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Infection rates with Cowdria ruminantium of nymphs and adults of the bont tick Amblyomma hebraeum collected in the field in Zimbabwe
Veterinary Parasitology, 36 (1990) 277-283 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
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I n f e c t i o n R a t e s w i t h C o w d r i a r u m i n a n t i u m of N y m p h s and A d u l t s of the B o n t T i c k A m b l y o m m a hebraeum C o l l e c t e d in the Field in Z i m b a b w e R.A.I. NORVAL, H.R. ANDREW and C.E. YUNKER
University of Florida/USAID/Zimbabwe Heartwater Research Project, Veterinary Research Laboratory, P. O. Box 8101, Causeway (Zimbabwe) (Accepted for publication 23 January 1990)
ABSTRACT Norval, R.A.I., Andrew, H.R. and Yunker, C.E., 1990. Infection rates with Cowdria ruminantium of nymphs and adults of the bont tick A mblyomma hebraeum collected in the field in Zimbabwe. Vet. Parasitol., 36: 277-283.
Cowdria ruminantium (heartwater) infection rates of field populations of the bont tick, Amblyomma hebraeum, were determined at two locations in the southern lowveld of Zimbabwe. At Mbizi Quarantine Station, unfed adult males and females, and nymphs were collected at intervals over a 2-year period using traps. At Lemco Ranch, engorged nymphs were collected on three occasions from weaner calves and allowed to moult to adults. The unfed ticks were fed in small pools on heartwater-susceptible sheep, some of which became infected. The infection rates of the ticks were then estimated statistically. Depending on the date of collection and locality, these rates were in the range 0.0-44.9% for males, 20.0-36.1% for females and 0.0-13.4% for nymphs. Most of these rates are considerably higher than those previously believed to occur.
INTRODUCTION H e a r t w a t e r is a rickettsial disease o f r u m i n a n t s , c a u s e d b y Cowdria rumin a n t i u m a n d t r a n s m i t t e d b y several species o f t h e tick genus Amblyomma. I n s o u t h e r n Africa, t h e m a i n v e c t o r is t h e b o n t tick A m b l y o m m a hebraeum K o c h . I n t h e rest of s u b - S a h a r a n Africa a n d t h e C a r i b b e a n , t h e tropical b o n t tick A m b l y o m m a variegatum ( F a b r i c i u s ) a s s u m e s this role. T h e e p i d e m i o l o g y of t h e i n f e c t i o n is p o o r l y u n d e r s t o o d , p r i m a r i l y b e c a u s e of t h e lack of precise i n f o r m a t i o n on i n f e c t i o n rates in v e c t o r p o p u l a t i o n s . It was generally a s s u m e d t h a t i n f e c t i o n r a t e s were low b e c a u s e susceptible r u m i n a n t s could r e m a i n u n i n f e c t e d in e n d e m i c areas for m a n y m o n t h s or even y e a r s ( A l e x a n d e r , 1931; B o n s m a , 1944; N e i t z a n d A l e x a n d e r , 1945; Uilenberg, 1971 ). T h e s e a s s u m p t i o n s were s u p p o r t e d b y later studies on infection rates in field
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populations ofA. hebraeum in South Africa (Du Plessis, 1985; Du Plessis and Malan, 1987) and A. variegatum in the Caribbean (Camus and Barre, 1987). However the method used to detect infection in ticks in these studies was an indirect one, which depended on serological conversion of mice that had been inoculated with homogenates of adult ticks removed from cattle. In the present study, we derived infection rates statistically, based on the frequency with which field-collected unfed nymphs and adults of A. hebraeum infected sheep. The method was devised by Chiang and Reeves ( 1962 ) to estimate arbovirus infection rates in mosquito populations and was subsequently used to determine the prevalence of viral agents in ticks (Yunker et al., 1977, 1979). MATERIALSAND METHODS Ticks were collected from two localities in the southern lowveld of Zimbabwe, Mbizi Quarantine Station and Lemco Ranch, where A. hebraeum commonly occurs and heartwater is endemic (Norval, 1983). Mbizi Quarantine Station (31 ° 05' E, 21 °25'S)
Unfed nymphs and adults of A. hebraeum were attracted to traps emitting CO2 andthe male-produced aggregation-attachmentpheromone (AAP) (Norv a l e t al., 1989). The source of AAP was either fed males of A. hebraeum removed from cattle or ether extracts of fed males that had been applied to filter paper. Unfed ticks were collected by hand as they approached or entered the traps. Tick collections were made in and around six pens (100 m 2) in a 450-ha paddock in which 57 undipped Brahman xAfricander steers were kept. The steers were herded into a pen at approximately 17:00 h each day and released at approximately 07: 00 h the following day. Each pen was used continuously for a period of 1 month and then kept free of cattle for 5 months. Protected sites for the moulting of detached ticks were constructed in the pens and tree or brush branches were stacked outside the pens for the same purpose. The pens were sampled in August and October 1987, and January, July and October 1988. Pens were sampled only after a period of at least 6 weeks had elapsed since they had been used by cattle. Lemco Ranch (29 ° 55'E, 21 ° 25' S)
Engorged nymphs were collected by hand on three occasions (October 1987, March and April 1988) from weaner calves < 9 months of age which had never been treated with acaricides. Only one engorged nymph was removed from any one calf. The nymphs were returned to the laboratory where they were held in an incubator at 26 ° C and 85% relative humidity, and allowed to moult to adults.
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Transmission of C. ruminantium to sheep Merino sheep were obtained from a farm near Harare, an area that is free of
Amblyomma ticks and heartwater (Norval, 1983 ). Pools of small numbers (210) of unfed nymphs, adult males or adult females were placed in bags glued to shaved areas on the backs of sheep, and were allowed to attach and feed. Laboratory-reared fed males that were known to be uninfected were placed with the females to facilitate attachment and engorgement (Norval, 1974). The numbers of ticks that attached and fed were recorded. In all experiments, the rectal temperatures of the sheep were recorded daily. Heartwater infection was confirmed by the demonstration of colonies of C. ruminantium in brain smears, made either upon death or from brain biopsies taken according to the method of Synge (1978). All sheep found negative for heartwater by brain biopsy were subsequently challenged to test their susceptibility. The ability of single ticks to transmit infection was tested for nymphs, adult males and adult females. Two replicates were tested for each sex or stage. The ticks used had engorged in the preceding stage on sheep that were reacting to heartwater.
Estimation of infection rates The infection rate in each collection of unfed ticks was estimated using the following formula (Chiang and Reeves, 1962), which was run on an IBM PC computer.
['n_ X'~ lIra /5:1- [-~--) where iO= estimated infection rate; n = number of pools tested; m = average number of ticks per pool; X = number of pools positive. RESULTS Thirty-nine pools of ticks from Mbizi Quarantine Station were fed on sheep, 18 of which became infected with heartwater. The estimated infection rates were 0.0-44.9% in collections of adult males, >_-20.0-36.1% in adult females and 0.0-13.4% in nymphs (Table 1 ). Twenty-two pools of ticks from Lemco Ranch were fed on sheep, 14 of which became infected with heartwater. The estimated infection rates were 9.6>i 25.0% in collections of adult males and >t 20.0-34.1% in adult females (Table 2 ). The 29 sheep that failed to contract heartwater after pools of ticks had been fed on them were all found to be heartwater susceptible on subsequent challenge.
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TABLE 1 E s t i m a t e d infection rates with Cowdria ruminantium of unfed n y m p h s and adults of Amblyomma hebraeum collected at Mbizi Quarantine Station, Zimbabwe Date of collection
Average no. of ticks per pool (m)
No. of pools positive/No, of pools tested ( X / n )
E s t i m a t e d infection rate (t5) 1
Standard error 2
Males 8.87 10.87 1.88
2 3 5
1/2 5/6 0/2
29.3% 44.9% 0.0%
0.25 0.17 0.00
Females 8.87 10.87 1.88
3 4 5
2/2 5/6 2/2
/>33.3% 36.1% >/20.0%
0.15 -
Nymphs 10.87 1.88 7.88 10.88
2 4 10 4
1/4 0/7 2/2 0/6
13.4% 0.0% i> 10.0% 0.0%
0.13 0.00 0.00
1Estimated infection rates for collections in which all pools were positive (X/n = 1 ) are the lowest rates possible based on a single tick of each pool being positive (see text). 2Asymptotic standard error of/5.
TABLE2 E s t i m a t e d infection rates with Cowdria rurninantium of unfed adults of Amblyomma hebraeum collected at Lemco Ranch, Zimbabwe Date of collection
Average no. of ticks p e r p o o l (m)
No. of pools positive/No, of pools tested (X/n)
E s t i m a t e d infection rate (/3)
Standard error
Males 10.87 3.88 4.88
4 5 4
2/2 1/1 3/9
> 25.0% >~20.0% 9.6%
0.05
Females 10.87 3.88 4.88
5 5 3
2/2
>/20.0% >_-20.0% 34.1%
0.13
1/1 5/7
1See footnotes for Table 1.
The three pairs of sheep on which single laboratory-infected nymphs, adult males and adult females were fed, all contracted heartwater.
INFECTION RATES WITH C. RUMINANTIUMOF T H E BONT TICK
281
DISCUSSION The majority of infection rates estimated in this study are considerably higher than had been recorded or estimated for either A. hebraeum (Du Plessis, 1985; Du Plessis and Malan, 1987) or A. variegatum (Uilenberg, 1971; Camus and Barre, 1987). Nevertheless, the present estimates are considered to be conservative for two reasons. (1) The method of testing for infection could yield false negatives if single infected ticks failed to cause clinical disease. Whereas we showed experimentally that single ticks which had fed on reacting animals transmitted heartwater, it is not known if the same applies to ticks fed on carrier hosts. (2) In a number of instances, the Chiang-Reeves estimate was not applicable because all pools of a given collection proved positive (Tables 1 and 2 ). In these cases, estimates are expressed as the lowest possible percentage based on one infected tick being present in each pool. Conceivably, the actual rates could be much higher than those estimated. At Mbizi Quarantine Station, the infection rates were consistently higher in unfed adult males and females than in unfed nymphs. A possible explanation is that the larvae of Amblyomma ticks (which moult to nymphs) have a wider host range than the nymphs (which moult to adults) and consequently feed on a higher proportion of non-carrier hosts (Uilenberg, 1983). The cattle on which the ticks had fed at Mbizi Quarantine Station were heartwater immune and non-reacting. High infection rates in ticks from nonreacting cattle probably reflect the existence of a carrier state, previously demonstrated in both wild and domestic ruminants (Andrew and Norval, 1989). The calves from which engorged nymphs were taken at Lemco Ranch were at weaning age. It is possible that some of these calves had recently reacted to heartwater, but none exhibited signs of illness at the time of collection or in the weeks that followed. The reason for collecting only one engorged nymph per calf at Lemco Ranch was to prevent bias caused by the sampling of several either infected or uninfected ticks from single animals. The finding of high infection rates in unfed nymphs and adults of A. hebraeum is of importance in our understanding of the epidemiology and control of heartwater. It means that these ticks are an important reservoir of infection. In A. hebraeum, unfed nymphs and adults have long survival periods (Norval, 1977 ) and Neitz (1968) reported that a single generation may remain infective for up to 3 years. Ilemobade (1976) observed that C. ruminantium can survive in A. variegatum for at least 15 months. Another consequence of high infection rates is that the disease organism will be perpetuated by low numbers of vectors, hence the maintenance of enzootic stability is unlikely to require a large vector population. This is in contrast to Babesia bovis and Theileria parva, which have low infection rates in field populations of their vectors (Dallwitz et al., 1986). High infection rates in A. hebraeum probably make the eradica-
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tion o f h e a r t w a t e r in s o u t h e r n Africa impossible w i t h o u t the e r a d i c a t i o n of the vector. A m o r e positive effect of high v e c t o r i n f e c t i o n rates is t h a t it facilitates the m a n a g e m e n t of h e a r t w a t e r . In f a r m i n g situations, w h e r e the e r a d i c a t i o n of the disease m a y be impossible, t h e o n l y m e a n s o f c o n t r o l l i n g h e a r t w a t e r is to ensure t h a t all d o m e s t i c r u m i n a n t s are i m m u n e . In n o r m a l c i r c u m s t a n c e s , this is m o s t easily a c h i e v e d b y e n s u r i n g t h a t all y o u n g a n i m a l s in e n d e m i c areas are e x p o s e d to i n f e c t e d ticks d u r i n g t h e p e r i o d in which t h e y are p r o t e c t e d b y v i r t u e of a g e - r e l a t e d r e s i s t a n c e ( N e i t z a n d A l e x a n d e r , 1941 ). T h e n u m b e r s of ticks r e q u i r e d to e n s u r e i n f e c t i o n of all y o u n g a n i m a l s will obviously decrease as i n f e c t i o n s rates increase. ACKNOWLEDGEMENTS T h i s w o r k was f u n d e d b y t h e U n i t e d S t a t e s A g e n c y for I n t e r n a t i o n a l Develo p m e n t ( C o n t r a c t No. A F R - 0 0 0 0 - C - 0 0 - 6 0 0 3 - 0 0 ) t h r o u g h the U n i v e r s i t y of Florida ( P r i n c i p a l Investigator, M.J. B u r r i d g e ) a n d b y t h e G o v e r n m e n t of Zimbabwe. W e t h a n k Dr. R.W. S u t h e r s t a n d J.D. K e r r of C S I R O L o n g P o c k e t L a b o r a t o r i e s , Indooroopilly, Qld., Australia, for d e v e l o p m e n t of t h e c o m p u t e r p r o g r a m for e s t i m a t i o n o f i n f e c t i o n rates. T. C r a w f o r d a n d M. Visagie are t h a n k e d for t h e i r a s s i s t a n c e w i t h t h e collection of ticks in t h e field.
REFERENCES Alexander, R.A., 1931. Heartwater. The present state of our knowledge on the disease. 17th Report of the Director of Veterinary Services and Animal Industry, Union of South Africa, Part II, pp. 89-150. Andrew, H.R. and Norval, R.A.I., 1989. The carrier status of sheep, cattle and African buffalo recovered from heartwater. Vet. Parasitol., 34: 261-266. Bonsma, J.C., 1944. Hereditary heartwater-resistant characters in cattle. Farming S. Afr., 19:71 96. Camus, E. and Barre, N., 1987. Epidemiology of heartwater in Guadeloupe and in the Caribbean. Onderstepoort J. Vet. Res., 54: 419-426. Chiang, C.L. and Reeves, W.C., 1962. Statistical estimation of virus infection rates in mosquito vector populations. Am. J. Hyg., 75: 377-391. DaUwitz, M.J., Young, A.S., Mahoney, D.F. and Sutherst, R.W., 1986. Comparative epidemiology of tick-borne disease of cattle with emphasis on modelling. In M.J. Howell (Editor), Proc. 6th Int. Cong. Parasitology, Aust. Acad. Sci., Canberra, pp. 629-637. Du Plessis, J.L., 1985. A method for determining the Cowdria ruminantium infection rate of Amblyomma hebraeum: Effects in mice injected with tick homogenates. Onderstepoort J. Vet. Res., 52: 55-61. Du Plessis, J.L. and Malan, L., 1987. The application of the indirect fluorescent antibody test in research on heartwater. Onderstepoort J. Vet. Res., 54: 319-325. Ilemobade, A.A., 1976. Study on heartwater and the causative agent Cowdria ruminantium (Cowdry, 1925) in Nigeria. PhD Thesis, Ahmadu Bello University, Zaria, Nigeria, 299 pp.
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Neitz, W.O., 1968. Heartwater. Bull. Off. Int. Epizoot., 70: 329-336. Neitz, W.O. and Alexander, R.A., 1941. The immunization of calves against heartwater. J.S. Afr. Vet. Med. Assoc., 12: 103-111. Neitz, W.O. and Alexander, R.A., 1945. The immunization of cattle against heartwater and the control of the tick-borne diseases, redwater and gallsickness. Onderstepoort J. Vet. Sci. Anim. Ind., 20: 137-157. Norval, R.A.I., 1974. The life cycle of Amblyomma hebraeum Koch, 1844 (Acarina: Ixodidae ). J. Entomol. Soc. S. Afr., 37: 357-367. Norval, R.A.I., 1977. Studies on the ecology of the tick Amblyomma hebraeum Koch in the Eastern Cape Province of South Africa. II. Survival and developement. J. Parasitol., 63: 740-747. Norval., R.A.I., 1983. The ticks of Zibabwe. VII. The genus Abmlyomma. Zimbabwe Vet. J., 14: 3-18. Norval, R.A.I., Butler, J.F. and Yunker, C.E., 1989. Use of carbon dioxide and natural or synthetic aggregation-attachment pheromone of the bont tick, Amblyomma hebraeum, to attract and trap unfed adults in the field. Exp. Appl. Acarol., 7: 171-180. Synge, B.A., 1978. Brain biopsy for the diagnosis of heartwater. Trop. Anim. Health Prod., 10: 45-48. Uilenberg, G., 1971. Etudes sur la cowdriose a Madagascar. (Premiere partie). Rev. Elev. Med. Vet. Pays Trop., 24: 239-247. Uilenberg, G., 1983. Heartwater (Cowdria ruminantium infection ): Current status. Adv. Vet. Sci. Comp. Med., 27: 427-480. Yunker, C.E., Clifford, C.M., Thomas, L.A., Keirans, J.E., Casals J., George, J.E. and Parker, J.C., 1977. Sunday Canyon Virus, a new ungrouped agent from the tick Argas (A.) cooleyi in Texas. Acta Virol., 21: 36-44. Yunker, C.E., Clifford, C.M., Keirans, J.E., Thomas, L.A. and Rice, R.C.A., 1979. Aransas Bay Virus, a new arbovirus of the Upolu serogroup from Ornithodoros capensis Neumann, 1901, (Acari: Argasidae) in coastal Texas. J. Med. Entomol., 16: 453-460.
277
I n f e c t i o n R a t e s w i t h C o w d r i a r u m i n a n t i u m of N y m p h s and A d u l t s of the B o n t T i c k A m b l y o m m a hebraeum C o l l e c t e d in the Field in Z i m b a b w e R.A.I. NORVAL, H.R. ANDREW and C.E. YUNKER
University of Florida/USAID/Zimbabwe Heartwater Research Project, Veterinary Research Laboratory, P. O. Box 8101, Causeway (Zimbabwe) (Accepted for publication 23 January 1990)
ABSTRACT Norval, R.A.I., Andrew, H.R. and Yunker, C.E., 1990. Infection rates with Cowdria ruminantium of nymphs and adults of the bont tick A mblyomma hebraeum collected in the field in Zimbabwe. Vet. Parasitol., 36: 277-283.
Cowdria ruminantium (heartwater) infection rates of field populations of the bont tick, Amblyomma hebraeum, were determined at two locations in the southern lowveld of Zimbabwe. At Mbizi Quarantine Station, unfed adult males and females, and nymphs were collected at intervals over a 2-year period using traps. At Lemco Ranch, engorged nymphs were collected on three occasions from weaner calves and allowed to moult to adults. The unfed ticks were fed in small pools on heartwater-susceptible sheep, some of which became infected. The infection rates of the ticks were then estimated statistically. Depending on the date of collection and locality, these rates were in the range 0.0-44.9% for males, 20.0-36.1% for females and 0.0-13.4% for nymphs. Most of these rates are considerably higher than those previously believed to occur.
INTRODUCTION H e a r t w a t e r is a rickettsial disease o f r u m i n a n t s , c a u s e d b y Cowdria rumin a n t i u m a n d t r a n s m i t t e d b y several species o f t h e tick genus Amblyomma. I n s o u t h e r n Africa, t h e m a i n v e c t o r is t h e b o n t tick A m b l y o m m a hebraeum K o c h . I n t h e rest of s u b - S a h a r a n Africa a n d t h e C a r i b b e a n , t h e tropical b o n t tick A m b l y o m m a variegatum ( F a b r i c i u s ) a s s u m e s this role. T h e e p i d e m i o l o g y of t h e i n f e c t i o n is p o o r l y u n d e r s t o o d , p r i m a r i l y b e c a u s e of t h e lack of precise i n f o r m a t i o n on i n f e c t i o n rates in v e c t o r p o p u l a t i o n s . It was generally a s s u m e d t h a t i n f e c t i o n r a t e s were low b e c a u s e susceptible r u m i n a n t s could r e m a i n u n i n f e c t e d in e n d e m i c areas for m a n y m o n t h s or even y e a r s ( A l e x a n d e r , 1931; B o n s m a , 1944; N e i t z a n d A l e x a n d e r , 1945; Uilenberg, 1971 ). T h e s e a s s u m p t i o n s were s u p p o r t e d b y later studies on infection rates in field
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populations ofA. hebraeum in South Africa (Du Plessis, 1985; Du Plessis and Malan, 1987) and A. variegatum in the Caribbean (Camus and Barre, 1987). However the method used to detect infection in ticks in these studies was an indirect one, which depended on serological conversion of mice that had been inoculated with homogenates of adult ticks removed from cattle. In the present study, we derived infection rates statistically, based on the frequency with which field-collected unfed nymphs and adults of A. hebraeum infected sheep. The method was devised by Chiang and Reeves ( 1962 ) to estimate arbovirus infection rates in mosquito populations and was subsequently used to determine the prevalence of viral agents in ticks (Yunker et al., 1977, 1979). MATERIALSAND METHODS Ticks were collected from two localities in the southern lowveld of Zimbabwe, Mbizi Quarantine Station and Lemco Ranch, where A. hebraeum commonly occurs and heartwater is endemic (Norval, 1983). Mbizi Quarantine Station (31 ° 05' E, 21 °25'S)
Unfed nymphs and adults of A. hebraeum were attracted to traps emitting CO2 andthe male-produced aggregation-attachmentpheromone (AAP) (Norv a l e t al., 1989). The source of AAP was either fed males of A. hebraeum removed from cattle or ether extracts of fed males that had been applied to filter paper. Unfed ticks were collected by hand as they approached or entered the traps. Tick collections were made in and around six pens (100 m 2) in a 450-ha paddock in which 57 undipped Brahman xAfricander steers were kept. The steers were herded into a pen at approximately 17:00 h each day and released at approximately 07: 00 h the following day. Each pen was used continuously for a period of 1 month and then kept free of cattle for 5 months. Protected sites for the moulting of detached ticks were constructed in the pens and tree or brush branches were stacked outside the pens for the same purpose. The pens were sampled in August and October 1987, and January, July and October 1988. Pens were sampled only after a period of at least 6 weeks had elapsed since they had been used by cattle. Lemco Ranch (29 ° 55'E, 21 ° 25' S)
Engorged nymphs were collected by hand on three occasions (October 1987, March and April 1988) from weaner calves < 9 months of age which had never been treated with acaricides. Only one engorged nymph was removed from any one calf. The nymphs were returned to the laboratory where they were held in an incubator at 26 ° C and 85% relative humidity, and allowed to moult to adults.
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Transmission of C. ruminantium to sheep Merino sheep were obtained from a farm near Harare, an area that is free of
Amblyomma ticks and heartwater (Norval, 1983 ). Pools of small numbers (210) of unfed nymphs, adult males or adult females were placed in bags glued to shaved areas on the backs of sheep, and were allowed to attach and feed. Laboratory-reared fed males that were known to be uninfected were placed with the females to facilitate attachment and engorgement (Norval, 1974). The numbers of ticks that attached and fed were recorded. In all experiments, the rectal temperatures of the sheep were recorded daily. Heartwater infection was confirmed by the demonstration of colonies of C. ruminantium in brain smears, made either upon death or from brain biopsies taken according to the method of Synge (1978). All sheep found negative for heartwater by brain biopsy were subsequently challenged to test their susceptibility. The ability of single ticks to transmit infection was tested for nymphs, adult males and adult females. Two replicates were tested for each sex or stage. The ticks used had engorged in the preceding stage on sheep that were reacting to heartwater.
Estimation of infection rates The infection rate in each collection of unfed ticks was estimated using the following formula (Chiang and Reeves, 1962), which was run on an IBM PC computer.
['n_ X'~ lIra /5:1- [-~--) where iO= estimated infection rate; n = number of pools tested; m = average number of ticks per pool; X = number of pools positive. RESULTS Thirty-nine pools of ticks from Mbizi Quarantine Station were fed on sheep, 18 of which became infected with heartwater. The estimated infection rates were 0.0-44.9% in collections of adult males, >_-20.0-36.1% in adult females and 0.0-13.4% in nymphs (Table 1 ). Twenty-two pools of ticks from Lemco Ranch were fed on sheep, 14 of which became infected with heartwater. The estimated infection rates were 9.6>i 25.0% in collections of adult males and >t 20.0-34.1% in adult females (Table 2 ). The 29 sheep that failed to contract heartwater after pools of ticks had been fed on them were all found to be heartwater susceptible on subsequent challenge.
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TABLE 1 E s t i m a t e d infection rates with Cowdria ruminantium of unfed n y m p h s and adults of Amblyomma hebraeum collected at Mbizi Quarantine Station, Zimbabwe Date of collection
Average no. of ticks per pool (m)
No. of pools positive/No, of pools tested ( X / n )
E s t i m a t e d infection rate (t5) 1
Standard error 2
Males 8.87 10.87 1.88
2 3 5
1/2 5/6 0/2
29.3% 44.9% 0.0%
0.25 0.17 0.00
Females 8.87 10.87 1.88
3 4 5
2/2 5/6 2/2
/>33.3% 36.1% >/20.0%
0.15 -
Nymphs 10.87 1.88 7.88 10.88
2 4 10 4
1/4 0/7 2/2 0/6
13.4% 0.0% i> 10.0% 0.0%
0.13 0.00 0.00
1Estimated infection rates for collections in which all pools were positive (X/n = 1 ) are the lowest rates possible based on a single tick of each pool being positive (see text). 2Asymptotic standard error of/5.
TABLE2 E s t i m a t e d infection rates with Cowdria rurninantium of unfed adults of Amblyomma hebraeum collected at Lemco Ranch, Zimbabwe Date of collection
Average no. of ticks p e r p o o l (m)
No. of pools positive/No, of pools tested (X/n)
E s t i m a t e d infection rate (/3)
Standard error
Males 10.87 3.88 4.88
4 5 4
2/2 1/1 3/9
> 25.0% >~20.0% 9.6%
0.05
Females 10.87 3.88 4.88
5 5 3
2/2
>/20.0% >_-20.0% 34.1%
0.13
1/1 5/7
1See footnotes for Table 1.
The three pairs of sheep on which single laboratory-infected nymphs, adult males and adult females were fed, all contracted heartwater.
INFECTION RATES WITH C. RUMINANTIUMOF T H E BONT TICK
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DISCUSSION The majority of infection rates estimated in this study are considerably higher than had been recorded or estimated for either A. hebraeum (Du Plessis, 1985; Du Plessis and Malan, 1987) or A. variegatum (Uilenberg, 1971; Camus and Barre, 1987). Nevertheless, the present estimates are considered to be conservative for two reasons. (1) The method of testing for infection could yield false negatives if single infected ticks failed to cause clinical disease. Whereas we showed experimentally that single ticks which had fed on reacting animals transmitted heartwater, it is not known if the same applies to ticks fed on carrier hosts. (2) In a number of instances, the Chiang-Reeves estimate was not applicable because all pools of a given collection proved positive (Tables 1 and 2 ). In these cases, estimates are expressed as the lowest possible percentage based on one infected tick being present in each pool. Conceivably, the actual rates could be much higher than those estimated. At Mbizi Quarantine Station, the infection rates were consistently higher in unfed adult males and females than in unfed nymphs. A possible explanation is that the larvae of Amblyomma ticks (which moult to nymphs) have a wider host range than the nymphs (which moult to adults) and consequently feed on a higher proportion of non-carrier hosts (Uilenberg, 1983). The cattle on which the ticks had fed at Mbizi Quarantine Station were heartwater immune and non-reacting. High infection rates in ticks from nonreacting cattle probably reflect the existence of a carrier state, previously demonstrated in both wild and domestic ruminants (Andrew and Norval, 1989). The calves from which engorged nymphs were taken at Lemco Ranch were at weaning age. It is possible that some of these calves had recently reacted to heartwater, but none exhibited signs of illness at the time of collection or in the weeks that followed. The reason for collecting only one engorged nymph per calf at Lemco Ranch was to prevent bias caused by the sampling of several either infected or uninfected ticks from single animals. The finding of high infection rates in unfed nymphs and adults of A. hebraeum is of importance in our understanding of the epidemiology and control of heartwater. It means that these ticks are an important reservoir of infection. In A. hebraeum, unfed nymphs and adults have long survival periods (Norval, 1977 ) and Neitz (1968) reported that a single generation may remain infective for up to 3 years. Ilemobade (1976) observed that C. ruminantium can survive in A. variegatum for at least 15 months. Another consequence of high infection rates is that the disease organism will be perpetuated by low numbers of vectors, hence the maintenance of enzootic stability is unlikely to require a large vector population. This is in contrast to Babesia bovis and Theileria parva, which have low infection rates in field populations of their vectors (Dallwitz et al., 1986). High infection rates in A. hebraeum probably make the eradica-
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tion o f h e a r t w a t e r in s o u t h e r n Africa impossible w i t h o u t the e r a d i c a t i o n of the vector. A m o r e positive effect of high v e c t o r i n f e c t i o n rates is t h a t it facilitates the m a n a g e m e n t of h e a r t w a t e r . In f a r m i n g situations, w h e r e the e r a d i c a t i o n of the disease m a y be impossible, t h e o n l y m e a n s o f c o n t r o l l i n g h e a r t w a t e r is to ensure t h a t all d o m e s t i c r u m i n a n t s are i m m u n e . In n o r m a l c i r c u m s t a n c e s , this is m o s t easily a c h i e v e d b y e n s u r i n g t h a t all y o u n g a n i m a l s in e n d e m i c areas are e x p o s e d to i n f e c t e d ticks d u r i n g t h e p e r i o d in which t h e y are p r o t e c t e d b y v i r t u e of a g e - r e l a t e d r e s i s t a n c e ( N e i t z a n d A l e x a n d e r , 1941 ). T h e n u m b e r s of ticks r e q u i r e d to e n s u r e i n f e c t i o n of all y o u n g a n i m a l s will obviously decrease as i n f e c t i o n s rates increase. ACKNOWLEDGEMENTS T h i s w o r k was f u n d e d b y t h e U n i t e d S t a t e s A g e n c y for I n t e r n a t i o n a l Develo p m e n t ( C o n t r a c t No. A F R - 0 0 0 0 - C - 0 0 - 6 0 0 3 - 0 0 ) t h r o u g h the U n i v e r s i t y of Florida ( P r i n c i p a l Investigator, M.J. B u r r i d g e ) a n d b y t h e G o v e r n m e n t of Zimbabwe. W e t h a n k Dr. R.W. S u t h e r s t a n d J.D. K e r r of C S I R O L o n g P o c k e t L a b o r a t o r i e s , Indooroopilly, Qld., Australia, for d e v e l o p m e n t of t h e c o m p u t e r p r o g r a m for e s t i m a t i o n o f i n f e c t i o n rates. T. C r a w f o r d a n d M. Visagie are t h a n k e d for t h e i r a s s i s t a n c e w i t h t h e collection of ticks in t h e field.
REFERENCES Alexander, R.A., 1931. Heartwater. The present state of our knowledge on the disease. 17th Report of the Director of Veterinary Services and Animal Industry, Union of South Africa, Part II, pp. 89-150. Andrew, H.R. and Norval, R.A.I., 1989. The carrier status of sheep, cattle and African buffalo recovered from heartwater. Vet. Parasitol., 34: 261-266. Bonsma, J.C., 1944. Hereditary heartwater-resistant characters in cattle. Farming S. Afr., 19:71 96. Camus, E. and Barre, N., 1987. Epidemiology of heartwater in Guadeloupe and in the Caribbean. Onderstepoort J. Vet. Res., 54: 419-426. Chiang, C.L. and Reeves, W.C., 1962. Statistical estimation of virus infection rates in mosquito vector populations. Am. J. Hyg., 75: 377-391. DaUwitz, M.J., Young, A.S., Mahoney, D.F. and Sutherst, R.W., 1986. Comparative epidemiology of tick-borne disease of cattle with emphasis on modelling. In M.J. Howell (Editor), Proc. 6th Int. Cong. Parasitology, Aust. Acad. Sci., Canberra, pp. 629-637. Du Plessis, J.L., 1985. A method for determining the Cowdria ruminantium infection rate of Amblyomma hebraeum: Effects in mice injected with tick homogenates. Onderstepoort J. Vet. Res., 52: 55-61. Du Plessis, J.L. and Malan, L., 1987. The application of the indirect fluorescent antibody test in research on heartwater. Onderstepoort J. Vet. Res., 54: 319-325. Ilemobade, A.A., 1976. Study on heartwater and the causative agent Cowdria ruminantium (Cowdry, 1925) in Nigeria. PhD Thesis, Ahmadu Bello University, Zaria, Nigeria, 299 pp.
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Neitz, W.O., 1968. Heartwater. Bull. Off. Int. Epizoot., 70: 329-336. Neitz, W.O. and Alexander, R.A., 1941. The immunization of calves against heartwater. J.S. Afr. Vet. Med. Assoc., 12: 103-111. Neitz, W.O. and Alexander, R.A., 1945. The immunization of cattle against heartwater and the control of the tick-borne diseases, redwater and gallsickness. Onderstepoort J. Vet. Sci. Anim. Ind., 20: 137-157. Norval, R.A.I., 1974. The life cycle of Amblyomma hebraeum Koch, 1844 (Acarina: Ixodidae ). J. Entomol. Soc. S. Afr., 37: 357-367. Norval, R.A.I., 1977. Studies on the ecology of the tick Amblyomma hebraeum Koch in the Eastern Cape Province of South Africa. II. Survival and developement. J. Parasitol., 63: 740-747. Norval., R.A.I., 1983. The ticks of Zibabwe. VII. The genus Abmlyomma. Zimbabwe Vet. J., 14: 3-18. Norval, R.A.I., Butler, J.F. and Yunker, C.E., 1989. Use of carbon dioxide and natural or synthetic aggregation-attachment pheromone of the bont tick, Amblyomma hebraeum, to attract and trap unfed adults in the field. Exp. Appl. Acarol., 7: 171-180. Synge, B.A., 1978. Brain biopsy for the diagnosis of heartwater. Trop. Anim. Health Prod., 10: 45-48. Uilenberg, G., 1971. Etudes sur la cowdriose a Madagascar. (Premiere partie). Rev. Elev. Med. Vet. Pays Trop., 24: 239-247. Uilenberg, G., 1983. Heartwater (Cowdria ruminantium infection ): Current status. Adv. Vet. Sci. Comp. Med., 27: 427-480. Yunker, C.E., Clifford, C.M., Thomas, L.A., Keirans, J.E., Casals J., George, J.E. and Parker, J.C., 1977. Sunday Canyon Virus, a new ungrouped agent from the tick Argas (A.) cooleyi in Texas. Acta Virol., 21: 36-44. Yunker, C.E., Clifford, C.M., Keirans, J.E., Thomas, L.A. and Rice, R.C.A., 1979. Aransas Bay Virus, a new arbovirus of the Upolu serogroup from Ornithodoros capensis Neumann, 1901, (Acari: Argasidae) in coastal Texas. J. Med. Entomol., 16: 453-460.