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LEPTOSPIROSIS IN FREE-LIVING ANIMALS IN NEW ZEALAND, HITH PARTICULAR
REFERENCE TO THE POSSUN (TRICHOSURUS VULPECULA)
A the s i s presented in par tia l ful f i lment of the requir ements for the degree of Doc tor of Philos ophy in Veter inary Pathology and Pub lic Hea l th a t Massey Univers ity.
S tephen Cl ive Hathaway 1 9 7 8
ABSTRACT
A serologica l survey of 600 pos sums (Trichosurus vu lpe�ula ) f rom f armland environments in the sou thern hal f o f the Nor th Is land o f New Zealand revealed tha t t i tres agains t lep tosp ires of the Hebdomadis s er o group were p resent in 52% of sera . Bacteriological studies iden t ified the causative o rganism as Leptospira interrogans s erovar balcanica. This serovar had previously been iso lated only in Eas tern
Eur ope ; from man , cattle and pigs .
Isolation o f lep tospires from k idneys was aided by the use of a system invo lving whole kidney homogenisa t io n in y-sterilised p lastic bags in a ' Colewor th Stomacher ' . Comparison o f cultur a l and serological resu lts demons tra ted tha t , when titr es were read at low minimum serum dilutions , the s erological prevalence was an accurate ind ica tor of the bac ter iological prevalence�
Field surveys and experimental s tudies demons trated that the possum was a typical maintenanc e ho s t for balcanica . Experimental infec tion was easily es tablished and 50% of animals were s till lep tosp irur ic one year after infection . Experimental inf ec tion with hardjo could no t be
es tab l ished , and it is cons idered tha t previous r eports of endemic haPdjo infec tion in po ssums in New Zealand could be incorrect .
There was a marked d if ference in the age-sp ecif ic prev al ence of balcanica inf ec tion in possum popula tions , with infe�t ion being
restric ted to s exual ly-mature animal s . Evidenc e is presented to suppor t the hypo thes is tha t infection becomes e s tablished following behavioural changes assoc ia ted with the onse t of s exual maturity . It is sugges ted tha t the transmiss ion o f balcanica wi thin a population is dependant on direc t con ta c t b etween animal s rather than env ir onmental contamina tion . No correlation coul d be shown between the p revalence of inf ec t ion in diff er en t p opulations and the nature of the hab i tat .
A cons is tent paradoxical reac t ion to hardjo was found in sera from pos sums infected with balcanica . Chromatographic s tudies revea�ed that this was due to he tero logous ag g lutinating ac tiv ity of antibodies of the IgM clas s . Chromatographic s tudies a lso ind ica ted that , f o llowing
i i
i i i
infe c tion , the trans ition o f the pr edominant agglu t inati11g a c t iv ity i n sera from antibodies of the IgM class t o antibo d ies o f the IgG c las s was cons iderably s lower than in euther ian mammal� .
Balcanica was f ound to haemolys e r ed b lood cells o f several spec ies . An in vitro haemolysin tes t for the differentia tion of balcanica and hardjo i so la tes is descr ibed . Haemolytic activ i ty has no t been prev ious ly demons tra ted in o th er memb er s of the Hebdomad i s s er ogroup .
Inves tigat ions of o ther small free-l iv ing mammals revealed tha t serogroup Ballum infec t ion was endemic i n ship rats (Rattus rattus ) , house mice (Mus musculus ) and hedgehogs (Erinaceus europaeus ) in natural and syanthropic biotopes . The Norway rat (Rattus norvegicus ) was also shown to be capab le of maintaining a focus of Ballum sera
gro up infec tion when the populat ion dens ity was high . No l ep tosp ires f rom o ther than the Ballum ·serogroup were isolated from the roden ts examined .
Field evidence sugges ted that ther e was no predator-cha in transm is s ion of lep tos p iral inf ec tion from rodents to free- l iv ing carnivores . �irds were s imilarly found to be unimportant in the e p idemio logy of l ep tospirosis in the ecosys tems stud ied .
The nidality o f lep tosp iros is was inves tiga ted in an int en s ive farming environment for a per iod of tuo years , and wel l-def ined maintenance-ho s t p arasi te relationships f or dif ferent s erovar s were def ined in both domes t ic and free-liv ing species . No cul tural and v ery l i t tl e s erolog ical ev idence of interspecies transf er was f ound , d es p i te the apparent oppor tunity for trans fer of i nfection .
The concept o f a maintenance hos t for a p ar ticular l ep to s p iral serovar was investiga ted us ing a laboratory mouse model . As a
consequence of thi s inves t igation and field s tudies , the cha racter istics of a maintenance ho s t for a lep tospiral s erov ar are redef ined .
iv
ACKNOHLEDGEHENTS
The research pr oj ect descr ibed in thi s the s is was funded p rima r i ly by the Womens D iv is ion of Federa ted Farmers and I woul d l ike to gratefully acknowledg e their support . The continued interes t shovm by the members o f this organisa t ion throughout the proj e c t was very gra tifying . I woul d also l ike to acknowledge the f inancial suppor t of the Accident
Compensation Commission .
Special thanks are due to my superv isors , Professor D . K . Blackmore and Dr R. B . Mar shal! , for unbridled enthu s ia sm and encouragement at al l
times , both i n the laboratory and in the f ield . The adv ice and a s s is tance o f Profes sor B . W. Manktelow, Professor R. E . Munf or d , Dr vl. A . G . Char l es ton, Dr R . E . Harris and Dr K . M . Mo r iar ty has also been greatly apprec iated .
The capture of free- living species in the f ield was an important p ar t of this r es earch proj ect and many people p rovided ass is tance and she lter , bo th during favourab le and adverse conditions . I would l ike to collectively thank all these peop l e . In par t icular , I would like to ment ion the l a te Mr N . L ee, Superviso r of the South Hawke ' s Bay Pes t
D e s truction Board , and 0. Harr is , his successo r . Night-shoo t ing operat ions w i thout their help would no t have b een po ssible . Par ti cu lar thanks are
due to Mr J . Harris , Mr R. Perry ,Mr R.Ferr i s and Mr J . Innes . P ermi s s ion to sample p os sums in fores t ar eas was g iven b y Hr I . Logan of the New Z ealand Fo res t S e rvice and Mr J . Marya t t of the Well ing ton Reg ional Wat er Boar d . The many hours spent sha rp shoo ting of Norway ra ts at rupb ish tips b y Profes sor D . K. B lackmore is also acknowledged .
Cheerful and competen t technical a s s i s t ance was g iven b y Mr s Lyn B el l and Ms Barbara Wilton . I am indeb ted to them for their co-opera tion and willingnes s a t a l l t imes . I would a l so l ike to thank Ms Sandra
Roxburgh and Ms Lynley F ray for prepara ti on o f media , Mrs Jan Schrama f or preparation o f g lasswar e , Mr P . Wildbore for adminis trative assistance , Mr T . Law f or pho tographic work and Mr R . F au ld ing for servic ing o f
t echnical equi pment .
V
The typ ing o f thi s thesis was carried out by Mrs Alla in Sco tt ,
Mrs F ay Wicher t s and Ms Kathie Whit e , and their help is much appre c ia ted .
The resea r ch described in this thes is has undoub ted ly been enriched by the companionsh ip and cons truc t iv e criticism o f my eo-workers , John Hel ls trom and Terry Ryan . The mora l suppor t p rov ided in t ime of need is greatly apprecia ted .
Finally , I would l ike to thank my wif e Jul i e and daughte r The a , to whom a deb t o f time will hopefu lly b e repayed .
. '\ "
Frontspiece : The Aus tralian b rushtailed possum (Trichosurus vulpecula)
TABLE OF CONTENTS
Ab s tract
Acknowledgements Tab le of Cont ents Lis t o f Figures L i s t of P lates Cha p ter I
Chap ter I I
Chap ter Ill Chap ter IV Chap ter V Chap ter VI
Chap ter VII Chap ter VIII
Chap ter IX
Chap ter X
Chap ter XI
Lep tosp irosis in wildlife , ep id emiologica l c onsidera tions and a summary o f lep tospirosis in New Zealand .
The natural his tory o f the possum , Trichosurus vulpecula .
General method s .
Serological survey of pas toral possum populaticns . Development o f cul tural techniques .
Cultural and s erological inves tigations o f pastora l possum popula t ions .
Exper imen tal inf e c tion in po s sums .
Some aspects o f the ep id emiology of ba lcanica infection in possums .
The immunological response of possums to lep tospiral infection .
An in vitro tes t for the diff erentia t ion o f serovars balcanica and hardjo.
Natural his tory of smal l mammal s in New Zealand
vi
Page number ii
iv
v i
viii xii 1
32
42 6 1 85 107
14 1 1 84
2 1 3
244
273 and the me thods o f cap ture used in the p re seht s tudy .
Chap ter XII Lep to spirosis in ra ts. 2 86
Chap ter XIII
Chapter XIV
Chap ter Y01 Chap ter XVI
vii
Page ntl!!lb e r
Lep tospiro s is i n sma l l mammalian specie s , f er al ungula tes a nd pukeko ' s (Porphyria me lanotus ) in New Zealand .
The nidali ty o f l e p to s p iros is in an intens ive f arming environmen t .
The maintenance hos t . General dis cu s s ion .
References Appendices
319
335
366
40 1
408 435
"Leptospira interrogans serovar balcanica f rom a pos sum" .
follo\ving page
"The serologic and cul tural prevalence of Leptospira interrogans serovar ba lcanica in pos sums (Trichosurus vulpecula ) in New Zealand" .
4�0
F igur e 4 . 1
4 . 2
4 . 3
6.1
6 . 2
6.3
6.4
6.5
6.6
6.7
6 . 8
6 . 9
viii
LIST OF FIGURES
Page number
Loca tions from which pas toral possum samples were taken .
Prevalence of hardjo ti tres and geometr ic mean t i tr es , by age group and sex, in pas toral possums .
Frequency dis tribution o f hardjo t i tres by age gr oup in pastoral possums .
Preva lence of ba lcanica and hardjo titres , G . M . T . ' s and bac teriological prevalences for mature adul t and trans i t ional-s tage adul t pos sums .
Frequency dis tribu tion of hardjo t i tres in ma ture adult possums .
Frequency distribution o f balcanica ti tres in mature adult possums .
Frequ ency distr ibut ion of hardjo t i tres in trans i t ional-s tag e adul t possums .
Frequency dis tribution of balcanica t it res in t rans i t ional- s tage adult po ssums .
Frequency o f isolations f rom ma ture adult possums with different hardjo titres .
Frequency of isola tions from ma ture adult pos sums with dif f er ent balcanica t itres .
Frequency o f isola t ions f rom transi t ional-s tage adult possums wi th d if ferent hardjo t i tres .
Frequency o f isola tions from trans i tional-s tage adult possums with differen t balcanica t i tres .
65
7 5
77
1 2 2
1 2 4
1 24
124
1 24
1 2 7
1 2 7
1 27.
1 2 7
7.1
7.2
I 7.3
7.4 7.5
7.6 7.7 7.8
7.9
8.1
8.2
8.3
9.1
9.2
ix
Page numbe r
Hebdomadis serogr oup titres in pos sums experimentally infe cted w i th ba lcanica
( Experiment 2 ) .
Experimental infec tion with balcanica in pos sum E4 . 2 . Experimental infection wi th balcanica in possum E4 . 4 . Experimental inf ection with balcanica in possum E4 . 5 . Experimental infec tion with balcanica in pos sum E4 . 6 .
Exper imental inf ection with balcanica in pos sum E4 . 7.
Experimen tal infection with balcanica in pos sum E4 . 8 .
Log GMT of hardjo and ba lcanica titres in experi
mentally-inf ec ted· possums (Experiment 4 ) .
• Parameters o f infection with balcanica in r�ms ters inoculated with s erial samples of possum urine in Experiment 4 .
Geographical locations f rom which possums inhab iting deep forest� forest-pas ture boundary and coa s tal s eaface ecosys tems were sampled .
Growth curve of j uvenile possums and prevalences o f balcanica t i tr es i n dif ferent ag e groups .
Balcanica t i tres in j uvenile pos sums o f less than 1 9 0 days of age.
Dis tribut ion of ba lcanica and hard;jo agglutinins in f ractionated s er a f rom Joey 205 , Joey 254 and the dam o f Joey 2 54 .
Mean p ercentage d is tr ibution and s tandard errors o f balcanica and hard;jo agglutina ting ant ibodies o f the
!gM and IgG class in exper imentally-inf ected possums .
1 5 2
1 5 6 1 57 1 5 8 159
160
1 6 1 1 64
1 7 0
188
201
202
224
234
9 . 3
9 . 4
1 0 . 1
10 . 2
1 2 . 1
1 2 . 2
1 2 . 3
1 2 . 4
14 . 1
1 4 . 2
X
Page number
Mean distrib·Jtion o f ba lcanica and hardjo
agglutinins in f rac t i�nated s er a from expe rimentally
infected possums .
Changes in mean agglu tina t ing ac t iv i ty o f ant i
balcanica and ant i-hardjo IgM and IgG immune
globulins with time in possums experimentally infected wi th ba lcanica .
Haemoly tic ac t ivity of thr ee , s ix and twe lve day ba lcanica cul tures f or washed human and bovine red b lood cell s .
Haemoly tic a c t iv i ty of three , s ix and twelv e day balcanica cul ture s f or unwa shed human and bovine red b lood cells .
Per centage d is tr ibu t ion o f weight-range groups o f Norway r a ts from rubbish tips and the prevalence o f Ballum serogroup infe c t ion i n differen t weight-range g roup s .
235
237
262
263
302
Percentage d is tr ibu tion of weight-range g roup s of ship 3 04 rats and the p revalence o f Ballum serogroup infect ion in d if ferent weight-range g roup s .
Regression o f prevalence o f Bal lum serogroup infection 307 in Norway rats on s ub j e c t iv e e s t imate o f rela t ive
abundance .
Epidemiology o f Ballum s erogroup infec tion in the Norway and ship rat .
316
Massey Pig Res earch Centre and No . 1 Dairy Farm showing 3 3 7 trapping and environmental sampling loca tions .
Geographical d is tr ibu tion o f lep tosp irosis in domestic 3 6 1 animals a t the Hassey P i g Research Centre and No . 1
Dairy Farm .
1 5 . 1
15 . 2
1 5.3
1 5 . 4
1 5 . 5
Serial passage o f balZum, pomona, and baZc�nica in weanling hams ter s .
P revalence o f lep tosp iruria in groups of s ix mice infected with baZ Zum, pomona and baZcanica at a dose rate of 1 08 leptosp ires .
xi
Page number 3 74
3 79
P revalence and intens ity of leptospirur ia a t dif ferent 380 times pos t-ino cula tion in groups of six mice infec ted
with baZZum and pomona.
Transmiss ion of baZZum within family groups of l abora tory mice .
Transmiss ion of pomona within family groups of labo ra tory mice .
387
38 8
/
xii
LIST OF PLATES
Fol lowing page
Frontspiece : The Aus tralian brush- ta iled possum (Trichosurus vuZpecuZa ) .
V
3 . 1
3 . 2 3 . 3
8 . 1 8 . 2 8 . 3 9.1
1 1 . 1 1 1 . 2 1 1 . 3 1 2 . 1
1 4 . 1
S teel trap of the typ e used for the sampl ing of s ome possum populations .
Cyanide. pa s te bait ea ten by a possum .
Modified Co rnwal l syringe for the d ispens ing of antigen.
Coas tal seaface e cosys tem . Deep fore s t ecosystem .
Fores t-pas ture boundary eco sys tem .
Immunoelectrophore t ic reac tion o f concentrated gel f il tration fra c t ions of possum serum with r abb i t antipos sum I gM and IgG .
Ship ra t cap tured in a snap tr ap .
45
45 53
1 9 1
1 9 1 191 220
283
Cage trap for cap ture of ra ts . 283
"Katch All " automa tic mous e trap . 284 Hi gh dens i ty popula tion o f Norway rats at Feilding 3 1 3 dump .
Double-chambered duck trap in u s e at the Mas s ey P ig 339 Research C en tr e .
