen and shewed an increase in volume of axoplasm. Focal loss of myelin was also observed

along damaged axis cylinders. Dendrites were swollen and edematous".

Carter (1968) and Cerva (1971b) observed N. favleri as an occasional intracellular parasite of neural cells.

Treatment

Only the anti-fungal agent anphotericin B has proven to be highly effective in vitro (Carter, 1969; Duma et al. 1971) and occasionally effective in vivo. Anphotericin B was proven to protect mice frcm

N. favleri infections by Culbertson et al. (1968) and Carter (1969).

TWo human cases in Virginia (Duma et al., 1971) and two frcm Australia 15

were treated with this drug without success. Apley's (1970) two

unconfirmed cases of N. fcwleri infections were treated with anphotericin B and survivied. The first conclusive proof of anphotericin B's effectiveness as a cure for naeglerial-caused meningoencephalitis was reported by Carter (1972) and Anderson and

Jamieson (1972).

Epidemiology Fcwler.and Carter (1965) suspected an intranasal route of infection for N. fowleri in humans. Butt (1966) inplicated lakes and sewage effluents as areas harboring the etiological agent for primary amoebic

\j meningoencephalitis. Cerva et al.(1968) established a heated indoor swimming pool as the site of sixteen infections frcm 1962-1965. Lakes have been implicated in 22 cases frcm Florida, Virginia, and Georgia.

Heated swimming pools were considered sites of infection for three cases in Belgium and sixteen cases in Czechoslovakia. A California hot springs, mud puddles in Britain, and an open swimming pool in

Czechoslovakia were implicated in three deaths. In Australia, Anderson and Jamieson (1972) isolated N. favleri frcm a municipal water supply system.

Prevention Cerva (1971a) proposed measures designed to prevent human exposure to N. fowleri that included emptying and cleaning the swimriing pool daily, steam cleaning of the sand filters, and reduction of free 16

surface area within the water. Jadin et al.(1971) suggested restriction

of the pool area to swimmers, hot showers and antiseptic foot baths

required before gaining access to the pool area, and limitations on the number of swimmers at one time.

N. fowleri apparently can survive and be infectious at chlorine

concentrations of 0.3 mgm/liter. Bromidization does not offer any advantages oVer tihlorination (Jadin et al., 1971). Anderson and Jamieson (1972) reported that salination of swimming pool water to

0.7 per cent weight/volume eradicated N. favleri. Lakes offer even more of a problem for protective measures

against infection with N. fowleri. Ihe Virginia epidemic was only curbed by isolating three lakes as the sites of infection and closing them by court order.

Visceral Naegleriosis

Culbertson et al. (1972) discovered that guinea pigs were susceptible to experimental subcutaneous and intramuscular infections

of his HB-1 (Culbertson et al., 1968) isolate of N. fowleri. Ten thousand amoebae were injected into each guinea pig. Symptoms noted were a general loss of body weight and strength. Death ensued within

thirty days post exposure. Gross examination of infected animals

revealed lesions at the site of injection; and on the liver, lungs,

and small intestine. The spleen was enlarged. Upcai microscopical examination the muscle and fatty tissue showed a mixture of acute 17

abcess, fat necrosis, and fibrous tissue proliferation. Amoebae were clustered around arteries and veins. The vessels were usually thrombosed. The liver was a site of a large number of amoebic lesions.

Focal lesions were found in the small intestine and nodular granulo matous lesions appeared throughout the lungs. Amoebae were also seen in the glomerular capillaries. The central nervous system was not infected.

This information prompted Culbertson et al. (1972) to warn of possibility of wounds being infected with N. favleri. Though no conclusive evidence has been reported for human cases of visceral naegleriosis, S.S. Browne decribed in Carter's (1972) review rare bodies ressembling slime amoebae in histological sections of a granulomatous skin rash occuring after a penetrating wound. Mice appear incapable of being infected by N. fowleri by subcutaneous or intramuscular routes (Culbertson et al., 1968; Carter, 1970).

Host Immune Response

Mammals have both natural and acquired immune systems to combat infection. An aggressive form of natural immunity is the inflammatory response. It is an immediate and non-specific response to host tissue damage. Cellular components of the inflammatory response may include polymorphonuclear leucocytes and macrophages. These white blood cells accumulate at the site of inflammation and often ingest damaged host cells, cell debris, and foreign particles. The acquired immune system consists of both humoral and cellular 18 responses. The humoral response consists of the production of specific immunoglobulins by the B-lymphocytes in response to a specific antigen. Acquired cellular immunity is mediated by a T-lymphocyte/ macrophage system.

Acquired cellular iiimunity, though stimulated by a specific antigen lacks specificity in its expression (Mackaness, 1964). Therefore, the immunity induced in mice by Toxoplasma gondii is effective in the same animal against infections of Besnoitia jellisoni (Raskin and

Remington, 1969). This ncai-specific response has been observed to cross phylogenetic barriers. To mention a few, T. gondii or B. jellisoni infections in mice prevent further infections by the bacterial parasites Listeria monocytogenes and Salmonnella typhimurium; and Mengo's virus (Ruskin and Remington, 1969).

BOG (bacille Calmette-Guerin) is an attenuated strain of Mycobacterium bovis (Class rStihizomycetes; Order:Acrtinomycetes; Family:

Myoobacteriaceae). BCG immunization acts to non-specifically enhance cellular irrmune response. It confers protection in mice to the bacterial infections L. monocytogenes and Klebsiella pneumoniae (Coppel and Young, 1969), Brucella melitensis (Elberg et al., 1957}, and Salmonnella enteritides (Howard et al., 1959). Larson et al. (1971, 1972b) demonstrated the protective effects of BCG immunization in mice infected with Friend's Disease virus. Herpes type II viral infections were prevented in BCG immunized rabbits (Larson et al., 1972a). Studies by Gcble et al. (1963) and later by Smrkovski

(1974; unpublished results) demonstrated BCG's protective effect 19

against Leishmania donovoni infections in mice. In related studies

Bryoeson et al. (1970) were able to confer partial immunity in guinea pigs against Leisbmania enrietti using complete Freund adjuvant plus

soluble and insoluble antigen fractions. Complete Freund adjuvant was again used by Krahenbuhl et al. (1972) to protect mice and rabbits against T. gondii infections. Krahenbuhl et al. (1972) claimed that

the Freund adjuvant's ability to enhance non-specific cellular irrmune

response prevented the protozoan infections.

Most of the parasitic protozoan-host irtmune interactions studied have involved intracellular parasites. Generally, it has concluded that the cell immune response was the most effective system

for resisting the infection with humoral antibodies sometimes playing

a supporting role.

Since the course of primary amoebic meningoencephalitis is short and is apparently fatal upon first exposure, there appears to be only a non-specific cellular response in humans. Polymorphonuclear

leucocytes, mostly neutrophils, are found in high numbers both in the cerebrospinal fluid and blood circulation. Martinez et al. (1971)

described only a moderate inflammatory response within the brain tissues of experimentally infected mice. Polymorphonuclear leucocytes were present including eosinophils, and macrophages. Phagocytic

activity of N. fowleri by these white blood cells was observed. Cerva (1971b)observed naeglerial meningoencephalitis in guinea pigs.

There appeared to be cnly a non-specific response in infected brain tissue. Visceral invasion of N. fowleri elicits a non-specific 20 response in all sites of infection (Culbertson et al., 1972). Specific humoral response in guinea pigs to naeglerial meningoencephalitis was demonstrated by CervaV (1971b) using complement fixation techniques. Delayed hypersensitivity, indicitive of an acquired cellular immune response, has not been tested in cerebral or visceral infections by N. fowleri prior to this study. Delayed hypersensitivity is characterized by a non-specific immediate hypersensitivity which peaks at between four and six hours after antigenic stimulation. Generally, polymorphonuclear leucocytes are the dcrainent white blood cell type at the lesion site. However, lymphocytes pass through the inflammation site. The lymphocytes sensitized to the antigen injected remain at the site and secrete a cytotoxin thus increasing the inflammation. Macrophages infiltrate the site of increased inflammation and are inhibited from leaving by a macrophage inhibition factor secreted by the sensitized lymphocytes. This delayed and specific reaction peaks at between twenty-four and and forty-eight hours after antigenic stimulation. It is characterized by mononuclear (lynphocyte/Vnacrophage) cell type invasion of the lesion. Statement of Problem

The purposes of this study were to determine if BOG immunization confers protection to guinea pigs infected subcutaneously with N. fcwleri and to determine if delayed hypersensitivity develops in guinea pigs exposed subcutaneously to N. fowleri.

21 CHAPTER II

MATERIALS AND METHODS

Experimental animals

Outbred laboratory mioe were obtained from the breeding stock in the Department of Zoology, University of Montana. Thirty-one male and five female guinea pigs employed during this study were purchased from the Rosecrans Company; Hamilton, Montana. Upon receipt, the male guinea pigs weighed approximately 500 grams, and the females, 700 grams. Infected animals were- maintained in cages separate from uninfected control animals. No more than five animals were kept in each cage. All animals were housed together in isolation from casual human or animal contact and fed and watered ad libitum. Three groups of five male guinea pigs were designated Group I (cages C,D, and E). Group II also consisted of three groups of five male guinea pigs (cages A,B, and F). Three female guinea pigs to be infected with N. fcwleri were designated Group III (cage G). Two females (cage H) and two males (cage J) served as untreated, uninfected controls.

Imrnunization

Group I guinea pigs frcm cages D and E and Group II guinea pigs frcm cages A and B were immunized with BCG (Paris strain) vaccine. The vaccine consisted of an average of 1.32X10® viable units per ml.

Dubos liquid medium. The vaccine was administered IP (dosage to each guinea pig on each occasion:0.2 ml. of undiluted, 200 Klett BCG).

The number of injections were varied in order to obtain a graded

22 23

degree of sensitivity to BCG irrmunization. The number and times of vaccinations were as indicated in Tables 3 and 4.

Skin tests were performed at different times on all guinea pigs to determine relative sensitivity to BCG inmunization. The antigen employed was BCG protoplasm prepared as described by Larson et al. (1968). Intradermal injections of the antigen were made in a shaved part of the mid-lateral body region of the guinea pig. Dosages and times administered are recorded in Tables 3 and,4. Skin test lesion volumes were recorded at twenty-four hours post injection according to the method outlined by Larson et al.(1968).

N. fowleri culture used An HB-1 isolate of N. fowleri was employed during the course of this study. The history of isolation and maintenance of the HB~1 culture was reported by Culbertson et al. (1968) and Butt et al. (1968). Cultures of N. fowleri during this study were maintained at

35 C in liquid axenic medium of Chang (1974) modified by using rabbit or sheep whole blood. The cultures were kept in 15 ml. screw-capped test tubes and transferred at 30 day intervals. Vero (secondary monkey kidney), SIRC (rabbit cornea) and mouse melanoma tissue cultures were used to grew fowleri for inoculation. Tissue cultures infected with N. fowleri were maintained in 30 ml. Falcon flasks (Becton Dickenson Company; Qxnard, California) at 35 C in Medina 199

(GIBOO; Grand Island, New York) plus 10 percent calf serum (obtained frcm a local abattoir). 24

Infective inoculum of N. fcwleri

N. fowleri were collected from axenic medium by centrifugation (1000 rpm/5 min.^ . The amoebae were instilled intranas ally into two anesthesized (phencbarbitol-sodium; 5 mg./kg. body wt.) laboratory mice.

Infection procedures were those described by Culbertson et al.(1968). Within six days, both mice died. N. fowleri were collected from brain

tissue, then transferred to mouse melanoma and SIEC cell lines. The tissue culture medium was poured off and replaced daily. Three days

after tissue culture inoculation, N. fcwleri were collected by centri fugation and counted by haemocytcmeter. Subcutaneous injections of 3X10^ N. fovleri in 0.1 ml. culture medium were administered to each guinea pig in Groups II and III. To test for infectivity of the amoebae in the inoculum, approximately 1X10^ amoebae in 0.01 culture medium were injected intrace rebrally in a laboratory mouse. Reisolation of amoebae from infected mouse brain tissue was considered evidence of the presence of pathogenic N. fovleri.

Controls

To test for deaths in guinea pigs caused by variables other than N. fovleri, Group I was designated the control. An inoculum was prepared similiarly to the method described previously to infect Group II and III, except that the inoculum was diluted (5:1) with fresh culture medium. A sub-lethal dose of approximately 1X10 N. fowleri in 0.1 ml. of culture medium was injected subcutaneously into two laboratory mice. Death of these mice and reisolation of the amoebae confirmed the pathogenicity of the inoculum. 25

Procedures and observations

Groups I, II, and III guinea pigs were observed daily and weighed weekly. Animals dying during the course of the experiment

(sixty days post infection) were autopsied. Livers, kidneys, spleens, and lungs were removed from the animals and preserved in 10 percent

sodium phosphate-buffered formalin (pH:7.4). These tissues were later, embedded, sectioned, and stained for microscopical examination and

photography.

Tests to elicit delayed hypersensitivity

A soluble fraction of N. fowleri was prepared in the following manner. Trophozoites were collected in culture medium fron five day old infected Vero cell cultures. Massive doses of amoebae (1X10^) were instilled intranas ally into anesthesized laboratory mice. Upon

death, the mice were autopsied and N. fcwleri collected fron mouse brain tissue. The amoebae were washed three times by suspension and

oentrifugation in sterile physiological saline and mouse brain sediment discarded. The amoebae were then counted by haemocytaneter and killed by freeze-thawing three times in a dry ice-alcohol bath. Soluble and insoluble fractions were separated by centrifugation (1.9X10^ rpm/ 30 min.). The soluble fraction was sterilized by filtration through a

four millimicron filter and maintained, when not in use, between -45 and -70 C. The antigen processing was repeated on three different occasions in order to vary the concentration of antigen in solution. The first preparation of soluble antigen (Ag 2) consisted originally of 2.5X10^ 26

amoebae per ml. sterile physiological saline. The second process(Ag 3) used 3.0X104 N. fovleri per ml.; and the third (Ag 4) contained 4.8X104 amoebae per ml.

To determine if any delayed hypersensitivity elicited could be attributed to mouse brain, normal uninfected mouse brain was processed under identical circumstances as the amoebic preparation. This control antigen was maintained between -45 and -70 C until used for skin tests.

All antigen and control skin tests were accomplished by intradermal injections made in a shaved portion of the lateral mid-body region of the guinea pig. Dosage administered to each guinea pig on each occasion was 0.1 ml. undiluted antigen preparation. The Ag 2 fraction was injected on day 15 post infection. On days 30 and 36 post infection, Ag 3 was employed. Ag 4 was used on days 42,95, and 126. The mouse brain control was injected on days 15, 30, and 36. To determine if a correlation existed between senitivity to BOG and sensitivity to N. fowleri, 1 microgram of BCG protoplasm in 0.1 ml. sterile physiological saline was intradermally injected on day 42 post infection. All skin test lesion volumes were recorded at 24 hours post intradermal injection. The method for determining volumes were the same used to measure BOG lesion volumes described previously. Lesions caused by the soluble fraction of N. fowleri administered on day 30 were surgically removed from two guinea pigs (G1 and G2). These lesions were preserved in 10 percent sodium phosphate-buffered formalin. Later the tissues were embedded, sectioned, and stained to observe the cell types infiltrating the site. 27

Statistical analysis

A Chi-square test was used to determine if the number of deaths of BOG immunized guinea pigs differed significantly frcm that of infected control guinea pigs. The Spearman rank test was used to determine the correlation between percent weight loss and sensitivity to the soluble antigen fraction of N. fovleri. It was again used to determine correlation between sensitivity to BOG and sensitivity to

N. fowleri. CHAPTER III

RESULTS

BCG irrmunization

As recorded in Table 4, sensitivity to BOG in Group II guinea pigs varied frcm 295.0-37.1 ran , fourteen days after immunization.

Sensitivity to BOG peaks in guinea pigs at 21 days and is retained for at least six months (Larson; personal cottnunication). This increase in sensitivity is reflected in the BOG-elicited lesions conducted 42 days post-infection (Table 6). Therefore, the subcutaneous infection by

N. fcwleri in Group II occurred within a period of maximum sensitivity to BCG and was tested for any prolonged protection afforded by the vaccine.

The inocula used to infect Groups I, II, and III proved to contain viable, pathogenic N. fowleri. Mice infected with the amoebae collected from the inocula displayed symptoms characteristic of primary amoebic meningoencephalitis described by Carter (1970) and died within six days of infection. At autopsy, numerous N. fowleri were observed in mouse brain tissue. Within 28 days post infection; two BOG immunized (A4 and A5) and two non-immunized guinea pigs (F3 and F5) in Group II died of visceral naegleriosis. Symptoms noted were an overall body weight loss below initial weight. Animals F3 and A4 demonstrated large blackened necrotic lesions at the site of infection (Fig. 10). All animals appeared normally active until 24 hours before death when they were found listless and unable to move.

28 29

Upon death, these animals were autopsied. Gross examination shewed large diffuse yellcw lesions on the liver (Fig. 11, 14). The lungs contained numerous dark nodular lesions (Fig.llr 12). The spleen appeared enlarged and mottled (Fig. 11).

Microscopical examinition (Fig.13,16, and 17) showed numerous

N. fovleri in the liver and lungs of these animals.

Gross and microscopical examination of guinea pigs dead from visceral naegleriosis confirmed Culbertson et al.'s (1972) cbservations. None of the Group I guinea pig controls lost weifcfht during a sixty day period post infection. Group II and III deaths not attributed to visceral naegleriosis were those occurring 28 and 30 days post infection. B5 died of unknown causes. G1 died in post-operative care after surgical removal of skin test lesions. Neither animal shoved gross or microscopical pathology characteristic of visceral naegleriosis. No amoebae were observed in tissue examinations of either animal.

Fatal visceral naegleriosis did not only strike the runts in each cage. There appears to be no correlation bttween initial weights and deaths caused by N. fovleri. A Chi-square test was used to compare numbers of animals dead and surviving in the BCG immunized and non-immunized control groups. The test showed no significant protective value to BCG immunized guinea pigs subcutaneously infected by N. fowleri. It also appears from the results that within the range of sensitivity tested there is no gradation of protection. Guinea pig A4, with the highest sensitivity 30

to BCDG (295 mm 3 ), died frail visceral naegleriosis 25 days post infection. The last animal to die of visceral naegleriosis (F3) was a non-irrmunized control.

Delayed hypersensitivity tests Skin test lesion volumes elicited by soluble antigen fractions of N. fovleri are recorded in Table 5. Insoluble fractions were tested and found to elicit little response in guinea pigs. Guinea pigs

(Bl, B2, B3, Gl, and G2)consistently demonstrated high delayed hypersensitivity to the soluble fraction of N. fowleri. These animals also demonstated a percent weight loss between 7 and 21 days post infection. All Group II and III guinea pigs showing a percent weight loss during the same period of time demonstrated small to neglig- able sensitivity to the antigen. Skin test lesion volumes elicited by N. fcwleri antigen appeared to decrease over time. This may be due to the nature of the cell immune response or loss of antigenicity of the preparation.

The only animal skin tested subsequently dying of visceral naegleriosis was guinea pig F5. The negative results in this case are unexplained but may be due to the animal's weakened condition caused by the disease process. Guinea pig F5 died within 24 hours of the skin testing. G uinea pig Gl showed percent weight loss and delayed hypersensi tivity to the antigen; both indicative of visceral naegleriosis.

However, at autopsy, 30 days post infection, gross and microscopical examination showed no pathological damage and no amoebae were observed 31

in the tissues. Autopsies of the rest of the guinea pig survivors conducted four months after infection also shewed none of the pathology characteristic of visceral naegleriosis.

The soluble fraction of the N. fcwleri derived antigen appears to be specific in its response. Generally, little or no response was elicited by the antigen in uninfected controls (Hi, H2, Jl, and J2).

The lesion volume recorded for Hi on day 42 was probably due to non specific factors. Stained preparations of the lesions recovered frcm guinea pigs Gl and G2 on day 30 showed infiltration of macrophages and lymphocytes at the site of the lesion (Fig. 18-19)_. This further indicated that the lesions were caused by a specific cellular response to the antigen.

The mouse brain control antigen elicited little or no response in either infected or uninfected guinea pigs (Table 5).

BCG immunization apparently does not enhance or cross react with the N. fcwleri derived antigen. Table 6 records the BOG and N. fcwleri stimulated skin test lesion volumes conducted on day 42 post infection. A Spearman rank test shewed no correlation at the 0.05 level of significance. CHAPTER IV

DISCUSSION

Guinea pigs A4 and A5, challenged with BCG 38 days prior to a subcutaneous infection by N. fcwleri, died frcm visceral naegleriosis. Apparently the non-specific stimulation of macrophages by BOG imrnunization conferred no significant protection against the amoebic invasion. If BOG had been administered just before or after the subcutaneous injection of N. fowleri, the results might have been different. Perhaps the macrophages required a more recent challenge by BCG in order to maintain a heightened response against N. fcwleri.

But the projected purpose of this study was not to discover an immunization for an experimentally induced disease only reported in laboratory animals. It was an exploration considering possible protective measures against cerebral or visceral infections of N. fcwleri in human populations. By the time a human case was presented to a physician and correctly diagnosed, BCG challenge would be of little use.

Because BCG immunization was not found to cross-react or enhance the delayed hypersensitivity elicited by the antigen derived frcm N. fowleri or even reduce significantly the number of antigenically stimulated survivors; it appears that in the times and doses administered in this study BOG has little protecitve value against this extracellular parasite.

32 33

Though all guinea pigs in Groups II and III were apparently infected with the same number of viable N. fowleri, only four animals lost weight and died of visceral naegleriosis. Guinea pigs Bl, B2, B3, Gl and G2 demonstrated a weight loss indicating the disease process, but had apparently recovered frcm the infection as seen frcm autcpsy evidence. The cause for recovery is unknown, but perhaps specific and/ or non-specific iranune factors destroyed the N. fcwleri infection before irreparable damage occurred to the liver and lungs. There is no statistical evidence that BOG iirmunization assisted in the recovery process. N. fcwleri stimulated a specific cellular immune response as evidenced by delayed hypersensitivity elicited by the antigen. Weight gain and/or little response to the antigen indicated reduced immunogenic exposure and disease process in the rest of Group II and III guinea pigs. Perhaps the N. fcwleri infection was inhibited at the site of infection before spreading to the viscera. If natural resistance or other non-specific factors destroyed the amoebae soon after exposure, this would explain the reduced immunogenic response and symptomatology displayed by these animals. Cerva (1971b) reported similiar results in intracerebral infections of guinea pigs by N. fowleri. After intranasal instillation of the amoebae, a few of the guirea pigs demonstrated a high fever and subsequently died of naeglerial-meningoenoephalitis. A second group of guinea pigs also displayed a febrile state, but eventually recovered f jrcm the infection. These guinea pigs shewed high titers of humoral 34

antibodies specific for iJ. fcwleri as measured by complement fixation.

The rest of the guinea pigs though exposed with the same number of N. fowleri, never demonstrated a fever and subsequently did not demonstrate humoral antibody production.

The results gathered from cerebral and visceral infections of guinea pigs by i\[. fowleri possibly indicate degrees of immunogenic conpetency in these animals. One of the unanswered questions concerning human infections by N. fcwleri is how the organism can be so pathogenic and yet infect so few people. Only fatal human cases have been reported except for the single survivor tneraputically treated (Anderson and Jamieson, 1972; Carter, 1972). Yet thousands of people frequent a single lake or swiimdng pool where only two or three contract the disease. Humans, like guinea pigs, may have temporary or genetically determined degrees of imnunocortpetency.

Perhaps one portion of the human population has an immune deficiency and when exposed to 14. fcwleri succumb to the disease. A second portion of the population may be naturally resistant to infection by i£. fowleri. Somehow, the infectious agent is denied access to its potential host. A third portion of the human population may indeed contract the disease, but recover without proper diagnosis or therapy. The N. fowleri must in some way be arrested by non-specific and/or specific immune factors. Possibly, there is an antigenic stimulation of the host immune system. To test this hypothesis concerning non-fatal naegleriosis in man, humoral and/or cellular iinmune tests could be developed and administered 35

to human populations residing in endemic areas with a high probability of exposure. Such a location might be Adelaide, Australia where

fowleri was isolated from the municipal water supply system. If humans demonstrate an inmunogenic response specific for H. fowleri then it can be assumed that not all cases of naegleriosis in humans are fatal. The antigen preparation described in this study has demonstrated an ability to elicit a delayed hypersensitivity response in guinea pigs exposed to N. fowleri. This antigen might prove to be a useful diagnostic tool if human cases of visceral naegleriosis indeed exist. Whether this antigen can demonstrate a previous intracerebral exposure to N. fowleri in guinea pigs or humans has yet to be tested. CHAPTER V

SUMMARY

Ten BCG immunized and five non-immunized control guinea pigs were subcutaneously infected with 3.0X104 N. fowleri. Two BCG immunized and two non-irtmunized controls died fron visceral naegleriosis within thirty days post-infection. BCG immunization appeared not to confer significant protection against the infection. A soluble fraction derived from disrupted cells of N. fowleri was tested for antigenic properties. This preparation consistently elicited delayed hypersensitivity in guinea pig survivors of visceral naegleriosis. A third group of guinea pigs, though exposed to the same number of N. fowleri, showed none of the symptomatology characteristic of the disease and did not demonstrate delayed hypersensitivity to the antigen. A parallel was drawn between the apparent differences in imnuno- oompetence displayed by guinea pigs exposed to 1£. fowleri and human cases of primary amoebic meningoencephalitis.

36 37

TABLE 1

HUMAN CASES OF N. fowleri-CAUSED PRIMARY AMOEBIC MENINGOENCEPHALITIS

Country Where Year Number of Reported Reference Reported New Cases

Australia Fowler and Carter 1965 1 Carter 1968 3

Carter 1969 2

Carter 1970 1

Carter 1972 2 Anderson and Jamieson 1972

Belgium Jadin et al. 1971

Czechoslovakia Cerva et al. 1968 16

Cerva 1969 1

Great Britain Apley et al., 1970 Warhurst et al. 1970

New Zealand Mandal et al. 1970

United States

California Hecht et al. 1970 1 Georgia McCroan and Patterson 1970 1 38

TABLE 1 (cont.)

Country Where Year Number of Reported Reference Reported New Cases

United States (cont.) Florida Butt 1966 Butt et al., 1968 Culbertson et al. 1968

Chang (personal communication) 1974 1

Virginia Callicot et al. 1968 8 Duma et al. 1969 1 Duma et al. 1971 3 Dos Santos 1970 5 39

TABLE 2

POSSIHLE HUMAN CASES OF N. fowleri- CAUSED PRIMARY AMOEBIC MENINGOENCEPHALITIS

Country Where Year Number of Reported Reference Reported New Cases

Australia Derrick 1948

New Zealand Mandal et al. 1970

Great Britain Symmers 1970 Apley et al., 1970 Warhurst et al. 1970

India Pan and Gosh 1971

Africa Grundy and Powers 1970 40

TABLE 3

GROUP I (CONTROL) DATA:IMMUNIZATIONS, SKIN TESTS, AND WEIGHT CHANGES

BCG Immunization BCG Skin Test Weight Change Animal Schedule in Days Lesion Volumes from 7-21 Days Post Number Prior to Infection* (mm-5) Infection

D1 8, -1 21.0 1.135

D2 8 -1 16.2 1.295

D3 8 -1 95.6 1.176

D4 8 -1 32.4 1.106

D5 8 -1 48.6 1.209

El 8 18.8 1.092

E2 8 36.0 1.145

E3 8 9.0 1.156

E4 8 13.5 1.130 E5 18 26.5 1.136

CI none 0.0 1.111

C2 none 0.0 1.068

C3 none 0.0 1.086 C4 none 0.0 1.148 C5 none 0.0 1.052

* Skin test dosage of BCG protoplasm was 0.1 ug. in 0.1 ml. sterile physiological saline. This skin test was performed 14 days after initial immunization (the same day as control infection).

No Group I animal died during a 60 day period post infection with the sub-lethal dose (1X103) of N_. fowleri. 41

TABLE 4 GROUP II DATA: BCG IMMUNIZATION, BCG SKIN TESTS, INITIAL WEIGHTS, WEIGHT CHANGE, AND DAYS OF INFECTION

Animal BCG Immunization BCG Skin Test Initial Percent Wt. Infection Number Schedule in Days Lesion„Volumes Wei ght Change 7-21 Time for Prior to Infection (mm ) * (gm.) Days Post Fatal Cases Infection (days) A4 -74, -67, -38 295.8 879 0.844 25 /

B1 -38 191.2 . 878 0.951 --

A3 -74, -67, -38 164.3 583 1.062 --

A5 -74, -67, -38 143.4 852 ; 0.979 24

A2 -74, -67, -38 135.0 698 1.100 —

B3 -38 120.0 762 0.944 —

B2 -38 82.0 840 0.957 --

B4 -38 75.0 912 0.974 --

Al -38 41.2 865 1.061 -- B5 -38 37.1 747 0.994 28**

F1 none 0.0 891 1.035 —

F2 none 0.0 740 1.030 -- F3 none 0.0 679 0.971 28

F4 none 0.0 843 1.086 -- F5 none 0 .0 885 0.894*** 17

* Skin test dosage of BCG protoplasm was 1.0 ug. in 0.1 ml. sterile physiological saline. This skin test was conducted 17 days after the last BCG immunization (21 days prior to infection). ** Unlike the other deaths recorded in this table, guinea pig B5's death cannot be attributed to visceral naegleriosis. ***Guinea pig F5 died from visceral naegleriosis on day 17 post infect ion. The percent weight change represents differences between day 7- 17. TABLE 5 SENSITIVITY TO ANTIGEN IN GROUPS II AND III

Animal Wei ght Mouse Brain^ Soluble N. fowleri o Avg. Number Change Between 7-21 Antigen (mnr) Antigen Lesion Vol. (mm ) of Days Post-Infection (days P.I.) (days post-infection) 36-95 15 30 36 15 30 36 42 95 126 F5* 0.894 0.0 — -- 0.0 ------— B3 0.944 -- -- 0.0 — -- 819.0 315.0 100.8 25.7 411.6

B1 0.951 — -- 0.0 -- -- 436.0 202.5 207.0 117.0 281.8 G2 0.953 0.0 0.0 8.8 29.2 115.5 110.3 29.3 36.0 0.0 58.5 B2 0.957 -- -- 0.0 -- -- 133.9 234.0 252.0 200.0 206.6 B4 0.974 -- -- 0.0 -- -- 4.5 18.9 0.0 0.0 7.8 — Gl** 0.983 1.9 0.6 0.0 53.6 218.7 ------— F2 1.030 -- -- 0.0 -- -- 37.1 21.0 0.0 — 19.3 F1 1.035 -- -- 0.0 -- -- 6.6 63.0 7.9 — 25.8

G3 1.045 — — 0.0 -- — 16.2 66.2 0.0 -- 27.5 Al 1.061 -- — 0.0 — — 75.6 24.3 15.7 2.2 38.5 A3 1.062 — -- 0.0 — -- 2.7 4.5 0.0 5.4 2.4 F4 1.086 0.0 0.0 0.0 45.0 26.3 20.5 10.5 0.0 — 10.3 __ A2 1.100 — -- 0.0 -- 12.2 3.0 0.0 0.0 k 5.1

Uninfected Controls HI D.O 1.2 37.5 2.0 0.0 37.5*** 9.6 0.0 0.0 15.7 H2 D.O 1.3 D.O 0.0 2.2 0.0 0.0 0.0 0.0 0.0 J1 ------0.0 0.0 0.0 0.0 J2 -- — ------0.0 0.0 0.0 0.0 * Guinea pig F5 died of visceral naegleriosis 17 days post-infection. ** Guinea pig Gl died in post-op. 30 days after infection. *** This reading is probably due to non-specific inflammation or other factors unknown. 43

TABLE 6

COMPARISON OF SKIN TEST LESION VOLUMES

Animal Skin Test Lesion Rank Skin Test Lesion Rank Number Volume To N. fQwleri Volume to BCG Antigen (mm->) (mnr) B3 315.0 1 213.0 1 B2 299.0 2 127.0 5 B1 126.0 3 201.0 2 F1 63.0 4 0.0 TO G2 29.3 5 0.0 10 Al 24.3 6 64.4 7 F2 21.0 7 0.0 10 B4 18.9 8 107.0 6 G3 16.2 9 0.0 10 F4 11.0 10 0.0 10 A3 4.5 11 150.4 4 A2 3.0 12 162.0 3

A Spearman rank test showed no correlation (Z=0.64) through a 0.5 significance level between lesion volumes caused by BCG and lesion volumes elicited by the N. fowleri antigen. 0

microns

Figure 1. N. fovleri, amoeboid stage. Contractile vacuole (C); nucleus (N); food vacuole (F); pseudopodium (P).

44 Figure 2. N. favleri, flagellate stage. Contractile vacuole (C); nucleus (N); basal body (B); flagellum (F).

microns

Figure 3. N. fa^leri, cystic stage. Nucleus (N); cyst wall (C).

45 46

Figures 4-7. Naegleria spp. mitotic pattern (after Singh and Das, 1970). 4. Nucleus at prophase. Nucleus (N); nucleolus (Nu); chromatin granules (CG). 5. Nucleus at metaphase. Chromatin mater ial (CM); spindle fibers (S); polar masses (PM). 6. Nucleus at anaphase. Polar masses (PM); chromatin material (CM); daughter nucleus (DN); interzonal bodies (IZ); chromatin granules (CG). 7. Naegleria sp. at telophase. Polar masses (PM); interzonal bodies (IZ); chromatin granules (CG); daughter nucleus (DN). Nu PM

CG CM

CG CM -—IZ -

PM 6

IZ CG PM DN 48

Figures 8-9. Photanicrographs of unstained N. fowleri suspended in a hang drop preparation (X1000). 8. Amoeboid form. 9. Cystic form.

5 0

Figure 10. Guinea pig dead from visceral naegleriosis demonstrating blackened, necrotic lesion at the site of infection.

Figure 11. Comparison between various organs removed frcm an N. fcwleri-infected and uninfected control guinea pigs. The liver (d) and lungs (a) frcm the infected guinea pig demonstrate lesions characteristic of visceral naegleriosis. The spleen (f) of the infected animal is enlarged as ooipared to the uninfected animal's spleen (e). Control liver (c) and lungs (b) demonstrated no lesions.

Figures 12-13. Photomicrographs of hematoxylin-fast green stained lung tissue sections frcm N. fcwleri-infected guinea pig. 12. Tissues shewed large nodular lesions (X4). 13. N. fcwleri were observed in these nodular lesions (X1000).

Figures 14-17. Photcmicrographs of iron hematoxylin-eosin stained liver tissue sections recovered frcm N. favleri-infected guinea pigs. 14. Cross-section of liver tissue demonstrating lesion (X4). 15. Infiltration of white blood cells and disruption of liver sinesoids at site of lesions (X40). 16. Oil emersion view of the distinctive nucleus of N. fowleri (X1000). 17. Numerous N. fowleri were observed in the liver tissue surrounding the liver lesions (X400). dfc. s 54

Figures 18-19. Photomicrographs of a hematoxylin-eosin stained section of a skin lesion elicited by antigen derived frcm a soluble fraction of N. fowleri. 18. Lesion showing infiltration of white blood cells into perivascular spaces .(X40). 19. White blood cell types infiltrating the lesion site. Only mononuclear cell types were observed: small lymphocytes (SL); large lympho cytes (LL); and macrophages (M) (X1000).

LITERATURE CITED

Anderson, K. and A. Jamieson. 1972. Primary amoebic meningoenceph alitis. Lancet 7756:902-903.

Apley, J.C., S.K.R. Clark, A.P.C.H. Rocme, S.A. Sandry, G. Soygi, 3. Silk, and D.C. Warhurst. 1970. Primary meningoencephalitis in Britain. Brit. Med. J. 1:596-599.

Armstrong, J.A. and M.S. Pereira. 1967. Identification of the Ryan virus as an amoeba of the genus Hartmannella. Brit. Med. J. 1:212.

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