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.
LEPTOSPIROSIS IN NEW ZEALAND PIG HERDS An epidemiological s tudy and a computer simulation model
of endemic leptospiral infection in New Zealand pig herds,
with
p a r ticula r reference t o
Leptospira interrogans serovar pomona .
A thesis p resented i n p a rtial fulfilment of t h e requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University.
Ingeborg Bolt 1 990
ABSTRACT
A serological s urvey of pig sera from six regional areas throughout New Zealand indicated that 60% h a d titres to Leptospira interrogans serovar pomona and 1 3 % to serovar tarassovi. Pig sera fro m the North Island districts had higher titres to pomona than t hose fro m the South Island districts, however the converse was true for titres to tarassovi. A serological s urvey of bacon weight pigs a t slaughter revealed that 60% had titres to pomona, 53% to bratislava, while titres to tarassovi were undetectable. The¥e
was no significant linear association between the magnitude of corresponding pomona and bratislava titres. Pomona was isolated from 53% of pigs
kidneys, however attempts to isolate bratislava were unsuccessful. The median prevalence of infection in bacon pigs from fa rms with endemic pomona infection, a t the time of slaughter was 80%. A retrospective s tudy of farming practices revealed that vaccination of breeding pigs had no effect o n the infection status o f their grower pigs at slaughter. It was also found that farms which reared their grower pigs to pork weight for slaughter were free o f leptospiral infection, as were farms with less than fifty breeding sows.
Cross s ectional serological and cultural p revalence studies o f grower pigs on farms with endemic pomona infection revealed that pigs less than ten weeks o f age were not leptospi ruric and had low or undetectable ti tres to pomona.
Pigs b etween ten and twenty weeks of age showed an increasing p revalence o f both leptospiru ria and pomona titres. Fu rther p rospective studies indicated that piglets acquire passive immunity from their dams, which h as a half life o f a round sixteen d ays. The majority of pigs less than fou rteen weeks of age appear to be resistant to infection, thereafter the level o f their p assive immunity wanes and they become infected and leptospiruric. The weekly incidence of leptospiruria for pigs in an infected grower house was usually between 10% and 20%. Following infection, the intensity of leptospiruria was greatest in the first three to fou r weeks and it l as ted for at least s ix weeks . Infection is bel ieved to occur by both d irect and indirect transmission of leptospires between infected and susceptible pigs. I t was s hown that grower pigs are a t the centre of the endemic cycle o f infection which is perpetuated by the trans mission of infection fro m older infected pigs to you nger s usceptible pigs . This cycle o f endemic infection can persist independen tly o f the breeding herd.
iii Experimental evidence following the artificial exposure o f grower p igs to e i t he r serovar pomona or bratislava supported an hypothesis that the occurrence o f bratislava titres were associated with early i n fection of p igs w i t h serovar pomona . The heterologous t itres were believed to be a s erological cross reaction with homologous IgM antibody to which p igs had been exposed .
Experimental evidence demonstrated that leptospires could survive i n droplets of less t h a n 50 urn, however hamsters exposed to a leptospiral a e rosol containing droplets o f less than 50 urn failed t o become infected.
Infection via the intranasal route i n both hamsters and p igs showed that the i n fective dose of pomona was between 104 and 1 06 leptospires, i ndicating the intranasal route as a natural route for infection. Transmission o f infection could therefore occur d irectly by infective droplets lodging in the nasal cavity.
The vaccination of pigs, com m encing at ten weeks of age o n a farm with endemic pomona infection, revealed t h a t m ultiple i noculations o f a commercially available b acterin can be used to control the level of endemic i n fection within a grower pig herd. There was evidence to suggest t h a t persistent passive immunity i n young pigs could interfere w i t h t h e efficacy o f vaccination.
A computer s imulation model of endemic pomona infection in a pig herd [Simulated Leptospiral Infection within a Pig herd, S LIP89] was d eveloped using the results of invest igations described in this thesis by sequentially breo.king down the cycle of endemic pomona infection into a series o f logical events. The model repeats a number o f p redetermined i ndependent and dependant even ts for each pig within a simulated herd. The o u tcome of each even t is randomly determined fro m an appropriately selected probability distribution. Each cycle o f repetition represents one week in time. The o utcome generated by the s i m u l ation can be used to obse rve varying patterns o f i n fection which a re due to either the element of chance o r the alteration o f k ey variables within the model . The results generated by the SLIP89 must b e viewed with the s tructure and l imitations of the model i n mind.
ACKNOWLEDGEMENTS
I wish to acknowledge the fin a ncial s upport given by the Wom e n 's D ivision of Federated Farmers who funded this research, the P hyllis Irene Grey Fel lowship Fund which provided a computer and the Muriel Caddie Schol a rs h i p for personal funding. The p rovision of animal remedies fro m Coop ers Animal Health is appreciated .
I would l ike to thank my supervisors D r Roger Marshall for his encouragement, enthusiasm and patienc� throughout the period o f research and documentation, Professor Roger Mo r ris for his guidance throughout the development of the computer simulation model, and to Professor D avid Blackmore for his initiation o f the rese a rch p roj ect, his interest, constructive criticism and advice during his final years at Massey Universi ty.
Spec i a l tha nks to friends and eo-workers Ray Corner and Steve Flint for their help with the collection o f sam ples and for providing valid a rgument and d iscussion. I thank Wendy Gable for her support in the laboratory, Jan Schr a m a for her work i n the media preparation room, Peter Wildbore for ordering a l l materials, Pam Slack for the preparing h is to logical sections, Faris S h arpe for incinerating infective tissue and material, D r . Ram adass for the B R E NDA typing of leptospires, and the help of Fio na D icki nson with preparation of the final draft of this thesis.
I a m grateful to the management and employees of the Kiwi Bacon Comp any o f L ongburn for their co-operation in allowing the collection o f samp les fro m t h e i r abattoir. I wo uld l ike to thank t h e managers and workers o f all the p i g farms which were contacted througho u t t h i s study, with special thanks to P h i l Weber, N e i l Managh a n d A l a n Taylo r for their long t e r m assistance.
Finally, I wish to thank my husband B rad Bridges for his encouragement and support, his sense of humour and love, all o f which helped sustain me throughout this study. I am grate fu l that our son, Bear n a rd , who was born i n t h e later stages o f the prep aration of this thesis, proved to be a h a p py and contented child, and one who accept ed my divided attention.
V
TAB LE OF C O NTENTS
Abs tract . . . i i
Acknowledgements . . . iv
Table of contents . . . v
List of Tab les . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi
List of Figures . . . xi
Chapter O n e Introduction . . . 1
Chapter Two Chapter Three Chapter Fou r Chapter Five Materials and Methods. . . 24
Prevalence Studies of Lep tospiral Infection in Pigs . . . 48
Observational Studies . . . 74
Experimental Studies . . . 1 20 Chapter Six Vaccination Trials . . . 160
Chapter S even A Computer Simul ation Model of Endemic Appendix I Appendix 11 Appendix Ill Appendix I V Appendix V Appendix VI Leptospiral Infection i n a Pig Herd . . . 2 1 2 Preparation o f Media Conversion of Titres 253 257 Questionnaire to Farmers . . . 258
Procedure for using S LIP89 . . . 260
List of SLIP89 data files . . . 278
Program Units i n S LIP89 . . . 279
References . . . 3 2 1
Chapter O n e 1 . 1
Chapter Two 2 . 1
2.2
2 . 3
2 . 4
C hapter Three 3 . 1
3 .2
3 . 3
3 .4
3 .5
3 . 6
3 .7
LIST O F TABLES
Maintenance hosts for leptospiral serovars
found in New Zealand . . . 1 8
Fina l test dilutions o f s e r u m samples i n the
microtitre plates used for t he MAT . . . . . . . . . . . 3 7 Leptospiral serovars main tained for the
Microscopic Agglutination Test . . . 40
T h e o r e t i c a l n u m b e r of l e p t o s p i r e s inoculated into five cul ture dilutions of
u rine . . . . . . . 41 Number of th ree u rine cultures from which
leptospires were isolated a fter two and fou r
weeks o f incuba tion . . . 42
Serological results for serovar pomona o f pigs from s i x MAF admin istrative dist ricts
of New Zealand . . . 55 Serological results for serovar tarassovi of
pigs from s ix MAF administra tive dis tricts
of New Zealand . . . 56 Distribution of pomona a nd bratislava titres
of grower p igs at slaughter . . . 59 Serological and cultu ral res ults of twenty
two bacon weight p igs sampled at
slaughter . . . 60 A comparison of some farm p ractises and
the infection status o f their pigs, for twelve
farms . . . . . . . . . . . . . . . . . . . . . . . . 64 Sizes of twelve farms in comparison with the
infectious status o f their grower pigs at sla ughter and the vaccination s tatus of their
breeding pigs . . . 65 Number of fa rms wi thin each district with
near equal or u neven ratios o f
pomona:tarassovi titres . . . 67
Chapter Fou r 4. 1
4 . 2
4 . 3
4 . 4
4 . 5
4 . 6
4 . 7
4 . 8
4 . 9
4 . 10
Chapter Five 5 . 1
5.2
Serological and cultural prevalence o f i nfection with pomona i n g rower pigs fro m
vii
Farm B . . . . . . 85 Serological and cultural prevalence of infection
with pomona in grower pigs fro m Farm D . . . 86
Schedule of sero logical and cultural examination of fou r age cohorts over an
eleven week period . . . 90 Serological results to serovar pomona of two
sows and five of their piglets between five
and thirty-fo u r d ays of age . . . 9 1 Serological results to serovar pomona o f
pigs between six a n d fifteen weeks of a g e i n
the "weaner coho rt" . . . 93 Serological and cultural results to serovar
pomona of pigs between e leven and twenty
one weeks of age in the "young grower
cohort" . . . 95 Serological and cultural results to serovar
pomona of p igs between s ixteen and twenty
six weeks of a ge in the "older grower
cohort" . . . 98 Mixing and movement of pigs between pens,
and the infectious status of pigs in adj acen t pens . . . . Onset and duratio n o f leptospiruria i n grower p igs fro m Farm B
Culture results and location of gilts o n Far m
. . . 1 0 1
. . . 1 02 - 1 0 3
B a t the t ime o f exa mination . . . 1 04
Homologous pomona t itres of pigs exposed to serovar pomona by intramuscul a r
inoculation or v i a the i n tranasal route . . . 1 27 Heterologous bratislava t itres of p igs
exposed to serovar pomona by intramuscul a r
i noculation or via the i ntranasal route . . . 1 2 9
5 . 3
5 .4
5 .5
5 .6
5 . 7
5 . 8
5 . 9
5 . 1 0 5 . 1 1
5 . 1 2
5 . 1 3
Chapter Six 6 . 1
6.2
6.3
Geometric mean t i tres [GMT] of pigs
artificially exposed t o serovar pomona . . . 129 Homologo us bratislava titres of p igs exposed
to serovar bratislava by intramuscular
inocu l a tion or via the i n tranasal route . . . 1 3 0 Heterologous pomona t i tres o f pigs exposed
to serovar bratislava by intramuscular
inoculation or via the intranasal route . . . 1 3 0 Geometrical mean t it res [GMT] of pigs
artificially exposed to serovar bratislava . . . 1 3 1 Homogenous and heterologous heat treated
and untreated titres o f pigs exposed to
pomona and bratislava . . . 1 3 2 O n s e t , d u r a t i o n a n d i n te n s i ty o f
leptos piruria determined by cultural
examination . . . 1 3 3
Diameter o f droplets formed by spinning
disc . . . . 1 48 Diameter of droplets by nebuliser . . . . 1 49 Minimum infective dose of pomona for pigs
via the intranasal route . . . 150 Minimum infective dose of pomona for
hamsters via the i n tranasal route . . . 1 5 1
Results o f exposing o f h amsters t o pomona
via the res piratory route using an aerosol . . . 152
Regimes of inoculation tested in grower
pigs . . . . . . . 1 7 1
Number o f p igs examined i n each inoculation group fro m Farm B throughout the period o f s ampling and a t the time of
slaughter . . . 173
Reciprocal titres o f p igs inoculated with vaccine i n the first week [Group 1 ] which were raised o n the far m free of leptospiral
i nfection [Farm A] . . . 1 77
6 . 4
6 . 5
6.6
6.7
6.8
6.9
6 . 1 0
Chapter Seven
7 . 1 7.2
7.3
Recip rocal t i tres and kidney culture results for p igs inoculated with vaccine i n the first week [Gro u p 1] and u nvaccinated pigs [con trol group] which were raised on the farm with endemic pomona infection [Farm
ix
B] . . . 1 78 Recip rocal titres o f pigs inoculated with
vaccination in the first and fifth week [Group 2] which were r a ised on the far m
free of leptospiral i n fection [Farm A] . . . 1 82
Reciprocal t itres and kidney culture results for pigs inoculated i n the first and fifth week [ Group 2] and u nvaccinated pigs [contro l group] which were fro m the farm
with endemic pomona i n fection [Farm B] . . . 183 Reciprocal titres o f pigs inoculated with
vaccine in the first, third and fifth week [Group 3 ] which were from the farm free o f
leptospiral infection [Farm A] . . . 187 Reciprocal titres and kid ney culture results
for p igs inocul ated with vaccine i n the first, third and fifth week [Group 3] and unvaccinated p igs [co ntrol group] which were from the farm with endemic pomona
infection [Farm B] . . . 188
Reciprocal titres of pigs inoculated with vaccine in the first, second and third week [Group 4] which were from the farm free of
leptospiral infection [Fa rm A] . . . 192 Reciprocal titres and kidney culture resu lts
for pigs inoculated with vaccine in the first, second and third week [ Group 4] and u nvaccinated p igs [control group] which were from the fa rm with endemic pomona
infection [Farm B] . . . 1 9 3
Age related m ating frequency for boars . . . 22 1 The average p revalence of leptospiral
i n fection for grower p igs in SLIP89, using
the default sett ings . . . 23 9
S LIP89 results o f the average prevalence o f
7 . 4
7 . 5
7 . 6
7 . 7
leptospiral infection i n grower pigs, when
validating for weaning age . . . 240
SLIP89 results of the average prevalence of leptospiral infection in grower pigs, when
validating for the d uration of leptospiruria . . . . . . 242 SLIP89 results of the aver age p revalence of
leptospiral i nfection i n grower pigs, when
validating for the i ntensity of leptospiruria . . . 243 SLIP89 results of the average prevalence o f
leptospiral infection i n g rower pigs, when validating for the contamination division
factors . . . 245 SLIP89 results of the aver age prevalence o f
infection for validation o f contact between
pigs . . . 246
Chapter 2
2. 1
2.2
2.3 2 . 4
2 . 5
Chapter 3 3 . 1
3 .2
3 . 3
3 . 4
3 .5
Chapter 4
4 . 1 4 . 2 4 . 3 4 . 4
xi LIST OF FIGURES
Collection o f a blood sample by jugular
bleeding . . . 27 Collection of a midstream urine sample . . . . . . . 28
Kidn ey culture p rocedure . . . 3 2 Urine culture procedure . . . . 3 3 Equ ations to convert reciprocal titre to a
coded titre unit [CTU] and CTU to a
reciprocal titre . . . 3 8
The six MAF administrative districts o f New Zealand used in the serological survey o f pomona a n d tarassovi ti tres of pigs
thro ughout New Zealand . . . 52 Serological resu lts for s erovar pomona of
pigs from six MAF administrative districts
of New Zealand . . . 53 Serological resu lts for serovar tarassovi of
pigs from s ix MAF administrative districts
of New Zealand . . . . . . . 54 Frequency distribution of pomona and
bratislava titres in 140 b acon weight pigs at
slaughter . . . 58 Distribution of pomona t itres in grower p igs
at slaughter fro m farms which either do or
do not vaccinate their b reeding pigs . . . 63
Design of piggery o n farm B . . . . . . . . . . . . . 8 1 Design o f p iggery o n fa rm D . . . . . . . 82 Design of piggery o n farm E . . . 83 Intensity of leptospiruria in naturally
infected pigs over a ten week period o f
examination . . . . . . . 97
4.5
4.6
4.7
4.8
4.9
4 . 1 0
4. 1 1
4 . 1 2
Chapter 5 5 . 1
5.2
5.3
5.4
5 . 5
5.6
Geometric mean t itre of pomona i n grower pigs fro m Farm C with endemic pomona
infection . . . 97 Schematic representation of movement of a
grou p of pigs througho u t the grower shed
on Far m B over a period of 1 6 weeks . . . 1 05
Schematic representatio n of the movement of two individual p igs BG- 1 and BB-2 throughout the grower s hed o n Fa rm B for
a period of 1 6 weeks . . . 1 06- 1 07
Average prevalence of leptospiruria i n 3
groups of n aturally infected grower pigs . . . 1 08 Prevalence of leptospiruria i n the pig herd
fro m Farm B with endemic pomona
infection . . . 1 09 Decline of matern al antibody titres i n p igs . . 1 1 1 Age of onset of leptospiruria i n grower p igs
of Farms B and C . . . 1 1 2 Cycle of maintenance of infection with
pomona in a grower pig herd . . . 1 16
Pom ona and bratislava titres of pigs
artificially exposed to s erovar pomona . . . 1 28 Bratislava and pomona titres
artificially exposed to bratislava of p igs Homogenous and heterologous pomona and bratislava titres o f heat treated and u ntreated sera
Spinn ing disc used for the formation of
. . . 128
. . . 1 3 1
d rops greater t h a n 5 0 JL m i n d i ameter . . . 1 43 Nebuliser used for the formation of droplets
less than 50 JLm i n d i a meter . . . 144 S ize of droplets landing in pen adj acent to
pen which was spray hosed . . . 150
5 . 7
5 . 8
5 . 9 5 . 10
Chapter 6
6 . 1
6 . 2
6.3
6.4
6.5
6.6
6.7
xiii Theoretical m aximum n umber of leptospires
in a drop of known d i ameter . . . 1 54 Theoretical percentage o f drops formed
from two leptospiral concentrations which contain at least one leptospira . . . . Pigs "rooting" amongst i n fective effluent . . . Pigs "urine sniffing", a behaviour allowing
1 5 5 156
i nfection via the intranasal route . . . 1 57
Serological results o f p igs raised on the farm free of leptosp i ral infection [Farm A]
which were inocula ted with vaccine in the
first week only [Group 1] . . . 1 76 Serological resu lts o f pigs raised on the
farm with endemic pomona infection [Farm B] which were inocul ated with vaccine i n
t h e first week [Gro u p 1 ] . . . 1 76 Serological results of pigs raised on the
farm free of leptospiral infection [Farm A]
which were inocu l ated with vaccine i n the
first and fifth week [Group 2] . . . 1 8 1 Serological results of pigs raised o n the
far m with endemic pomona infection [Farm B] which were inocul ated in the first and
fifth week [Group 2] . . . 1 8 1 Serological resu l ts o f p igs raised o n the
farm fre e o f leptospiral i nfection [Farm A]
which were inocul a ted with vaccine i n the
first, third and fifth week [Group 3] . . . 1 86 Serological results o f pigs raised on the
farm with endemic pomona infection [Fa r m B] which were inoculated with vaccine i n
t h e first, third and fifth week [Group 2 ] . . . 1 86 Serological results o f p igs raised o n the
far m free o f leptospiral infection [Farm A]
which were inoculated with vaccine i n the
first, second and third week [Group 4] . . . 1 9 1
6 . 8
6 . 9 a - d
6. 1 0
6 . 1 1
6 . 1 2
6 . 1 3
6 . 1 4
6 . 1 5
6 . 1 6
Chapter 7 7 . 1
7 . 2 7 . 3 a
7 .3b
Serological results of p igs r aised on the farm with endemic pomona i n fection [Farm B] which were inoculated with vaccine i n
the first, second and third week [Group 4] . . . 1 9 1 Proportion o f kidneys from v accinated and
u nvaccinated p igs fro m which leptospires
were isolated . . . 1 94 Percentage of pigs i n a p opulation with
residual m a ternal antibody . . . 200 Nomograph indicating optimum times for
the commencement of vaccination of p igs with a pomona vaccine, based on the a ntibody ti tre of the dam and a half life of
s ixteen days . . . 201
" Windows o f s usceptibility" for a population o f pigs, indicating the optimum age r ange
for the commencement o f vaccination . . . 202 Distribution of pomona titres of p igs
i noculated once and of u nvaccinated pigs at
s laughter . . . 204 Distribution of pomona titres of pigs
i noculated twice, and of u nvaccinated pigs
at slaughter . . . 204
Distribution of pomona ti tres of pigs inoculated three times at two weekly intervals, and of u nvaccinated pigs at
slaughter . . . 205 Distribution of pomona titres of pigs
inoculated t hree times at weekly intervals,
and of unvaccinated pigs a t slaughter . . . 205
Pathways for the transm ission of infection between pigs in SLIP89 . . . . Flow D iagram of S LIP89 . . . . SLIP89 data records for sows and boars . . . SLIP89 d a t a records for grower pigs . . .
2 1 6 2 1 8
220 222
7 . 4
7 . 5 a 7 .5b
7 . 6
7 . 7
7 . 8
7 . 9
7 . 1 0
7 . 1 1
Appendix IV A4 . 1
A4.2
A4.3
A4.4
A4. 5 A4.6 A4. 7
A4.8
A4.9
XV
Floor plan of weaner and grower houses i n
SLIP89 . . . 223 Normal distribution o f post-infection titres
The distribution of post-infection titres after a pp lying minimum and m aximum titre
224
restrictions . . . 225 The simulated increase o f p igs titres
following infection in S LIP89 . . . 226
P a t hw a ys fo r d i re c t a n d i n d i r e c t t r ansmission of infection between pigs i n
SLIP89 . . . 229 The preval ence of infection in grower p igs
for SLIP89 d a t a and field d a t a . . . 236 The pomona t itres of grower pigs for
S LIP89 data and field d a t a . . . 237 The prevalence o f lep tospiruria for
popu lations of pigs i n S LIP89 data and field
d a ta . . . 238
P revalence of infection in grower p igs
following simulated vaccination . . . 250
Main menu and sub-menus of SLIP89 . . . 261 Reproductive variables s ubmenu option of
SLIP89 . . . 262 Epidemiological variables sub-menu o p tion
o f SLIP89 . . . 263 SLIP89 sub-menu option for executing
SLIP89 . . . 266 Output Files submenu o f SLIP89 . . . 270 Cohort Data submenu o f S LIP89 . . . 272 Time Series Display submenu option of
SLIP89 . . . 274
Age Data submenu option of SLIP89 275
Pen D a t a submenu option of SLIP89 276
Appendix VI A6. 1
A6. 1
Flow Chart of interaction between SLIP89
p rogram units . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 Summary of the interaction of program
p rocedures within the WKUPDATE and
R UNSLIP units . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307