Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. - ' r - In a I thermal poo s to avoid the possibility of developing the serious illness called AMOEBIC MENINGITIS. T is dosease can be caught in hermal pools if water enters the nose, while swimming or diving. ISSUED BY THE DEPARTMENT OF HEALTH Massey University Library New Zealand & Pacific Collection PATHOGENIC FREE-LIVING AMEBAE? OCCURRENCE IN NEW ZEALAND THERMAL REGIONS TOGETHER WITH INVESTIGATIONS INTO THEIR DISINFECTION, IMMUNITY AND VIRL?ENCE A thesis presented in partial fulfuilment of the requirements for the ?egree of Doctor of Philosophy in Microbiology at Massey University, New Zealand Raymond Thomas Michael Cursons 1978 t.l I 1? t n ID ? (ii) ABSTRACT Pathogenic free-living amebae (PFLA), of which Naegleria ?owleri was the predominant pathogenic species, were isolated from 6 out o? 10 pools sampled from the Hamilton, Rotorua and Gisborne Health Districts. The majority o? these PFLA contaminated-pools occurred in the Matamata-Taupo region, and this localized geographical distribution appeared to be influenced, in part, by the particular physical and chemical properties o? the pool. 'High-risk' pools, which exhibited a high incidence o? isolations o? PFLA, were shown to be natural pools, i.e. soil enclosures, as opposed to concrete constructed pools. PFLA were also isolated from the soil, and it was thought that soil acted as a reservoir o? PFLA. No sea? sonal distribution in the occurrence of PFLA in thermal pools was noticed. A comparative study on the disinfecting potential o? chlorine, chlorine dioxide, ozone and deciquam 222 for PFLA showed that all 4 disinfectants possessed amebicidal pro? perties, but only at higher levels than those normally used for disinfecting bacteria. 0? the 4 disinfectants, deciquam 222 proved to be the most effective amebicide, followed by chlorine, chlorine dioxide and ozone. An immunological survey of normal human sera for the presence o? antibodies to either Naegleria and Acanthamoeba spp. had a titre ranging between 1/5 pathogenic or non-pathogenic established that human sera - 1/20 for Naegleria spp. and between 1/5 - 1/80 for Acanthamoebae. No discrimination in titres was observed between blood groups or sexes and ?luorescein-label?rl class-specific immunoglobulins showed that these antibodies belonged mainly to both the Ig M and lg G classes. It was also shown that fresh adult human sera, as opposed to cord or specific hyperimmune-rabbit sera, contained a heat-labile neutralizing ?actor which inhibited the formation of cytopathic effects (CPE) in Vero cell &:c ,_, ? I ?; l .. ( iii) culture by Acanthamoeba culbertsoni, but not by N. ?owleri. Homologous, as well as heterologous antigens o? Naegleria spp. were however, shown to cross-react with both the in vitro, macrophage inhibition ?actor assay, and in vivo, delayed hypersensitivity, correlates o? cell-mediated immunity. Finally, this study also demonstrated that both patho? genic and non-pathogenic species o? Naegleria and Acanthamoeba secreted both an extracellular phospholipase 2 and lysophospholipase into their axenic cultures. The rela? tive production o? phospholipase 2 correlated with the for? mation o? CPE in Vero cell c ulture by either amebae, or by cell-free filtrates from axenic c ultures o? amebae. The relative level o? production of this enzyme appeared to influence the virulence and hence pathogenic -potential of these micro-organisms. (iv) ACKNOWLEDGEMENTS The production of this thesis was Qnly pQssible because of the generous help which I received during the course of this investigation. I am indebted to the Department of Microbiology and Genetics, Massey University for providing the opportunity and facilities ?oi thi?- - ? investigation, to the New Zealand Department of Health for financial support and co-operation, and, to the Medical Research Council for financial support for technical assis? tance for the survey. In particular I would like to thank my supervisor, Dr Tiro Brown, Prof. D.F. Bacon, Dr Adam Wilkins, Dr Vaughn Crow and other academic and technical staff of the Department of Microbiology and Genetics for their advice, help and encouragement. I would also like to thank: Dr Kevin Moriarty of the Department of Veterinary Pathology and Public Health for his help with Immunological studies. Dr John Tweedie and Mr John McLean of the Department of Biochemistry for help with biochemical investigations. Mr Des Till of NHI for his co-op eration and suggestions. Mr Doug Hopcroft, Ives, Keith and Al of the Electron? Microscope Department, Applied Biochemistry Division, DSIR for help with electron microscopy. The Medical Officers of the Hamilton, Rotorua and Gisborne Health Departments, and especially Mr Morrie Marks, Mr Charlie Barber, Mr Rod Findon, Mr Maurice Miles with other nameless health inspectors, for collecting samples and arranging bacterial counts and photographs. Without their generous help, the survey could not have been completed. (V) ?Irs Elizabeth Keys ?or her patience, efficiency and enco ur agemen t. .. T!WtGywn; "tiil, A!?'11iAtllii.?1CN?1? ??ltti;?a.n:? dofh@a? [f{ert' ? . :f r t:li!ei?dh@! ?'"' My parents . ?Irs Penny van Doorn ?or typing the draft. Bridget for typing the final copy . The Library sta?? o? Massey University ?or the numerous interloan requests. Robinson Designs, Auckland, ?or the supply o? ' whirl bags' . Maui Brothers ?or the gift of deciquam 222 . Jim, Les and the girls o? the Central Photographic Unit. (vi) PREFACE Since the first reported cases in New Zealand in 19 68 o? primary amebic meningo-encephalitis (PAM) (Mandal et al., 1970) the small free-living amebae o? the genera Acanthamoeba and Naegleria have been o? great interest to the New Zealand Department o? Health. Locally it is known as 'Hot-Pool Meningitis' and there have been 7 confirmed cases o? the disease all due to Nae0leria ?owleri and all contracted after swimming in thermal pools in the central North Island (Cursons et al., 19 78b). Subsequently, these pathogenic free-living amebae (PFLA) (Adam et al., 197 1) have been shown to be responsible ?or a number o? diseases ranging from chronic illnesses such as respiratory infec? tions (Martinez et al., 19 75) and humidifier ?ever (M.R.C. Symposium, 1 97 7) to blindness (Visvesvara & Jones, 1 9 75). The apparent ubiquity and ease o? isolation o? these amebae from the environment stimulated a program (initiated by the New Zealand Department o? Health and run by the National Health Institute) o? isolating amebae from specific thermal pools. Later this program was extended under con? tract to Massey University to cover 10 thermal pools in 3 different Health Districts, with the aim o? sampling the pools ?or the presence or absence o? PFLA and trying to cor? relate their presence or absence with such parameters as the chemical composition o? the waters, pH, temperature, and numbers o? bacteria. In addition, comparative disinfection tests were carried out using a variety o? compounds to ob? serve their respective amebicidal properties. Because o? the repeated isolations o? PFLA from some New Zealand thermal pools, an immunological study was also undertaken. It was hoped to explain the enigma o? the human population's appa? rent immunity to these amebae, despite their pathogenicity and distribution. Finally, the virulence o? these amebae in relation to their pathogenicity was also studied. TABLE OF CONTENTS ABSTRACT ? ACKNOWLEDGEMENTS PREFACE LIST OF TABLES LIST OF FIGURES LIST OF PLATES CHAPTER 1 : INTRODUCTION 1. 1 The History of Free-living Amcbae as Disease Agents 1. 2 Occurrence and Distribution 1. 3 Infection 1. 4 Immunity 1 . 5 Disinfection CHAPTER 2: S.Al\1PLING SITES 2. 1 Introduction 2. 2 Hamilton Health District 2. 3 2. 2. 1 2 . 2. 2 2. 2. 3 2. 2. 4 Waignaro Springs ? Moana-iti Springs Opal Springs Okoroire Springs . Rotorua Health District ? 2. 3. 1 Soda Springs 2. 3. 2 Butcher's Pool 2. 3. 3 Otumaheke Stream ? 2. 4 Gisborne Health District 2. 4. 1 Te Puia 2. 4. 2 Morere CHAPTER 3: MATERIALS 3. 1 Amebic Cultures used 3. 2 Plate Media 3. 2. 1 Ameba Saline Agar 3.2. 2 Ameba 1% Saline Agar ( vii) ii iv vi xiii XV XVll 1 1 4 5 6 7 9 9 1 1 1 1 12 1 2 1 3 1 6 1 6 1 6 16 20 20 21 25 25 30 30 30 ? ? ? Cont'd 3 . 2 . 3 3 . 2 . 4 3 . 2 . 5 3 . 2 . 6 3 . 2 . 7 3 . 2 . 8 3 . 2 . 9 N.M. Agar E.Y . Agar Lysolecithin Agar Haemolytic Agar Proteinase Agar DIFCO Standard Methods Agar DIFCO Violet Red Bile Agar . 3 . 3 Axenic Media ?or Amebae ? 3 . 3 . 1 Page's Amcba Saline 3 . 3 . 2 CYM Medium ? ? 3 . 3 . 3 CYr-1H M edi urn . 3 . 3 . 4 4% Ne?? Medium 3 . 4 Cell Culture Media ? 3 . 5 Lyflphocyte and Macrophage Culture Media 3 . 5 . 1 Lymphocytes . 3 . 5 . 2 Macrophages ? 3 . 6 Disinfection Solutions Used 3 . 6 . 1 Water 3 . 6 . 2 Chlorine 3 . 6 . 3 Chlorine dioxide . 3 . 6 . 4 Ozone 3 . 6 . 5 Deciquam 2 22 3 . 7 Polyacrylamide Gel Electrophoresis 3 . 7 . 1 Polyacrylamide Gels 3 . 7 . 2 Running Bu??er 3 . 7 . 3 Tracking Marker 3 . 7 . 4 Gel Staining Solution ? 3 . 8 Staining Solutions 3 . 8 . 1 Sudan black . 3 . 8 . 2 Ehrlich's haematoxylin 3 . 8 . 3 Glycerin-jelly 3 . 9 Chemicals and Serological Reagents 3 . 10 Experimental Animals CHAPTER 4 : METHODS 4 . 1 Miscellaneous Methods ( viii) Page 30 30 3 1 3 1 3 1 3 2 3 2 3 2 3 2 3 2 3 2 3 2 3 2 3 2 3 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 3 4 3 4 3 4 3 4 3 5 35 35 35 35 3 6 4 . 1 . 1 Chemical Analysis o? Thermal Pool Water 3 7- 3 7 3 7 ? ? . Cont'd 4 . 2 4 . 3 4 . 4 4 . 5 4 . 6 4 . 7 4 . 8 4 . 1 . 2 Scanning Electron Microscopy Identification o? Amebae and Bacteria ? 4 . 2 . 1 Identification o? Amebic Isolates 4 . 2 . 2 Total Coliform and Total Bacteria Count .Ar.lebic Culture Techniques 4 . 3 . 1 Cloning 4 . 3 . 2 Axenic Culture Cell Culture ( ix ) Page 3 7 3 7 3 7 3 9 40 40 40 40 4.4.1 Neutralization of Amcbae 1n Cell Cultures 4 1 Indirect Macrophage Inhibition Assay 4 . 5 . 1 Immunization of Guinea- pigs 4 . 5 . 2 Lymphocyte Culture and Production o? Macrophage Inhibition Factor . 4 . 5 . 3 Culture of Macrophages 4 . 5 . 4 Delayed Hypersensitivity Disinfection 4 . 6 . 1 Production o? Disinfectants 4 . 6 . 2 Chemical Analysis of Disinfectants 4 . 6 . 3 Disinfection Tests Polyacrylamide Gel Electrophoresis Cytochemical Staining 4 1 4 1 4 1 4 2 44 44 44 46 46 46 4 7 4 . 9 Preliminary Isolation and Characterization of Extracellular Enzymes of Pathogenic Free-living Ar.1ebae 4 7 4 . 9 . 1 Detection of Amebic Extracellular Enzymes 47 4 . 9 . 2 Thin Layer Chromatography ? 47 4 . 9 . 3 Haemolysis 48 4 . 9 . 4 Isolation of Phospholipases from the S upernatants of Naegleria fowleri ( MsT) 48 4 . 10 P rotein Determination 4 . 1 1 Sterilization 50 5 0 CHAPTER 5 : RESULTS 5 . 1 Survey of PFLA in New Zealand Thermal Pools 5 . 1 . 1 The Distribution o? PFLA in New Zealand Thermal Pools during the 1 9 7 6 Survey 5 . 1 . 2 The Occurrence o? PFLA in New Zealand Thermal Pools during the 1 9 7 7 Survey 5 2 5 2 5 2 5 8 ? ? ? Cont'd 5 . 2 T h e C omp a r a t i v e Us e of D i s i n f e c tan t s agai n s t F r e e- l i v i n g Amebae 5 . 2 . 1 The Use of C h l o r i n e a s a D i s infec tan t a g ai n s t F r e e - l i vi n g Amebae 5 . 2 . 2 The Us e of A l t e r n a t i ve D i s i nfec tan t s t o C h l o r i ne a g ai n s t F r e e - l i ving Amebae 5 . 3 I mmuni ty to PFLA 5 . 3 . 1 T h e P r e s ence o f An t i bodi e s t o PFLA 1 n ( x ) 6 2 6 2 6 3 6 5 Human Sera 65 5 . 3 . 2 Th e P r e sence o f a Spec i fi c Neut r a l i z i ng F a c to r agai n s t PFLA i n Norm a l Hum a n S e r a 7 6 5 . 3 . 3 Th e Reac t i on of t h e C e l l -M e di a ted Immune S y s t em to An ti gen s o f Naegl e r i a s pp . 5 . 4 Vi r u l e n c e o f PFLA ? ? 5 . 4 . 1 The P r e s e nc e of Ex t r ace l l u l a r Enzyme s i n t h e Sup e r n a t an t s o f Axenic C u l t u r e s o f F r e e - li v i ng Amebae 5 . 4 . 2 P r oduc t i o n of P hospho l i p a s e 2 in Axe n i c 80 83 83 Cul t u r e s of F r e e - l i vi ng Am ebae 9 1 5 . 4 . 3 . T h e P r e limina r y I so l a t i on o f a Pho spho l i p a s e 2 f r om Axeni c C u l t u r e s of N. fow l e r i (M s T ) 9 4 5 . 4 . 3 1 The I s o l a t i o n o f Pho spho l i p a s e 2 f rom S e r um - s upp l emented Axen i c Cu l t u r e s 9 8 5 . 4 . 4 Vi r u l en c e o f Amebae and C e l l - f r e e Axen i c F i l t r a t e s i n V e r o C e l l Cul tur e CHAPTER 6 : D I SCUSS I ON ? ? 6 . 1 Su rvey of New Zea l an d T h e r m a l Poo l s 6 . 1 . 1 The D i s t r i bu t i o n of PFLA i n New Z e a l and Therm a l Poo l s dur i ng the 1 9 7 6 S u r v ey 6 . 1 . 2 The Effe c t o f C h emi c al , Phys i c a l and B i o l o g i c a l P a r ame t e r s on the D i s t r i b u t i o n 1 0 5 1 1 8 1 1 8 1 1 8 of PFLA i n New Zea l and Thermal Poo l s 1 1 8 6 . 1 . 3 The I ncidence o f PFLA i n New Z e a l an d T h e rma l Poo l s 1 2 5 ? ? ? Con t ' d 6 . 2 T h e Compar a t i ve U s e o f D i s i nfec t an t s agai n s t F r e e - l i vi ng Amebae 6 . 2 . 1 The Us e o f C h l o r i n e as a D i s i nfec t an t agai n s t F r e e - l i v i n g Amebae . . 6 . 2 . 2 Th e Us e of Al t e r na t i ve D i s i nf e c t an t s to ( xi ) 1 3 1 1 3 1 C h l o r i ne ag ain s t F r e e - l i vi ng Amcbae 1 3 7 6 . 2 . 2 1 The U s e o f C h l o r i n e dioxide a s a D i s i nf e c t an t again s t F r e e - l i vi ng Amebae 1 3 7 6 . 2 . 2 2 The U s e o f Ozone a s a D i si nf ec t an t agai n s t F r ee - l i vi n g Amebae 6 . 2 . 2 3 The U s e o f Deciquam 2 22 as a Di s i nf e c t an t agai n s t Fr e e - l i vi ng 1 3 8 Amebae 1 4 1 6 . 3 I mm un i ty t o PFLA 1 44 6 . 3 . 1 Th e P r e s ence o f An t i bodi e s to PFLA i n Human S e r a 1 45 6 . 3 . 2 The P r e s en c e o f a Spe c i f i c N e u t r a l i z i ng F ac t o r agai n s t PFLA i n N o r m a l Human S e r a 1 47 6 . 3 . 3 Th e I nduc t i o n o f t he Ce l l -Medi ated Immune S y s t em 1 49 6 . 3 . 4 Immune R e s po n s e s i n R e l a t i o n t o I nfe c t io n wi th PFLA 1 50 6 . 4 V i r u l e nce o f PFLA 1 5 6 6 . 4 . 1 The P r e s en c e of EA t r ac e l l u l a r Pho spho l i p a s e s i n the S up e r na t a n t s of Axe n i c Cul t ur e s of F r e e - li ving Am ebae ? . 1 5 6 6 . 4 . 2 P r o duc t i o n o f Pho spho l i p a s e 2 i n Axe n i c Cu l t u r e s o f F r e e - l i vi ng Am ebae 1 5 7 6 . 4 . 3 P r e limi nary I s o l ation o f P ho spho l i p a s e 2 f r om Axen i c Cu l tu r e s o f N . fowl e ri ( M s T ) 1 60 6 . 4 . 4 Vi r u lence o f Am ebae and Ce l l - f r e e Axenic F i l t r a t e s in V e r o Ce l l Cu l t ure 6 . 4 . 5 Th e Si gni fi c an c e o f P ho spho l i p a s e 2 P r oduc t i o n and i t s R e l a t i o n s h i p t o t h e 1 6 1 P a tho geni c i ty of PFLA 1 6 3 ? ? ? Con t ' d APPEND I CES App endi x I D a t a ? r om t h e 1 9 7 6 S ur v ey App endi x I I D a t a ? r om the 1 9 7 7 S u r v e y App e ndi x I I I P ap e r s Pub l i shed B I BLICGRAPHY ? ? ( xi i ) P age 1 66 1 6 6 1 7 5 1 8 9 1 90 ( xi i i ) L I ST OF TABLES I . Ca ses o f P r imar y Amebic Meni ngo -enceph a l i t i s repo r te d afte r 1 9 74 2 I I . C hem i c a l and Ph y s i cal Ch a r ac te r i s t i c s o f The rma l Poo l s f r om t he Wai k a to He a l t h Di s t r i c t I I I . Chemi c a l and Phys i c a l C h a r ac t e r i s t i c s o f The rma l Poo l s f r om t h e Ro to r ua-T aupo Hea l th Di s t ri c t ? ? IV . Chem i c a l and Phys i c a l Ch a r ac te r i s t i c s o f The r m a l Poo l s f r om t he Gi s bo r ne Hea l t h Di s t r i c t 1 3 2 0 2 1 V . Ameba C u l t u re s U s e d 2 5 V I . Di s t r i bu t ion an d I nc idence of PFLA f r om New Ze a l an d The rma l Poo l s d u r i ng t he 1 9 7 6 Surve y 5 2 VI I . M on t h l y I so l a t i on s o f PFLA f r om New Ze a l an d The rm a l Poo l s dur i ng t h e 1 9 7 6 Survey V I I . T he r ange of t o t a l Co l i fo rm and B ac t e r i a l C o u n t s f r om New Ze a l and T herm a l Po o l s dur i n g the 1 9 7 6 Su rvey IX . The I nc idence of PFLA i n New Ze a l and The rmal 57 58 Poo l s du r i ng t he 1 9 77 S u r v ey 5 9 X . M o n t h l y I s o l a ti o n s o f PFLA f rom New Z e a l and Th e rm a l Poo l s du r i n g the 1 9 7 7 Su rvey ? XI . Bac t e r i o logi c a l Q u al i t y o f The r m a l Poo l s and Sur r oundi ng S o i l s du r i n g t he 1 9 77 Sur vey XI I . The Ame b i c i da l C apa ci t y o f C h l o r i ne XI I I . The Comp a r a t i ve Amebi c i d a l Cap ac i t y of C h l o r i ne di oxi de , O z o ne and Deci quam 222 XIV . P re s e nce of An t i bo di e s t o PFLA i n Human Se r a 60 6 1 6 2 6 4 f r om t he P a lme r s ton N o r th He a l th D i s t r i c t ? 6 6 XV . P re s e n ce of An t i bodi e s t o PFLA i n Human Se r a f r om the Hami l to n He a l t h Di s t ri c t 68 XVI . P re se n ce o f An t ibodies t o PFLA i n Human Se r a f r om t he R o t o r ua He a l t h D i s t r i c t XV I I . P re s ence o f C l as s - spe c i f i c An t i bodie s to PFLA 70 i n Human S e r a 7 5 XVI I I . Use o f Hype r immune R abb i t and N o rm a l Human Se r a i n Neu t r a l i zi ng N . fowl e r i (M sT ) and A . c u l be r t s o ni (A - 1 ) Ve r o Ce l l Cul t u re 7 7 C:nn t:' rl ( xi v ) XIX . C r os s - r e ac t i vi ty of Homo l o g o u s and He t e ro l o go u s An tigens o f Naegl er i a spp . as j udged by D e l ayed Hype r s en s i t i vi ty 8 0 XX XXI . C r o s s - r e ac t i vi ty o f Homo l og o u s and He t e r o - l o g ous j udged P r oduc ts An t i g ens of Na egl e r i a spp . as by I nhibi t i o n o f Enzyrn a t i c o f f\1ac rophages . . Hyd r o ly si s of Pho s - 8 2 pha t i dy l c ho l i ne by D i f f e r e n t Phospho l i pa s e s 8 4 XXII . Eff ect of Ro sen t h a l ' s I nh i b i t o r on Pho sp h o l i - p a s e 2 A c t ivi ty i n EY Agar XXI I I . P r oduc t i o n o f Pho spho l ip a s e 2 by Naeg l e r i a spp . XXIV . P roduc t i o n o f P ho s pho l i p a s e 2 by Acan t h amoeba spp . XA? . Eff e c t of P r o tein Co n c en t r a t i o n of the R e a c t i o n o f N . fow l e r i an d A . c u l be r t s oni Ce l l - f r ee fi l t r a t es i n EY Ag a r XXVI . R e l a t i v e fo rm a t i on o f Cy t op a t h i c Effec t s i n V e r o C e l l Cu l t ur e b y N aeg l e r i a spp . XXVI I . R e l a t i v e fo rma t i o n o f Cy t op a t h i c Eff ec t s 1 n 8 8 9 2 9 3 9 7 1 0 6 V e r o C e l l Cul t u r e b y A c an t hamoeba spp . 1 08 XA? II I . R e l a t i v e fo rma t i on o f Cyt op a t hi c Effe c t s i n V er o C e l l Cul t u r e b y axen i c Ce l l -f r e e Fi l t r a t e s XXIX . Addi t i o n o f N . fowl e r i { M s T ) axeni c fi l t r a t e t o V e r o C e ll Cul t u r e s i no c u l a t ed wi th N . fo wl e r i ( Ts - 1 ) XXX . I nhibi t i o n of the f o r ma t i o n o f Cy top a t h i c Effe c t s i n V e r o C e l l C u l t u r e s inocu l a t ed 1 1 2 1 1 3 wi t h N . fow l e r i by an t i - p h o spho l i p a s e s s e r um 1 1 6 XXXI . C ompa r i s o n of t he p r op e r t i e s of Chl o r i ne , Chl o r i n e dioxi de , Ozone and D eci quam 2 2 2 1 32 XXXI I . P r ev i o u s R epo r t s o f an t i bo di e s i n Human S e r a 1 46 L I ST OF F IGURES 1 . M ap s howi ng t he loc a t i o n of the rmal areas of t he N o r t h I s l an d o f New Ze a l and 2 . F l ow d i ag r am showi ng t he s t r a t e g y used fo r the i so l a ti o n and iden t i fi c a t io n o f PFLA 3 . Me t h od used f o r t he p r o duc t i o n o f c h l o r i ne di oxi de 4 . Me thod used for the p r o duc ti on of o zone 5 . F l o w di a g r am showing t he s t r a t eg y used for t he i s o l a ti o n o f pho spho l ip a s es f rom N. fow l e r i s upe rna t an t s 6. G r ap h s howi n g ave rage mon t h l y t emper a t u r e fo r N ew Ze al an d 7 . G r ap h s howi n g av er age mo n t h l y r ai nfal l fo r th e fvJa tama t a and Taupo r e g i o n s 8 . G r ap h s h owi n g ave r age mo n t h l y r ai nf a l l fo r t h e R o to r ua a n d Gi sbo r ne re g i ons 9 . Th i n - l aye r c hr omato g r am s of ne u t r a l l ip i ds and phospho l i p i d s f r om e x t r ac t s o f EY p l a t e s 10 . PAGE p r ofi le of the s upe r n a t an t f rom se r um ? s upp leme n te d axen i c N . fowle r i cu l tu re 11. PAGE p r ofi l e of 40 - 60% ( NH4 ) 2 so4 precipi ta t" ed f r ac t i o n of the se rum - s upp l eme n ted axenic N . fowle r i c u l ture ( M s T ) 1 2 . PAGE p r ofi l e o f t he XM 50 , 000 MW u l t r afi l t r a ti o n f r ac t i on o f s er um - s upp l emen t e d axen i c N . fow l e r i c u l t u re 1 3 . PAGE p rofi le of the SEPHADEX G - 100 f ra c t i o n o f s e r um - s upp lemented axen i c N . fowle ri cu l t u re 1 4 . PAGE p r ofi le o f se r um - supp l eme n te d axeni c c u l t ur e me di um 1 5 . PAGE p r ofi le o f to v i ne se r um . . ( xv ) 1 0 3 8 45 45 45 54 5 5 5 6 8 6 9 5 9 5 9 6 9 6 9 9 9 9 1 6 . PAGE profi le o f se r um - f re e axe n i c c u l ture medium ? 1 0 1 1 7 . PAGE p r ofi le o f the s upe r n a t an t f r om se r um - f ree a xen i c N . fo w1e r i c u l t ure ? ? 1 8 . PAGE p r ofi le o f SEPI1ADEX G - 100 f r a c t i on o f se r um? f ree axeni c N . fowle r i c ul t u re . ? 1 9 . Ge l -diffu s i o n p a t te r n u s i ng an t i -pho s pho l i p ase s se rum s e rnn 1 0 1 1 0 2 1 03 ? ? ? C on t 'd 20 . G r aph o f p r eda t i on b y Naegl e r i a spp . o n t h e bac t er i um En te robac te r c l o acae 2 1 . G r aph of p r edation by A c an t hamoe ba spp . on the b ac t e r i um En te robac t e r c l oacae ( xvi ) 1 2 1 1 2 2 22 . D i s t r i b uti on o f HOC l and OC l as a func t io n o f pH 1 3 4 23 . R e l a t i v e b ac t e r i cidal e f f i c iency o f c h l o r i n e c o n t aining der i va t i v e s 24 . Effec t? of ni tr o g en compo unds o n t h e c h l o r i n e dem and 2 5 . R e l a t i ve effec t i v ene s s o f c h l o r i ne dioxide as a di s i nfe c t an t 1 3 5 1 3 5 1 39 ( xvi i ) L I S T OF PLATES 1 . Wai gnaro therm a l poo l 2 . M o ana - i t i t h e rm a l poo l 3 . Op a l ' J ockey ' t he rm a l po o l 4 . Oko r oi re ' No . 41 thermal poo l 5 . S o da therm a l poo l 6. B u t che r' s therm a l poo l 7 . Uppe r O t umah ek e s t re am 8 . 1\l o u t h of O t umah eke s t re am 9 . O l d ' Whe y ' s o i l - s i te of O t umah eke s t re am 1 0 . Ho t - wa te r s o u r ce o f Te P u i a S p r i n g s 1 1 . Wo ode n t r ough u s e d to p i p e t h e rm a l wa te r to Te Pui a poo l 1 2 . M o r ere thermal b a t h s p i pe s upp l y 1 3 . H u t e nc l o s i n g M o r e r e t he rmal b a t h s 1 4 . S c anning e l ec t r o n mi c r o g r aph o f N . gr ube r i t r opho zoi t e 1 5 . S c anning e l e c t ron mi c r o g r aph of N . gr u b e r i f l age l l a te S c anning e le c t r o n mi c r o g r aph of N . grub e r i S c anning e l e c t r o n mi c r o g r aph of N . fowl e r i c y s t c y s t 16. 1 7 . 1 8 . S c ann ing e l e c t r o n mi c r o g r aph of A . cas t e l l ani i t r opho z o i t e 1 9 . S c anning e le c t r o n m i c r o g r aph of A . cas t e l l an i i cys t 2 0 . S c anni ng e l e c t r o n mi c r o g r aph of A . c u l be r t so ni c y s t 2 1 . P h o tomi c r o g r aph o f N . fow l e r i s t ai ne d b y I FAB 2 2 . P h o tomi c r o g r aph o? N . gr ube r i s t ained b y I FAB 2 3 . Pho tomi c r o g r aph of A . culbe r t s oni s t ained by I FAB 2 4 . Pho tomi c r o g r aph o? A . cas t e l l an i i s t ai ne d b y I FAB Page 1 4 1 4 1 5 1 5 1 7 1 7 1 8 1 8 1 9 22 22 2 3 2 3 2 6 2 6 2 7 2 7 2 8 2 8 2 9 7 2 7 2 7 3 7 3 2 5 . Pho tomi c r o g r aph o f s ec t i o n t h r o ugh ski n o f gui ne a - p i g immuni zed wi t h N . ?ow l e r i and s ki n - te s t ed wi t h s a l i ne 8 1 ? ? ? Co n t ' d (xvi i i ) 2 6-28 . P h o tomi c r o g r aphs o f s e c tions t h r ough ski n o f guinea-p i g immuni ze d wi t h N . fowle r i and ski n - te s ted wi th Naegle r i a s pp . an ti ge n s 2 9 . Pho tomi c r o g r ap h o f c o n t r o l mi g r a t i on o f PEC 30 . Pho tomi c r o g r ap h o f i nh i b i t i on o f mi g r at i on o f PEC 3 1 . C le a r i ng o f EY p l a t e s by ce l l - fre e fi l t r a te s f rom 8 1 8 2 8 2 exponen t i a l axen i c c ul tu r e s of amebae . ? 85 32 . C l e aring o f EY p l a t e s by ce l l - f r e e fi l t r a te s f r om s t a t i o n a r y axen i c c u l ture s o f amebae 8 5 3 3 . C le a r i ng o f EY p l a t e s b y c e l l - f r e e fi l t r a te s f r om 48 h r axe n i c cul t u r e s o? ame b ae 3 4 . Inhi bi t i o n o f c le ar i ng o f EY p la t es by ce l l - f ree fi l tr ate s f r om 48 h r axe n i c c u l t u re s of amebae 90 by Rose n t h a l ' s i nh i b i t o r 9 0 35 . C le a r i n g a n d t u r bi di t y o f e g g - yo l k afte r n a t i ve PAGE of p u r i fied pho spho l i pase s 1 0 4 3 6 . S c anning e le c t ronm i c r o g r aph o f c o n t r o l Ve r o Ce l l C u l ture mono l aye r 1 0 4 3 7 . Pho tomi c r o g r aph o f Ve ro Ce l l C u l t ure i nocu l a ted wi t h N . fowle r i s h owi n g e a r l y c y top a th i c e ffe c t s 38 . Sc anni n g e le c tr onmi c ro g r aph s h owing a t t achme n t 1 0 9 o f N. fowle ri t o Ve r o ce l l s . ? 1 0 9 3 9 . Pho tomi c r o g r a ph o f Ve r o Ce l l C u l t u re i no c u l a te d wi th N . fowleri s h owi ng p r onounced c y t opa t hie ef fec t s 40 . Sc anni ng e l e c t r onmi c ro g r aph o f V e r o C e l l Cu l tu r e i no c u l a te d wi t h N . fowle r i s howi ng p ronounce d 1 10 c y topa t h i c effe c t s 1 10 4 1 . Pho tomi c r o g r aph of c on t r o l Ve r o Ce l l C u l t u r e s t ai ned wi th Sudan b l ack 42 . Pho tomi c r o g r aph o f Ver o C e l l C u l ture i no cu l a ted 1 1 4 wi t h A . c ul be r t soni s t ai ne d wi th S u dan b l ack 1 1 4 43 . P h o tomi c r o g r aph o f Ver o Ce l l C u l t ure i no c u l a te d wi th N . fo wle r i and s t ained wi th S u dan b l ack 1 1 4