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THE EPIDEMIOLOGY OF INFECTIONS BY
STREPTOCOCCUS SUIS TYP ES 1 AND 2
A thes i s p resented i n pa r t i a l fulfi lmen t of the req u i rements for t he d e g r e e o f Doctor of Phi losophy i n Veterinary Patho logy and Pub l i c Heal th a t Mas sey Unive rsity.
Ian Duncan Robert son 1988
ABSTR ACT
An Ind i rect F luo rescent An t i body Tes t ( I . F. A . T. ) was developed for the ident i f i ca ti on of carriers of S t reptococcus sui s . This test wa s more a c c urate than the " t radi t iona l " cultural techniques . The sen s i t iv i ty f o r de tect ing S . suis type 1 f rom tonsilla r swabs was 62% , and 7 6% f o r S .• suis type 2 . Fo r nasa l swabs collected f r om live pigs the sens i t iv i ty was 48% and 60% r e s pectiv e ly .
F r om t h e e x a m i n a t i o n o f 9 5 9 n o n s p e c i f i c p a t hogen f r e e domes t i cated p i g s f rom 6 3 herd s , 2 6 3 specif i c pa thogen f ree ( SPF) pigs f rom seven herds and 9 6 fera l pigs i t is concluded tha t all non-SPF domes ti cated pigs older than five weeks of age are infected with bo th S . s u i s t y p e s 1 and 2 . Va r i a t i o n in t h e apparent prevalence of i nfection be tween dif f e rent herds was associated wit h the number of p i g s s amp l e d in e a c h herd r a t h e r t ha n a real var i a t i on in the prevalence and was s im i lar in bo th New Zealand and Aus t ralian non-SPF dome s t icated pigs. Three of seven SPF he rds tes ted i n Aust ralia were f ree f rom i nfect ion wi th S . s u i s types 1 and 2. It i s proposed tha t all S P F pigs a re deliv e red f ree f rom infec tion and tha t i nfect ion i s subs equen t ly i n troduced into the piggery .
S t reptococcus suis was detec t ed f rom nume rous t i s sues and fluids i ncluding the b lood of normal pigs . I t is sugges ted tha t the isolat i on of S . suis f rom "diseased" t i s sue does no t infer that S . suis was the pr imary aeti o l ogical agen t .
Al though both S . suis types 1 and 2 we re readily detect ed i n the env i ronment of pigs housed in an i n t ensive p iggery , the mai n routes o f t r a n s m i s s i on we r e c o n s i d e r e d t o b e d i re c t con t ac t o r neona tal infect ion of piglets bo rn to sows wi t h vagi nal i nfect ions .
B a s e d o n s e r o l o g i cal r e s ul t s f r om u s i ng a n Enzyme Li nked Immunosorbent Assay ( EL I SA ) , the pa t t ern o f i nfect ion due t o S. sui s types 1 and 2 was s imilar in dome s t i c pig s . I n fera l pigs the pa t tern of i nfect ion due to S. suis type 2 was s imilar to tha t of dome s t i c pig s , but in t h e case of S . s u i s type 1 the p revalence and t i t res were consi d e rably lower in f e ra l pig s . It i s concluded from these resul t s ,
i i
that b oth domestic and feral pigs are true long term carriers of s. suis type 2 whi l st s. suis type 1 i s ma i ntained with i n a population by a s e ries of rei nfections dependent on the populati on density.
There wa s no evidence o f ascend ing uro-genital infection o r s igns o f c l i ni cal d i sease in 24 bacon weight pigs i noculated with S. sui s type 1 o r 2 by e i ther the i ntravag i nal o r i ntrapreputial route s . When two l i tt e r s o f p r evi ous ly non- i nf e ct e d p i g s we re i ntranasa l ly i noculated with s. suis , no pigs developed c li ni cal s igns . The results of thi s experiment showed that the infective dose was less than lOO o rgan i sm s by thi s route . Only one o f eight pigs developed "classical "
c l inical s igns after i ntravenous i noculation with a porcine isolate o f
s. sui s type 2 . Some p i g s remained apparently normal even though high numbers of o rgani sms were present in the blood for up to two weeks . Only two o f fourteen pigs i noculated with S. suis type 2 i nto the c e re b r o-spinal f luid ( C . S. F . ) d eveloped s i gns of c l i ni cal diseas e . Both these pigs were i noculated with a human strain o f S . suis type 2 . To explain the se results i t i s proposed that strai n s of S . suis o f d if f e rent pathogenicity exist. Thi s hypothe s i s was suppo rted b y the f i nd i ng s that thi s human strain was a lso pathogenic for mice , rats and r abbits whi lst the po rcine i solate was pathogeni c only f o r rabbits.
A c ross-s ecti onal s tudy of various occupational g roups showed a positive correlation between contact with pigs and being seropos itive to S . suis type 2 . No veterina ry students , 1 0 . 3% of meat inspectors , 1 4 % o f d a i ry farmers who also kept p igs and 2 1 . 4% of pig f arme rs were seropos i tive . In exposed pig farmers the a nnual i nc i dence of sero
conve r s i on could be as high as 30%. It is believed that subclinical i n f e c t i on o f human s , a s w e l l a s p i g s , occurs and that isolates pathogenic for pigs are also pathogenic for other s peci e s of animal s i nc luding man .
I t i s hypoth e s i z e d that s . s u i s i s endem i c i n p i g s a t a preval ence rate approaching 1 00% i n all countries where pigs are kept . The pathogenicity of i s o lates can vary and thi s could account for variations i n the seve rity o f d i s ease repo rted throughout the world . It i s conc luded that S . suis i s usua l ly a commensal and i s i nvolved i n a d isease process only when pathogenic stra ins are present and afte r some othe r pr ima ry i nsult .
Hi
ACKNOWLEDGEMENTS
The work repor ted in thi s t he s i s would no t have been po ssible w i t hout the encouragement , suppo r t and f i nancial as s i s t ance of many people and o rgani z a t i on s .
Financial suppo r t was gratefully received f rom the P i g Research Council ( Aust ra l i a ) ; the Ve terina ry Research Fund , Ma ssey Univers i ty , N e w Z e a l an d ; and t he Queens land Depa r tment of Primary Indus t r ies . Professor B . W . Mank t e low kindly made ava i lable the fac i l i t i es of the D e p a r t me n t of Ve t e r i na ry P a t h o l o g y and P u b l i c H e a l t h , Ma s s ey Univers i ty .
The a s s i s t ance , cons t ruct ive c r i t i c i sm , enthus i a sm , f ri end ship and enq u i ri ng mind of my mai n supe rv i sor P rofessor Davi d Blackmo re we re s i ncerely appre c i a t ed . My se cond supe rvi sor , Dr. Roger Ma rshal ! , provided further suppo r t and ideas for thi s proj ect .
Many people con t r i buted t o make my s t ay a t Massey Unive rs i ty an e n j oya b l e , f r i e nd l y , y e t f u l f i l l i ng t i m e . The s e i n c luded Lynne Cul linane and Robe r t Holdaway who , wi th t he i r technical advice , put ex t ra fun i n t o mi c robiology ; Jan Schrama who pre pa red the medi a , g l asswa re and cheered up the day ; Dr . Dav i d Hampson and Zhenfang Fu ( pos t-graduat e s tudent ) who helped wi t h t he hand ling of pigs i n t he cohor t s tudy , appra i sed ideas and o f f e red he lpful advice ; Lynley Denby who ass i s ted with t he ELI SA ; Aki Shimada ( pos t-g radua t e s tuden t ) who gave adv i ce on the i n terpreta t i on o f hist opa t ho logi cal s e c t i ons ; Pe t e r W i n t e r who p e r f o rm e d t h e ba c t e r i a l r e s t r i c t ion endonuclease DNA analy s i s ( BRENDA ) ; L i z Davies who ass i s ted wi t h the haemat o logy ; Peter Wi ldbore who ordered goods and helped spend t he money ; Tom Law who t o ok p ho t og raphi c evidence of t he work ; Brian Pi cke t t who offe red advice on t he i n t ri cacies and anoma l i es of compute rs and Mr. R i ck We b s t e r ( Se n i o r V e t e r i na ry O f f i c e r ) who gave inf orma t i on on t he disease i n Aus t rali a .
Wi t h the expe rimental anima l s , Dr . Kei t h Lapwood and Mr . Boyd J o n e s ( C l i n i ca l Ve t e r i na r i a n ) o f f e r e d a dv i ce o n t he s u r g i c a l p rocedures and J rene Ha l l capably and cheerful ly a s s i s t ed wi t h the
i v
anaes thes i a o f the s e pigs . Professor David Blackmore and Dr. Linda Scholium mus t also be thanked for a l lowing the use of s e ra collected f rom humans w i th d i f f e rent occupa t i on s and f rom feral pigs .
Speci a l thanks also mus t go t o Phi l Webe r , K i t Jameson , Graham P earson , D r . B i l l Smi t h and D r . Paul Moughan who made ava i labl e the f aci l i t i e s of Massey P iggery , he l ped and assis ted with handli ng of pigs and calm ly coped wi t h t he chaos I c reated. The s taff of Kiwi Bacon Fac t o ry , KR Dar l i ng Downs , Queensland Bacon and The Met ropo l i t an Publi c Aba t to i r and the i nspect o r s of the Mini s try of Ag ricul ture and F i s he r i e s , New Zea land and the Commonwea l th Depa rtment of Primary I ndus t r i e s and Ene rgy , Aus t ra l i a a re also t hanked for the way they included me into thei r eve ryday ro u t i ne and a l lowed the collec t i on of so many s amp le s .
A speci a l thanks mus t go t o my wife Cathy and children Mike and Jenny f o r being so understanding when I ' ve come home sme l l i ng of pigs , spent hours being an t i-social on the comput e r , for s t ruggling to hold experiment a l pigs whil s t they were being bled and mos t impo rtan t ly f o r t he i r love and suppo r t .
V
Abstract
Acknowledgements
Table of Contents
List of Figures
List of Plates
List of Tables
Chapter I
Chapter I I
Chapter I I I
Chapter IV
Chapter V
Chapter VI
Chapter VI I
Chapter VI I I
Introduction
Li terature Review
Development of Microbiological and serolog ical TeChn iques
Surv ival of Streptococcus suis
The Prevalence of Streptococcus suis in Pig s . The Results of a Cross-sectional survey .
A LOng itud inal survey of Infection wi th Streptococcus sui s .
Experlinental Infection with Streptococcus sui s
Publ ic Heal th Aspects o f Streptococcus suis type 2
i i iv vi
vi i i
x i x i i
l
3 36
91
122
149
175
251
Chapter IX
Chapter X
References
Append ices
The Prevalence of Streptococcus suis in Queensland Pigs
General Discussion
267
278
289
315
Figure
3 . 1
3 . 2 3 . 3
3 . 4
3 . 5
. ..
3 . 6
3 . 7
3 . 8
4 . 1 4 . 2 4 . 3 4 . 4 4 . 5
4 . 6
LIST OF FIGURES
pOsi tion of organisms and antibiotic d iscs for sens itivity tests
Antibiotic sens itivity of S . suis
Compar ison of ELISA read ings at d i fferent d i l utions of pig sera and conj ugate against antigen of s. suis type 1 and "no-antigen"
controls
Compar ison of ELISA read ings at d i fferent d i lutions of pig sera and conj ugate against antigen of s. suis type 2 and "no-antigen"
controls
Compar ison of ELISA read ings at d i fferent d i lutions of human sera and conj ugate against antigen o f s. suis type 2 and "ne-antigen"
controls
Compari son of ELISA activi ties at d i fferent
bacterial antigen (S . suis type l ) concentrations against d i fferent p1g serum d ilutions
Compar ison of ELISA activ ities at d i fferent
bacter ial antigen ( S . suis type 2) concentrations against d i fferent p1g serum d ilutions
Compar ison of ELISA activities at d ifferent
bacter ial antigen (S . suis type 2) concentrations against d i fferent human serum d ilutions
The effect of pH solutions on the survival o f a f ield isolate of s. suis type 2
The effect of pH solutions with 5% faeces on the surv ival of a f ield isolate of s. suis type 2 The effect of pH solutions on the surv ival o f a f ield isolate of s. suis type 1
The effect of pH solutions with 5% faeces on the surv ival of a f ield isolate of s. suis type l The effect of temperature on the survival of a f ield isolate of s. suis type 2 in physiolog ical sal ine
The effect of temperature on the survival of a strain cul ture of s. suis type 2 in physiolog ical sal ine
vi i i
page Nuober
44
45 83
8 4
8 5
8 6
87
88
104
104
105
105
107
107
Figure 4 . 7
4 . 8
4 . 9
4 . 10
4 . 11
4 . 12
4 . 13
4 . 14
5 . 1 6 . 1 6 . 2
6 . 3
6 . 4
6 . 5
6 . 6
6 . 7
6 . 8
The effect of temperature on the survival of a f ield isolate of s. suis type 1 in physiolog ical sal ine
The effect of temperature on the surv ival of a strain culture of s. sui s type 1 in physiolog ical sal ine
The effect of temperature on the surv ival of S . suis type 2 in physiolog ical sal ine wi th 5%
faeces
The effect of temperature on the surv ival of s. suis type 1 in physiolog ical sal ine wi th 5%
faeces
The effect of sal ine solutions on the surv ival of a field i solate of s. suis type 2
The effect of sal ine solutions wi th 5% yeast on the surv ival of a field i solate of s. suis type 2 The effect of sal ine solutions on the surv ival of a field i solate of s. sui s type 1
The effect of sal ine solutions with 5% yeast on the surv ival of a field i solate of s. suis type 1 Range of t i tres to S . sui s types 1 and 2 in pig s Handl ing o f pigs in l i tters under study
The temporal pattern of infection with s. sui s type 1
The temporal pattern of infection with s. suis type 2
The proportion of carr iers of s. suis types 1 and 2 in pig s housed under d i fferent conditions
Infection and the development of antibodies to s. suis type 1
Infection and the development of antibodies to s. suis type 2
Change in antibody titre to s. suis type 1 i n a cohort of pig s
Change in antibody titre t o s. suis type 2 in a l i tter of pigs
lX
Page Nmlber 108
108
109
109
111
111 112
112
135 152 156
157
159
164
164
166
167
Figure
7 . 1
7 . 2
7 . 3
7 . 4
7 . 5
8 . 1
10 . 1
Spread o f infection o f S . suis types 1 and 2 i n a l i tter intranasally infected with 109 organi sms Spread of infection of s. suis types 1 and 2 in a l itter intranasally infected with 100 organi sms TOtal whi te cel l counts of pigs intravenously infected with s. suis type 2
Antibody titre to s. suis type 1 in pigs intravenously infected w1th s. sui s type 2 Antibody titre to s. suis type 2 i n pigs
intravenously i nfected w1th S . sui s type 2
The range of ti tres to s. suis type 2 in different occupational groups
Temporal var iation of prevalence and incidence of infection wi th s. suis in a group of pigs
X
198
199
222
224
225
256
282
Plate
3 . 1
3 . 2 3 . 3
3 . 4 7 . 1 7 . 2
7 . 3 7 . 4
7 . 5
7 . 6
7 . 7
7 . 8
7 . 9
LIST OF PrATES
Page Nlinber
The haernolyi s of s. suis type 2 on sheep blood agar
A Gram stain of S . suis type 2
The moi sture chamber used in the linnunofluorescent tests
s. suis type 2 stained with an I . F.A.T.
Cannulae used for j ugular catheter i zation
Di recting the cannula from the jugular vein to the dorsal midl ine
Flushing of the cannula with hepar inized sal ine The pig fol lowing j ugular catheter i zation wi th a stretch-stocking to prevent the cannulae
Rad iograph of the pig head for positioning of the needle into the subarachnoid space
pos i tio n i ng of the need le in the subarachnoid space
Pig with signs of paddl ing and opisthotonus after injection wi th the human i solate of s. suis type 2 into the C . S . F .
Men i ng i t i s observed in a pig wi thout cl inical signs infected wi th the human isolate of s. su i s type 2 into the C . S . F .
A gl ial nodule i n a pig wi thout cl inical s igns after infect ion with a porcine isolate of s. sui s type 2
47
47 60
60 212 212
213 213
231
231
236
238
238
x i
Table
2 . 1
2 . 2
2 . 3
2 . 4
2 . 5
2 . 6
2 . 7
3 . 1
3 . 2
3 . 3 3 . 4
3 . 5
3 . 6
3 . 7
3 . 8 3 . 9 3 . 10
LIST OF TABLES
Page NUDber
Proportion ( % ) of di fferent Lancefield groups of streptococci isolated from pig s
Repor ted morbid ity and mortal ity due to s. suis infection in pigs
MaJor patholog ical lesions (%) of pigs associated wi th infection by s . suis type l
Major patholog ical lesions (%) of pig s associated wi th infection by S . suis type 2
Isolation o f other bacter ia from pneunonic lungs from which s . suis had been isolated (%)
Repor ted i so l a t i on rates of Streptococcus su is from various sites in pigs
Repo r ted i so l a t i on rates of Streptococcus sui s type 2 from herds fed antibiotics (Cllfton-Hadley and Alexander , 1984a)
Biochenical and physiolog ical character istics of streptococcus suis types l and 2
The b i ochem ical and gr owth character istics of streptococcus suis types 1 and 2
sens i t ivity of s . suis to antibiotics
F l uo r e scence o f S . su i s type 1 at var ious d ilutions o f conj ugate a na ant isera
Fl uorescence of s . su i s type 2 at d i f ferent concentrations of group R conj ugate and antiserum Speci f icity of the I .F . A . T . for s. suis type 2 with g roup R antiserum
Speci ficity of the I . F . A . T . for s . suis type 1 with group S antiserum
Compar ison of cultural and linnunofluorescent tests ( I . F . A . T . ) for the detection of s. suis type 2 Compar i son of cultural and linnunofluorescent tests for the detection of s . suis type 1
Compar ison of cultural and linnunofluorescent tests on nasal swabs for s. suis type 2
5
8
12
13
14
19 25
38
48
50 59
59
62
62
63
63
64
xii
Table 3 . 11
3 . 12
3 . 13
3 . 14
3 . 15
3 . 16
4 . 1
4 . 2
4 . 3 4 . 4 5 . 1
5 . 2 5 . 3
5 . 4 5 . 5 5 . 6 5 . 7 5 . 8
Page Nunber
Compar i son of cultural and inmunofluorescent tests on nasal swabs for S . suis type 1
Compar i son of nasal and tons i llar swabs for detecting S. suis type 2 wi th the I . F .A.T.
Compar ison of nasal and tons illar swabs for the detect ion of s. suis type 1 wi th the I .F . A . T . Compar i son o f 5% sheep blood agar with Etiwards med ium for the detection of tons illar carr iers of
S. sui s type 2 us ing I .F . A . T .
Compa r ison o f 5% sheep blood agar with Etiwards med ium for the detection of tons illar carr iers of s. suis type 1 us ing I . F . A . T .
The number o f pig s to sample to be 95% confident o f de tec t i ng the presence o f infect i on wi th s. suis
De t e c t i o n o f s. s u i s t ypes 1 and 2 b y immunofluorescence from the piggery environment Detection (%) of S . suis type 2 by I .F .A. T . from pa l a t i ne to n s ils s t o r ed a t 4 oc a nd r o om temperature
Detection (%) of S . suis types 1 and 2 by I .F . A . T . from palatine tons ils stored at -2ooc
capaci ty-use disinfectant test results for s. sui s incorporating 5% yeast
Isolation rate of s. suis type 2 from the palatine tonsi l s o f slaughtered pigs
Isolation rate of s. sui s type 1 from the palatine tonsils of slaughtered pigs
Age ( class ) spec i f ic ra tes o f in fection wi th s. su i s in the palatine tonsils of slaughtered pig s
Isolation of s. suis from samples other than the palatine tonsil collected from slaughtered pigs The prevalence of uter i infected with s. sui s detected at the meatworks
The prevalence of nasal chambers infected wi th s. suis in l ive pigs
I sola tion of s. sui s from s ites other than the nose in l ive pigs
Isolation of s. suis from the vag inas of l ive pigs
64
66
66
67
67
73
93
98
98
114
125
126
129
130
131
131 133 133
x i i i
Table
5 . 9
5 . 10 6 . 1
6 . 2
6 . 3
6 . 4
6 . 5 6 . 6
7 . 1 7 . 2
7 . 3
7 . 4
7 . 5
7 . 6
7 . 7
7 . 8
7 . 9 . 7 . 10
7 . 11
Page Nunber
prevalence of bacteraemia due to s. suis in l ive pigs
Geometr ic mean titres to s. sui s in feral pigs The reproductive history and detection of s. sui s i n sows used i n the cohort study
The detect ion o f s. su i s type 1 in nasal and vag inal swabs of sows after farrowing
The de tec t i on o f s. su i s type 2 in nasal and vag inal swabs of sows after farrowing
The mean age at which pigs f irst became infected with s. sui s types 1 and 2
I .F . A . T . results from piglets examined post mortan I so l a t i o n of s. su i s types 1 and 2 from the env ironment of the study piglets
Summary of transmiss ion exper iments of Swe ( 1976) Summary of transmission exper iments of Cl ifton
Hadley and Alexander ( 1981)
Isolation of s. sui s type 1 from the reproductive tract of artif1c1ally infected pigs
Isolation of s. suis type 2 from the reproductive tract of arti f1c1ally infected pigs
The recovery of s. suis type 2 from the blood and l iver after intravenous inoc ulation
Nunberl? o f s. suis type 2 isolated fram the blood after intravenous infection
Macroscopic lesions detected in pig s infected wi th s. sui s type 2 v i a the intravenous route
Microbiolog ical find ings from pigs infected wi th s. sui s type 2 via the intravenous route
Histopatholog ical find ings in pigs infected wi th s. sui s type 2 by the intravenous route
C l i n i ca l , m i c r ob i o l og i c a l and patholog ical find ing s of subarachnoid inoculation with s. suis type 2
A stmnary of the find ings in laboratory animals inoculated with s. sui s
134
137 154
155
155
160
161 163
181 182
187
189
206
216
218
219 220 233
244
x iv
Table
8 . 1
8 . 2
8 . 3
9 . 1
9 . 2
9 . 3
9 . 4
9 . 5
9 . 6
Prevalence o f posi tive titres to S . suis type 2 in di fferent occupational groups
The relationship between potential risk factors and titres to s. suis type 2
The number of positive titres to s. suis type 2 in different age and occupational groups
Isolation rate of s. suis type 1 from the palatine tonsils of domesticated non-SPF Australian
slaughtered pigs
Isolation rate of s. suis type 2 from the palatine tonsils o f domesticated non-SPF Australian
slaughtered pigs
Isolation rate of S. suis type 1 from the palatine tonsils of SPF slaughtered pig s
Isolation rate of s. suis type 2 from the palatine tonsils o f SPF slaughtered pig s
The prevalence of infection wi th s. suis in pigs of d i f ferent or ig in
Age (class) speci fic rates of infection with
s. suis in the palatine tonsils of slaughtered pig s
XV
257
258
26 1
269
270
27 2
272
273
27 4
