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private study only. The thesis may not be reproduced elsewhere without
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TOGEI'fiER l"TITH PRELIMIN .... ,RY INVESTIGATIONS OF PUBLI c HEALTH rm.i\SURES
... i. tLesi s J.)resented in ~)artial fulfilment of the
requirements for the de:;ree of Master of Science in Microbi olot;y at
Massey University, New Zen.13.!ld
Raymond Thomas Michael Cursons
1974
lill.STRltCT
The tG.Xonomy of the o.etioloJj cal .:J.[;ents of l rimary .'\mebic Meninc;o- ence;_)hali tis (Pt.M) was investic;ci,ted to det ermi ne the reliabi l i ty of the
common fentures of the three current schemes. It i s conclud.ed that the scheme of Sin::;.h & Dc..s ( 1970) is the most suitable end should be cenerally adopt ed. The acceptance of one scheme will remove much of the confusion whi ch charact erizes the classifi cation of these orc;anisms.
Current iclentification methods that differentiate between Naeeleria r;ruberi (the !1.0n·-pathor_;en) and I!9-_e .. {!;.~:zj..D: fo~~ (the pathot;en) were also investiGat ed over a wider rani:~e of parameters than j_)reviously, to establish their relative usefulness. The conclusions of this investi- gation are ~!resented in Tables XXII and. XXIII. The controversial identification of the 1968 lrow Zealand C<:!.ses (isolates BH & BL) as a Myxomycete by Mandal ~!. .~l.. ( 1970) was re-exClillined. Evidence is pre- sented to demonstrate that they are N. ~~~e~_?:.·
It i:u1s establishecl that there ':Jas no ;cneral sel ection for the non-patho,zen over the IJatho:~en at
37°c
::rn inclico.ted 1:Jy their respective Qo2 v;:i,lues <.~t27°c
and37°c.
That there is potenti al for adaptationto a rnni_;e of te:·nperc:::.tures wi.s shuwa.
The failure 8f chlorine as a di sinfectant for these soil--amebae was al so cx2!lli ned. The ineffectiveness of normal levels of chlorination was confirmeC. and therefore the use of NaCl :.md the b.:1sic dyes Mala-
chite Green and Brillinnt Green investigated. It was found that no runebae could survive a concentration of 1.
5%
(W/V) of NaCl in axenic cul ture7 of 1.5 pg/cm3
of Malachite Green and of 3.0pg
of Brilliant Green.The author is indebtec.~ to the De~_,artment of Microbiolot-;y and Genetics, Massey University for ;_::irovidinc.; the opportunity and facili t- i eo for this inve3tic;a ti on.
In ~;articular I would like to thank:
My supervisor, Dr. T. J. Brown; Pr·Jfessor D.F. Bacon, Dr. J .K. Clarke, Dr. B. D. Jarvis, Mr. L. W. Smith, u.nd other acndemic and technical staff of the Department of TiicrobioloGY and Genetics for their r.;eneral help and encourar;ement.
r.Ir. A.F. Green for his help in serology and cell cultures.
Dr. R.A. Robinson cmcl tf, e No.tionnJ Fieo.l th Institute, Wellinc;ton for the r;ift of cultures, the use of their fncili ti es for the mouse-
~1athoceni city tests end also for their iJ,clvice and L;eneral cooperation.
I.frss N. Davies for tLe ::;i ft of cultures BH and BL.
Mrs. j"ean Kini; for tlie excellent typinc.
Miss Anne D::irber for the nuclear division cli w.;rruns.
Mrs. Christine Gradolf for the help with the little-bi ts.
Gla.x.o Laboratories for their cenerous donation of Griseofulvin.
The Central Photocraphic Unit, Massey' University.
PREFACE
Primary Jimebic I"1eningo-ence;:ihali tis (PfiJ'.n is a normally fatal dis- ease of the central nervous system (CNS) usually involvii113 youn51
heo.lthy individuals with a recent history of contact with fresh-water.
It was first recoi:;nisecl by Fowler in 1lustralia in January 1961 and since then1 about 74 di c:l£nosed cases have occurred in various parts of the world (Table I).
On purely histoloGical evidence, c.11 reported cases of PAM, prior t o 1968 \vere attributed to members of the Hartmannella/Acanthnmoeba eroup of amebae (Culbertson et nl., 1961i Fowler & Carter, 1965).
However, three reports in 1968 (Cerva & Novak; Cerva, Novak & Culbert- son; Carter) sugc;ested that the amebae were much smaller in histologi- cal sectionf:l than for the formerly known ~Jo.thor;enic Bartmannella/
!1canthLl!Tloeba strains, and probo.1..:lly belon~;ecl to the related e;enus Nae- gleria. Cul tureJ. verification of this su,:_;.;estion soon followed (Butt:
Baro & Knorr, 1968i Culbertson, Ens:ninc:er & Overton, 1968; Calicott
et al., 1968) ::i..'1,'~ the .:unaebae v-1ere subsequently clo.ssified as !ae3leri9;
.G~· Then, in 19701 on the basis of morpholoL:ical, cultural, a..11.d vathoGeni ci ty differences, Carter renQ!Tled the p~tho~onic NaeGleria, Naer;leric. fo~1leri. To elate, there are two other synonyms for the path- Oi~eni c Naec;leria:
£! ·
~9_~i-~ (Sini::;h & Das? 1970) .J.nd N •. ~1::Yo..de~.( Cho..rl(j 1 1971 ) •
The first New Zealand co.ses of PAM occurred in the late autumn of 1968 (Manda.l -~~al., 1970) cd'ter the victims had bathed in a Matwnata hot-spring. Al though the amebae were oric;inally considered to belong to the genus Nae{:Sleria, they were later reclassified. a.s a slime mould probably belonGinc to the genus Echinostelium, Mandal ~ ?l• The fifth case occurred in May 1972, after the victim had also swum in a thermal pool, but in this case the aetioloGical a{;ent was identified as
!!·
fowleri (Nicoll, 1973). Since then the Adelaide Amebic Research Unit has consistently isolated pathogenic Naegleria and Hartmannella/Acanthamoebae from New Zealand sources (Robinson, 1974).
Unfortunately, the classification of these organisms is still a controversial matter (Carter7 1970; Culbertson, 1971). With three
different classification schemes existin0 in the literature (Paee, 1967;
Sinsh
&
Das, 1970; Chang, 1971) all of which overlap to some consider--, '
able extent crentini:; confusion in the selection of a. name, it was con- sidered necesuary to review the classification to est::i.blish the clec;ree of relia"bili ty which could be :i.tt~checl to each of the characters cited.
It is essential as a prelude to the cli.nc;nosis of P1\M that there are reli2.ble characters on which to ;;ase 2...'1 identification, since b.v its very nature, iclentificntion :;resup;ioses that classification hi.ls
already distin[-;uished the species, ancl that rn::in1es hnve been assigned to them. As i.1 follow-up, it Wi.1S consiclerecl essential to review current identification methods over a wider rMt;e of experimental parmneters than have previously '::leen exemined, ns well as investicatinc new methods of identificc..tion. Usin-:; these methods, it was then decided to re- assess the controversial identificntion by Mnndal ~ al. ( 1970) of the causative .'J.Cents of the early New Zeal 2nd cases.
ABSTRAC'r ••
ACKHOWLEIGEJl:iEi'JTS ••
PREFACE
LIST OF PIGURES LIST OF TJJ3LES LIST OF PL.lTES
. .
C HilPTER ONE: INTRO DU CTI OU
TABLE OF COUTEJ.IJTS
. .
1. The History of Primary .l\mebic Menin{:;o-ence_;_Jhali tis
1. 1 Discovery
. .
1. 2 Classification and Identification 1. 3 Epi clemio loc;y
1.4 Patho[~eni city 1.
5
Diacnosis 1. 6 Treatment 2. Control Me.::i,sures3. Aims of the Investi~:~ation
CHAPTER TWO: NN~ERIALS
1 • Juneba Cultures Used 2. Plate Media ••
Axenic Media
:').. Cell Culture Media Vero Cell Culture 4.1
4.2 Chick flnbryo Kidney Cell Culture
s.
Miscellaneous Solutions. . . .
CHAPTER THREE: MEI'HODS 0 0
..
1 • Sterilization
. .
2. Culture Techniques • •
2.1 Cloning ••
. . . .
2.2 11.xeni zi nG
..
2.3 Flaeellation
. .
3. Serology ••
. .
3.1 Preparation of 1\ntigens ••
. .
. . . .
. . . .
. . . .
..
..
..
.. . . ..
. .
. .
..
..
•
. . . . . . ..
Pa.ge ii iii iv ix x .xi
1 1 1 2 6 8 11 12 15 16
17 17 21 23 26 26 26 28
29 29
30 30 30 30 31 31
4 . 5.
3.2 Preparation of Antisera ••
3. 3 l1r:;cl utinu.tion
3.4
Indirect FluoreGcent Antibody3.5
Gel-Diffusion (Ouchterlony) ••House Patho,_:enici ty Cell Culture ••
5.1 Vero
5.
2 Chi ck :Embryo Kidney •• 6. Disinfection ••..
32 32
32 33 34
35 35 35 37
6. 1 Disinfection Tests
37
6. 2 Effect of Different Cell Numbers on the Chlorino.tion
Level
37
6.3
Effect of a Maintgined Chlorination Level on a KnownConcentration of fimebae..
37
6 . 4
M1alysis of Chlorine37
7.
Staininr; ••7.
1 The Fet.:.ll;en Reaction7.
2 Iron-1-Jaematoxylin7.3
TrichomeCHAPTER FOUR: RESULTS
1. Classification c:w."ld Identifi cntion of NaeL:;leriu. and Hartmann-
39
ella/i1canthrunoeba Isolates
3 9
2.
1.1 Nucl ear Division 1. 2 Plaque Morpholo,::;y
1. 3 Diacnostic PhysioloGical Characters ••
1o4 Serolof5Y
. .
A!;csl utination 0 •
1. 4 . 2
Indirect Fluorescent Antiboey Titres ••1. 4 .3
Gel-Diffusion Analysis (Ouchterlony) ••1.
5
Mouse Pathosenici ty ••. . . .
..
1. 6
Vero Cell Culture Respiration Rate (Qo2) Respiration (Q10)and the Temperature Coefficient of
. . . .
Di sinfec ti on ••
. .
3. 1
Use of NaCl ••. .
3.
1. 1 In Ffonoxenic Culture ••. . . . . .
39 43 47
48
49
5153 54
59
61 6162
3.2
3.3
3. 1. 2 In Axenic Culture Bc.si c II'Jes."
3.2.1 f.1alachi te Green ••
3.2.2 Brilliant Greem •• Chlorine
3. 3. 1 '.:ffect of Different Cell Numbers on the Chlorin-
p~
62
6464 66 69
ation Level..
69
3.3.2 ~ffect of a Maintained Chlorinntion Level
69
CHAPTER FIVE: DISCUSSION 72
1. Classification of Ji';:unilies of iimebae ;\ssoci::ted with Prima.ry
1'.mebi c Meninc;o-encephali tis (PAM) 72
2. Identification of the ,letioloci cnl A:·; en ts of Primo.ry June bi c
M eni n:~:o-encephali tis
77
2.1 Identification Bnsecl on I-tioriJholoc:y-.. 77
2. 2
2. 1. 1 2. 1. 2 2. 1. 3 Growth 2. 2. 1 2.2.2 2.2.3 2. 2.,.i
The Trophozci te Sta:~e
The Flacellnte Stace The Cyst 3tt1(;e
1llll Physiolo .. ;y Plaque MorJholo:;y S'J.l t Tolernnce Tern;1er<1.turc ••
Respiration Ra.te
(Qo2)
and the r.rempero.ture Co- efficient of Res~iro.tion (Q10)2. 3 Serology
3. Clabsification ancl Id8ntifica.tion of the Wn.ika.to Isolates BH
77 80 80 81 81 82 82
84
85 88
o.nd BL of
1968 90
4 .
Disinfection92
5.
4 .1
The Fa.ilure of Chlorine as a Disinfect2.!1t.+ .
2 The Use of Na.Cl and the Basic llfes rnal achi te Green ::i.nd Brilliant Green as Potential Disinfecta.nts for theControl of Primary /i.mebic MeninGo-encephali tis ••
Conclusions ••
. . . .
BIBLIOGRAPHY
..
92
94
97
99
1 •
15 . - 27.
28. ·- 33.
34.
35 . 36 .
37.
40.
Li S'l' Ol<' FIGW."8S
Schenwiic (.:'..ia-.:;rom of c•J factorv rnucos<..;, .::~ncl ;:,djacent
~itosis of Eurtra~nnelliQC,e
Com11arnti V8 L;el--d~ ffusion .-:.c.'1.::.lysi s
c.xeni c cul-ture
GreE-E
Green
Effect of diffe:rent cell !rnmbe:·::; on tho=:• totnl avail- able chlorine level as ,:;, consequence of time. "
r.Jnintai:1ed chlorine level or.
105
Naec;leria/c:n3 o.G :::i.function of time ••
•I 1
•-j-'
)2
60
60
.·• '7 J ,
() 7
70
I l . IIL IV,
v .
VI.
VIL VIII.
x.
XE.
XIlI.
X IV.
X~· v 0
XVII .
DTIII.
XIX.
XXI.
XXII.
XXIII.
LIS.t OP Tf\BLES
c~ses cf P1;;-·1 iE tlw T,i tera.tm:·e
·.:::0m~_Qri.:;on of 1::.~j)hote:r:'icin B c..nd. Clotrimazole in vitro
im 1;ur~i7-::.t.ion ScLec~u_i_c
Cor.i;?::.rison of Pl::1que Char'.:cteristics
~)i:f£cr0ntio.l Di~:_;:o.ostic Cho..racterc of Naecleriu ••
f::~lu ;enicit:r :·. 1 VCl\) Cell Culture
I'ho o_ .(' Ilalr...::l:i tc Green a.s o. Disinfectant '11110 -:;;ffoctiv(;:1c::;•j ~f Brilli.-1.nt Grei::n r..s L. tlsinfectc.nt
'i'}l'1 'i'o-~·-,l ::\;,1.il:::~;1 0 C!1lorinc LE•veJ. .-:.,o c.. C1Jnscque!'lcc of Diffor\.::nt c~.J.l !Iu.111Jers ••
Com;.1~J. ::tj -..-0 C.1~.r;titn:an:i; Chlorine Levels on .:-.. Knoim Con·-
Cl.}ll~J::.1.::.·t·J Q_ 0 ~ l:iL1·::1)J..'3
Cc1:1p::i.r.::i:~i.vC:: Irm.nt."l.c~lcctro;:.horctic ;~nc..lysis of Different G-::nor1. of ;,rns'b.::.8 ••
,\~n:olute Fe~tnrer: Ust:.d in the Differentic.tion of N. :;;::-u12_;:_ri fror.i J~> .~owl o·~i.. ".
Supportin,·; Fe<.-.tura3 'J3ed in the Differentiation of £.!.
~ru~~ frorr. N. }~~E~·
•
j
5
u
1 ','
.) .
39
)_
,..
.; '
11
,. , ...
'"
LIST OF PLATES
1. Trophozoi te stage of N. gruberi 1 Nr;-27 ••
2. Cyst sta{~e of N • .';jruberi, Nr;-27
Trophozoite sta;3e of N. fowleri, NHI
..
Trophozoite stai:se of BL
. .
5.
Trophozoite st~se of BH6. Plaque morpholoGY of _!i. cruberi, Nc- 27 .c~rown on NM :::i.gar
with E. coli
7.
Plaque morpholocy of.!'!·
fowleri, NBI c;rown on NM LJGD.r with E. coli8.
Centred-plaque morpholo.:.:;;y offl·
fowleri, NHI Grown on NM ar;ar withE.
cloacae..
9.
Plaque morpholotzy ofE ·
fowleri, PA14 Grown on NM ~Jar withE. cloacae ••
10. 3 day normal Vero cell culture monoln,yer
..
11. 3 day cytopathic effects of~· fowleri, HB-1 in Vero cell
culture
. . . . ..
12.
& 3
day cytopathic effects of~· fowleri, NTH in Vero cell13.
culture/stain trichome ••. . . .
Pu.ge
18
18
19
20
20
44
45
45
46 56
56
57