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A thes i s pre sented i n par tial fu l f ilment of the requirements for the d egr ee of Master of Philsophy in Veter inary Scie:1ce
at Ma ssey Univer s i ty
P H ILIP BERNARD HcKENNA
November 1978
ABSTRACT
Studies were undertaken to establish for the first time the identity and prevalence of coccidian parasites of New Zealand cats and dogs and to determine the effect of various factors on the activation of Isospora fe_Ii s and Isospora ri vol ta sporozoites. The associations bebveen these protoz.oa and such organisms as Toxopla sma, Sarcocysti s, Besnoi t i a and other related genera are examined and the literature concerning their life cycles, nomenclature and general biology reviewed.
Examination of faecal samples from 508 cats and 4 8 1 dogs from North
IsJand localities revealed that 1 55 ( 3 0 . 5% ) and 307 ( 6 3 . 8% ) respectively, contained coccidia. The majority o f infected Sillnples were found to
contain a single coccidian but in total four valid coccidians from cats and four from dogs, as well as several pseudoparasitic coccidia, were recorded and described. The identities and prevalences of these valid coccidians were:
i
(a,). Cats Isospora fel i s ( 17 . 5 % ) Isospora ri vol ta ( 2. 2. 9.s)
Toxopla sma gondii ( 0 . 98 % ) 5.3L'COcys t i s sp. ( 16 . 9%)
(b) Dogs Isospora can i s 4 . O!?o) Isospora ohi oensi s ( 9 . 2% ) Harrunondi a heydorni 2 . 7 % ) Sarcocysti s sp. ( 58 . 8 % )
The sex o f the host had no significant effect on the prevalence of infection. The effect of other factors, such as season, host age and host origin, however, was found to vary from coccidian to coccidian and appeared to be explicable in terms of differences in routes of transmission, host immunity and intermediate host specificity.
Levels of sporocyst shedding in cats and dogs naturally infected with Sarcocystis sp. tended to be lmv with the majori·t:y excreting 200
sporocysts per gram of faeces or less. The specific identities of such sporocysts are unknown but at least some from cats were d emonstrated, by mouse infection, to be those of S . muris . Attempts to induce similar
Sarcocystis infection in mic e , u s ing i sola tes o f I . felis recovered from the faec e s of na tur a l l y i nfected cats "''e're unsuccessfu l .
Aft.er completion of the ma i n survey , a furtl.�e:r cocc idian showing
sim i l a r i ties to Besnoi t i a wal l acei wa s recov�red from the faeces of one of f ive feral cat s . The feed ing of sporula ted oocysts of this coc c i d i a n to mic e , ra ts , rabbits a nd guinea p ig s re sulit.ed in the forma tion of typica l Besnoi tia c ysts in a l l ho sts exc ept lt:ltle la st.
Stud i e s on the ac tiva t ion o f I . fel i s a nd I. r.ivol ta sporozo i tes revea l ed tha t , a l t:-tough some d i f f erenc es wer e apparent. be tween the two , both wer e capabl e of activa tion over a wide r ange of cuEdi tion s .
Ac t iva tion o f both species wa s observed to take pla c e i n trypsin and bile between temper a tur es o f 210 and 43 oc ( t'Jw: range tested ) a nd to occur rapidly at 3 9oc . Whi l e the presence o f bile appea red to be essent i a l for t h i s proc ess that of trypsin d id not . I� genera l , ne i ther the conc entration of bile (abov e 5%) nor the typ.e· of bile wa s found to have any marked e f fect on the level of ac t iva tion attained whi l e hydrogen ion conc e ntration (pH range 5.0 to 10.0) a l so a ppeared to have l i ttle i n f lu enc e .
Unl ike many species of cocc idia which have b.e�n stud ied , pretreatment o f ooc ysts before expo sure to trypsin a nd b i l e •�s found not t o b e a n
e s sential prerequ i site for the activa tion of I. fel i s a nd I . ri vol ta . However , higher l evel s of a c t iva tion were a ttained when pr e trea tment wa s used than when i t wa s not a l though for I. rivol ta a t least , the l ev e l of a c t ivation appear ed to be l e ss dependent on pretreatment for oocysts
stored in sulphur ic acid than for tho se storEd in pota s s ium d ichroma te.
The proc ess of a c t ivat ion a nd exc ysta t ion of both spec i e s wa s observed to be e s sentia l l y similar to tha t desc r ibed £�r other spec i e s of
cocc id ia which a l so lack e i ther sporocyst stieda bod ies or oocyst
micropyl es . Sporozoites e scaped fol lowing the col lapse o f the sporocyst wal l a nd were observ ed to complete excysta tion ·through identations a nd frac ture s a t one or both end s of the oocyst .
ACKNOWLEDGEMENTS
The present the sis cou ld not have been completed without the he l p and a s s i stance of numerou s people. Of these , some require spec ia l mention.
These i nc lude; my supervi sor , Dr \v . A . G . Char le ston of the Veterinary Fac u l ty of this univer sity , for his ever helpful advic e and continuou s encouragement throughout the course o f th i s study ; Dr R.E. Harris of the Veterinary Facu lty for his advice on sta ti stical a na lyses and
Mr G.H . Col l ins of the same Faculty for pelTii ssion to quote unpubl i shed f indings from his own wor k .
Spec ial thanks are a l so due to the former a nd pre sent Superintendents o f the Pa lmerston North Anima l Heal th Laborator y , Drs D . E . Gard ner a nd B . H. Simpson, for t�he ir toleranc e a nd understa nd ing of my pr eoccupation with thi s the s i s , par ticularly over the last two yea r s . Others to ':.·lhom spec i a l thank s are due includ e Ms . B.G . �;ie n s and Mr T . G . Law of the Ve ter inary Facu l ty , for thei r un stint ing a s si stance in the printing of a l l photogr aphic plates and graphs and to Mr s J . L. Simpson for the prepara tion of F igure l . 3. I wou ld a l so l ike to expre ss my gra titude t o Mr s C . R . Whitcombe , M r s J . Shore , M i s s A . Punch and M r M . Sou thern , who a l l , a t various t imes , a s sisted wi th the c ar e and ma intenanc e of
exper imenta l anima l s . I a l so gra tefuJ.ly acknowledge the a s si stance of people, too numerous to mention , who provided m e with faecal samp l e s from c a t s and dogs, in par ticular Professor D . K . Blackmore of the Veter inary Facu lty for providing samples from fera l cats and Mr s E .M.
Husba.nd and sta f f of the Pa lmer ston North S.P . C . A . who were total l y co�opera tive a t a l l times . The c o -operation of Mr R . W . Mof fitt ,
Supervis ing Meat I n spector , a nd sta ff of the Manawatu Abattoir , F e i ld ing i s also appreciated.
Other s to whom my thanks are due inc lude the typ i s t s of t.he Mini stry of Agr icu l ture a nd Fi sher ies , Pa lmer ston North , for prepara tion of the initial dra f t of this thesis a nd to Mr s F . S . Wicherts for her mo s t
e f f i c ient a n d expert typing of t h e f inal dra ft . Fina l ly I would l ike t.o express my apprec iation a nd gra t i tude to the Direc tor of the Animal Heal th D ivi sion , Ministry of Agriculture a nd Fisheries , for perm i s s ion to undertake thi s cour se of study.
i i i
1 .
2.
TABLE OF CONTENTS
ABSTRACT
ACKNOWLEDGEMENTS
I NTRODUCTION AND LITERATURE REVIEW 1.1 Systematic s of the cocc idia 1.2 Coc c id ia of c a ts and dogs
1.2.1 Eime rian oocysts of cats and dogs 1.2.2 I so sporan oocysts of cats a nd dogs
( a ) H i storic a l
( b ) Current spec ies and problems o f nomenc l a ture
1.3 Diagnosis and ident i f ication l . 4 Life cyc l e s
1.4.1 Genu s Isospora 1.4.2 Genu s Toxoplasma 1.4.3 Genu s Besnoi tia 1 .4 . 4 Genus Hammondia 1 .4.5 Genu s Sarcocysti s
1.5 D i ssemina tion of i nfec tion and tran sport hosts 1 .6 Re s istanc e a nd survival
l. 6 .1 Oocysts
1.6.2 I ntermediate host stages 1 .7 Pathogenic i ty
1.7 .1 Definit ive ho sts 1.7.2 Inte rmedi a te hosts 1.8 Immunity
1.8.1 Definitive hosts 1 . 8.2 I ntermedi a te ho sts 1.9 Trea tment
1.9.1 D e f initive hosts 1.9.2 I nte�Tied i a te hosts
THE IDENTITY AND P�/ALENCE OF COCCIDIAN PARASI TES IN NEW ZEALAND CATS AND DOGS
2.1 I ntrodu c t io n
Page
i iii
1 1 1 3 3 3
8 1 1 14 1 6 1 7 2 1 24 25 26 28 28 3 0 3 1 3 1 32 3 4 3 4 3 6 3 7 3 7 3 8
4 0 4 0
3 .
4 .
2 . 2 Materials and me thod s 2 . 2 . 1 Faecal samples
2 . 2 . 2 De tection of cocc idia 2 . 2 . 3 Sporulation o f coccidia
2 . 2 . 4 Morpho log ical charac teristic s
2 . 2 . 5 Identi fication of Toxoplasma-like oocysts 2 . 2 . 6 Oocyst and sporocyst counts
2 . 3 Results
2 . 3 . 1 Ident i ty and morphology ( a ) Fel i ne coccidia ( b) Can ine cocc idia
(c ) Pseudoparasitic cocc idia 2 . 3 . 2 Preva l e nce
( a ) General preva l e nce (b) Age preva lenc e ( c ) Sex preval ence
( d ) Town and cou ntry preval ence ( e ) Seasonal preva lenc e
( f ) Level s of i nfection 2 . 4 Discussion
THE EXPERIMENTAL INDUCTI ON OF SARCOCYSTIS INFECTIONS IN MICE
3 . 1 Introduc tion
3 .2 Materia l s a nd methods
3 . 2 . 1 Recovery of oocysts and sporocysts 3 . 2 . 2 Experimental anima l s
3 . 2 . 3 I nfection a nd exam ination o f rats and mic e 3.3 Experimenta l procedures and resu l t s
3 . 3 . 1 Experiments with I . fel i s
3 . 3 . 2 Experiments with fr ee sporoc ysts 3 . 4 Discussion
THE OCCURRENCE OF BESNOITIA SPECIES IN NEW ZEALAND CATS 4.1 Introduc tion
4 . 2 Material s a nd methods
Page
4 0 4 0 4 3 4 3 44 44 4 4 4 8 48 4 8 5 4 5 9 6 4 6 4 64 68 68 7 4 7 5 7 5
8 6 8 6 8 7 8 7 8 7 88 88 8 8 8 9 9 3
9 6 9 6 9 6
V
4 . 2 . 1 Fera l cats
4 . 2 . 2 Recovery of oocysts 4 . 2 . 3 Experimental a nimals
4 . 2 . 4 Infection and examination of intermediate hosts
4 . 3 Exper ime nta l procedures and r e su l ts 4 . 3 . 1 The initial i so late
4 . 3 . 2 Transmission experiments 4 . 4 Discussion
5 . S 'l'UD I ES ON THE ACTIVATION AND EXCYS 'rATION OF I . FELIS AND I . RIVOLTA SPOROZOITES
6 .
5 . 1 I ntroduc tion
5 . 2 Materia l s a nd methods
5 . 2 . 1 Collection and storage of oocysts 5 . 2 . 2 co2 pretreatment
5.2 . 3 S od ium hypochlor ite pr e treatment 5 . 2 . 4 Treatment with excysting med ium 5 . 2 . 5 De termination of ac t ivat ion 5 . 3 Exper imenta l proc edures and re sults
5 . 3 . 1 Effec t o f type and duration o f pretreatment 5 . 3 . 2 Effect of storage solution a nd storage
duration
5 . 3 . 3 Effect of duration of treatmen t i n excysting medium
5 . 3 . 4 Ef fect of bile and trypsin concentration 5 . 3 . 5 Effect of bile type or substi tute
5 . 3 . 6 Effect of treatment temperature 5 . 3 . 7 Effect o f hydrogen ion concentration 5.3.8 The e xcystation proc ess
5 . 4 Discussion
GENERAL DIS CUS S I ON
REFERENCES
APPENDICES
Page 9 6 9 6 9 6
9 7 9 7 9 7 98 lOO
1 04 1 04 1 0 5 1 05 105 1 0 5 1 0 6 1 06 1 06 1 06
1 0 7
1.12 1 12 1 1 3 1 1 6 1 16 1 1 7 123
129
1 3 4
1 5 5
Table
1 . 1
1 . 2
1 . 3
1 . 4
LIST OF TABLE S
Morphological charac teristics o f e imerian oocysts r ecorded from cats a nd dogs.
Reported preva lenc e s of eimerian oocysts in cat and dog faec es.
Coc c id ian spec ies o f cats
Coccid ian spe c i e s o f dogs
l. 5 Tentative key and i nvestigationa l sequence for the identif ica tion o f isosporan cocc idia i n cat and dog faeces.
1 . 6 Repor ted sporulation times of fel ine cocc idian spec ies
1 . 7 Reported sporulation t imes of canine coccid ian spec ies.
1 . 8 Reported prepatent and patent periods o f fel ine cocc idian spec ies.
1 . 9 Repor ted prepatent and patent periods o f canine cocc idian spec ies.
1 . 1 0
2 .1
2 . 2
2 . 3
2 . 4
2.5
Reported "e f f ec tive" treatments for general canine and fel ine coc c id io s i s .
Reported prevalences of coccidian spec ies in c at s .
Reported preval ences of cocc id ian spec ies i n dogs .
Sources o f faecal samples.
Results of a two-level nested anal ysis o f var iance to determine the significance of the d i f ferenc es in oocyst d i mensions between similar dog and cat cocc idia.
Resu lts of a two -leve l nested ana lysis of var ianc e to determine the signi f icanc e of the d ifferences in sporocyst dimensions between simi lar dog and c a t coccidia.
2 . 6 Preva lenc e o f c oc c id ia i n canine a nd feline faeca l samples.
2 . 7 Number and composition of coccidial i nfec tions i n individual f e l i ne faecal samples .
v i i
Page
4
5
1 2
l3
15 1 9 2 0
2 2
2 3
39
4 1 4 2
45
62
63
65
6 6
Tab l e
2 . 8
2 . 9
2 . 10
2 . 11
2 . 12
2 . 1 3
2 . 14
2 . 15
2 . 16
2.17 2 . 1 8
2 . 19
2 . 2 0
2 . 2 1
2 . 2 2
5 . 1
Number and compo sition of cocc idial infections i n individual canine faecal samples .
Prevalence of cocc idia in cats accord i ng to age .
Prevalenc e of cocc idia i n "kitten" and "adul t " cats .
Prevalenc e of cocc idia i n dog s accord i ng to age.
Prevalence of coc c i dia in "puppy" and "adu l t " dog s .
Prevalence o f coc c i d ia i n cats accord ing to sex .
Prevalence o f coc c id ia in dog s accord ing to sex .
Prevalenc e o f cocc idia in town and country ca ts.
Prevalence of coccidia in "kittens" and "adul t " tmvn and country cats.
Prevalence of cocc idia in town and country dogs.
Preva lence o f cocc id i a in "puppy" and "ad u l t " town and country dogs .
Seasona l prevalenc e of cocc idi a in cats.
Seasona l preva l ence o f coc c id ia i n "k i tten " and
"adul t " cat s .
Seasonal preva lence o f coc c id ia i n dog s.
Sea sona l preva l ence of coc c id ia in "puppy" and "adult "
dogs.
Effect of variou s pre trea tments on the activation o f I . fel i s and I . rivol ta sporo zoites
Pa ge
67
69 6 9
70
70
71
71
72
73
73
78
78 79
79
1 08
LIS'l' OF F IGU RES
F igure
1 . 1 An apicomplexan sporozo ite or merozoi te
1 . 2 A sporulated cocc i dian oocyst ( Eimeria sp . )
1. 3 Schema tic compa r i son o f the l i fe cyc l e s of the c a nine and f e l ine cocc id ia
2 . 1
2 . 2
2 .3
2 . 4
2 . 5
Geographica l d i str ibution of sampled cats
Geographical d istr ibution of sampled dogs
Fr equency d i str ibu tions of ooc yst d imensions of fel ine cocc idia
Frequenc y d i stribu t ions of sporoc yst d imensions o f feline coc c id ia
Frequency d istr ibutions of ooc yst d imensions o f canine cocc idia
2 . 6 Frequency d i str ibutions of sporocyst d imensions of canine cocc idia
2. 7 2 . 8
2 . 9
3 . 1
4 . 1
5 . 1
5. 2
The seasonal prevalence of coccidia i n cats
The seasona l preval enc e of cocc id ia in dog s
Frequency d i str ibution of Sarcoc y s ti s sp. counts i n cat and dog fa eces
Comparison of the d imension s of S. muri s sporocysts r ecovered from the faeces of exper imenta l l y i nfec ted kittens and Sarcoc y s ti s sp . sporoc ysts recovered from the faeces of natural ly infected cats .
Frequency d i str ibutions of oocyst and sporocyst dimensions of Besnoi tia sp .
Effec t of storage solution and storage duration on the ac tiva tion o f I. ri vol ta sporozoi te s
Effect o f duration o f treatment i n exc ysting med ium on the a c t ivation of I. fe li s sporozoites
ix
Page
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2 7
4 6
4 7
5 2
5 3
.57
5 8
76 77
8 1
9 5
1 0 2
1 0 9
1 10
;Figure 5.3 5.4 5.5 5.6 5.7
Effect of duration of treatment in excysting medium on the activation of I. rivol ta sporozoites
Effect of bile and trypsin concentration on the activation of I . fel i s and I. rivol ta sporozoites Effect of bile type or substitute on the activation of I. fel i s and I . rivol ta sporozoites
Effect of treatment temperature on the activation of
I . fel i s and I . ri vol ta sporozoites
Effect of hydrogen ion concentration on the activation of I. fel i s and I. rivol ta sporozoites
Page
111 114 115 1 1 9
120
LIST OF PLATES
P la te Page
2 . 1 Val id cov .. idia r ecovered from the faeces o f nat.ura lly infec ted cats
2 . 2
2 . 3
3 . 1
3.2
4. 1
5 . 1 5. 2
Val id coccidia recovered from the faec e s of natura l ly infec ted dogs
Sporulated oocysts of pseudopara si tic cocc id i a r ecovered from the faec es of na tural l y infec ted c ats and dogs
Ventral aspect of a labora tory mou se experime ntal l y infected with s. mu ris
Some l ife cyc l e stages of s. mu ris
Some l i f e cyc le stages of Besnoit ia sp.
The excystation process of I . fel is
The excystation proc ess of I . rivol ta
so
55
61
90 91
99
121 122
xi
