International Journal for Parasitology 29 (1999) 13211330

Biological control of rodents using Sarcocystis singaporensis

T. Jakel a, b, *, Y. Khoprasert c, S. Endepols d, C. Archer-Baumann a, K. Suasa-ard c, P. Promkerd c, D. Kliemt a, P. Boonsong c, S. Hongnark c
Division of Parasitology, Department of Zoology, University of Hohenheim, Emil Wol Str. 34, 70599 Stuttgart, Germany b German Technical Cooperation (GTZ), 65726 Eschborn, Germany c Agricultural Zoology Research Group, Entomology and Zoology Division, Department of Agriculture, 10900 Bangkok, Thailand d Bayer AG, 51368 Leverkusen, Germany Received 20 January 1999; received in revised form 31 May 1999; accepted 31 May 1999
a

Abstract Parasites have been identied as an important factor in regulating vertebrate populations. In replicated eld experiments (plots up to 4 ha) performed in Thailand we tested whether commensal and eld rodents could be artically infected and controlled with the host-restricted apicomplexan protozoon Sarcocystis singaporensis which is endemic in Southeast Asia. When bait-pellets containing high numbers of these parasites were consumed by rodents of three species (Rattus norvegicus, Rattus tiomanicus, Bandicota indica) in dierent agricultural habitats (chicken farm, oil palm plantation, riceeld), we observed a parasite-induced mortality ranging from 58% to 92%. Detection of merozoites of S. singaporensis in lung tissue samples of rats collected dead at the experimental sites using a species-specic monoclonal antibody conrmed that S. singaporensis was the causative agent of mortality. As observed with brown rats, the parasite's eect on the host was not related to sex. These experiments demonstrate for the rst time that articial infection of rodents with an endemic protozoon has the potential for eective population control. # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.
Keywords: Biological control; Sarcocystis singaporensis; Southeast Asia; Wild rats

1. Introduction The cause of cycles of vertebrate populations has for a long time interested biologists. It is now considered by many that trophic interactions rather than intrinsic mechanisms are the principal cause, and recent evidence suggests that parasites are an important factor [1]. Research on rodent
* Corresponding author: Tel: 49-711-459-3072: fax; 49-711459-2276, e-mail; tojack@uni-hohenheim.de

parasite interactions can be viewed from two principal perspectives. These are: rst, analysis and manipulation of the hostparasite relationship in the wild to better understand the natural situation; and second, exploitation of pathogenic eects of parasites for a practical purpose in rodent control. The latter will be discussed here. New approaches in the biological control of rodents which aim at a reduction of fecundity [2, 3] or a reduction of survival of rodents [4] are under development. Although

0020-7519/99/$20.00 # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 0 - 7 5 1 9 ( 9 9 ) 0 0 0 8 1 - 8

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microparasites have been associated with declines of rodent populations [57] direct demonstration of an eect on survival is lacking with the exception of certain rabbit viruses which, however, were introduced into the environment as alien organisms [8, 9]. Recently, the potential of an endemic virus was highlighted [10], and in the present study we examined whether an endemic apicomplexan protozoon, Sarcocystis singaporensis, could be used for population control. Sarcocystis singaporensis frequently occurs in rodents in Southeast Asia [11, 12] and has been found to be host-restricted [4, 13, 14]. It uses snakes (Python reticulatus) and rodents of the genera Rattus and Bandicota to maintain its life cycle. Sporocysts containing sporozoites, the stages which are infective for rats, can be obtained in large quantities from the snake host [4]. Its potential as a biocontrol agent was recognised [13, 15] because this normally apathogenic parasite induces a fatal pneumonia in rodents once infection with sporocysts exceeds a certain threshold. We then provided preliminary evidence that S. singaporensis increases mortality of wild rats in the eld [4]. In the present study, we tested in manipulative replicated eld experiments whether rodent populations in Thailand could be articially infected by high dosages of S. singaporensis. If so, would this provide a basis for control inside the parasite's natural distribution range in Southeast Asia? Changes in rat population size were measured mainly using comparative techniques such as bait consumption, footprint cover of tracking plates, or observation of activity at burrow entrances. Comparative indices can be related to actual numbers [16] and are a good indicator of population size [17].

from Thailand and produced in large numbers as described previously [4]. For application of parasites in the eld, 20 ml of a sporocyst suspension in PBS were injected into a 1 g bait-pellet. A bait was used which had proven to be highly attractive for wild rats in preliminary studies (unpublished). It consisted of wheat our mixed with broken corn, talcum, corn oil and various other ingredients including attractants like sh extract (for brown rats, Rattus norvegicus) or coconut extract (for Malaysian wood rats, Rattus tiomanicus). To protect against ants, pellets were sealed into small polyethylene sacchets or, for wood rats, into wax-coated paper. They were stored at 48C until use. 2.2. Detection of parasites in rats A mAb (SS11D5/H3) reactive against sporozoites and merozoites of S. singaporensis was generated according to procedures described elsewhere [18]. The antibody did not crossreact with acetone-xed air-dried stages of closely related parasites (unpublished observation) some of which frequently occur in wild rats [12, 19]. These included Sarcocystis zamani (sporozoites and merozoites; life cycle maintained at the University of Hohenheim), Sarcocystis cymruensis (bradyzoites; obtained from muscles of wildcaught brown rats from Thailand), Frenkelia glareoli (bradyzoites isolated from cysts in the brain of a wild-caught European bank vole, Clethrionomys glareolus), Toxoplasma gondii (sporozoites and tachyzoites of the strain GTP1; WHO Centre VPH, Hannover, Germany), and Cryprosporidium parvum (sporozoites; Public Health Center of the State of Baden Wurttemberg, Germany). Immunostaining of merozoites in lung imprints (200 mm2) of rats collected dead at the experimental sites was performed by indirect immunouorescence. Briey, imprints were xed onto glass slides with acetone for 10 min, washed in PBS, and incubated with undiluted SS11D5/H3 supernatant for 1 h at 378C. Primary antibodies were detected in two consecutive steps (30 min incubation time each at 378C) using a biotin-conju-

2. Materials and methods 2.1. Parasites and bait Sporocysts of S. singaporensis were originally isolated from a wild-caught reticulated python

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gated anti-mouse IgG antibody (Kirkegaard and Perry) diluted 1:100 in PBS and uorescein-conjugated streptavidin (The Binding Site) diluted 1:50 in PBS containing 0.01% Evans Blue. They were examined with a microscope equipped with epiuorescence. 2.3. Field experiment 1 An experiment with brown rats, R. norvegicus, was performed at three feed storage buildings on chicken farms in Bang Nam Prieo district, Chachoengsao province, Thailand. The buildings were surrounded by ponds which dened the borders of the study areas; they were 630 m2 (control), 645 m2 (site 1) and 820 m2 (site 2) large. As a rst index of rodent activity, consumption of dry oat akes before and after treatment of rats with parasites or placebos, respectively, was measured. Bait stations containing trays were placed at a density of 1/37 m2, 1/28 m2, and 1/ 36 m2, respectively. As a second index, the percentage of footprint cover on tracking plates covered with sand was measured using a transparent grid of 16 squares [20]. Plates were placed independently from bait stations on rat runs at a density of 1/24 m2, 1/26 m2, and 1/27 m2, respectively (>90% of plates showing rodent tracks before treatment). During treatment, parasite-pellets containing 1105 sporocysts each or placebos were oered to rats in excess inside bait stations. The time-scale of the experiment, including lag phases (no oat akes and measurement of consumption), to counter conditioning eects of census baiting is displayed in Fig. 1. All rodents found dead inside the study areas were weighed, inspected externally for the reproductive status, and dissected to take lung samples and inspect reproductive organs. To recover remaining rats from the sites after the experiment, they were kill-trapped for two consecutive nights followed by treatment with coumatetralyl-containing bait. 2.4. Field experiment 2 The second experiment was conducted in three rice elds located in Banglen district,

Thailand, which were mainly infested with bandicoot rats, Bandicota indica. Population size before and after two rounds of treatment (two nights each) was estimated by the number of reopened burrow entrances (active burrows, [21]) which had been closed with earth two nights before. Burrows were located on dikes which surrounded elds which were 4-ha-large each. Those elds had been selected which contained approximately 100 active burrows out of a total of 200 closed at a dike length of 800 m. All entrances were marked and monitored throughout the study. As a second index, the total number of footprints on tracking plates (2020 cm) marked crosswise with stripes of black ink was counted. Plates in each eld (n = 110) were placed independently from burrow-entrances every 68 m on rat runs in an alternating fashion at both sides of the dikes. Bandicoot rats received the same parasite-pellets and placebos as described previously. Pellets were evenly distributed on the dikes and placed close to burrows. Rats found dead in the study areas were dissected and tissue samples of the lungs taken for analysis by indirect immunouorescence. 2.5. Field experiment 3 The experimental plots were located inside an oil palm plantation in Thasae district, Chumporn province, Thailand. The Malaysian wood rat, Rattus tiomanicus, is the predominant rodent species in oil palm plantations in Thailand [22]. Four oil palm elds were randomly grouped to two parasite-treated and two control plots. Each eld was square, 3.24 ha large, and contained 400 palms which were 9 m apart. Parasite-bait or placebos were applied in each entire eld, whereas wood rats were captured and their activity measured inside a core area comprising 100 palms (0.81 ha). Parasitebait (a pellet contained 4105 sporocysts) or placebos were distributed in two rounds with a 15 days interval. Three pellets were placed under each palm tree during the rst round; two pellets per tree in the second round. Rodent numbers were determined before and

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Fig. 1. Time-course of the activity of wild brown rats, Rattus norvegicus, on three chicken farms before and after treatment with Sarcocystis singaporensis or placebos (control). Bait take (mean2S.E.M.) tray 1 day 1 and tracking plates expressed as the mean percentage 2S.E.M of footprint cover day 1. Values above the x-axis indicate the total number of parasite-pellets or placebos, respectively, consumed by rats.

after treatment using 100 drop-door live traps (one trap per tree) for three consecutive nights; the animals were released each following morning. Rodent activity was measured before treatment, and after the rst and second application of parasites; this was done using tracking plates as in the rst experiment. A total of 150 plates was used in each eld, at a density of 1/54 m2 (>90% covered with tracks before treatment). Eight to 15 days after each application of parasites, the core areas were extensively examined for presence of dead rats.

2.6. Computation of rodent mortality and statistical analyses Rodent mortality was calculated using the formula of Henderson and Tilton [23]: M = 100 [1-(t2 c1)/(t1 c2)], with M(%) = rodent mortality, t = treated population, c = control population, 1 = population before treatment, 2 = population after treatment. Where appropriate, the chi-square test using the Yates correction or the MannWhitney Utest were performed. Both tests are part of the

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statistical computer programme SigmaStat2 of the Jandel Corporation. 3. Results 3.1. Field experiment with brown rats Two colonies of brown rats on chicken farms were infected with the parasite. Rodent activity markedly declined at these sites 1013 days after parasite-bait had been disseminated, indicating an increase of rodent mortality (Fig. 1). Activity remained unchanged in the control which had received placebos. In contrast to the control, food consumption at the parasite-treated sites was signicantly greater in the pretreatment phase than in the post-treatment phase (Table 1) (MannWhitney U-test; site 1, P < 0.0001; site 2, U[n1 = 20,n2 = 23]=628, U[23,23] = 732, P < 0.0001; control, U[17,17] = 299, P = 0.97). Similarly, footprint frequency was signicantly decreased during the post-treatment phase when compared to the control (MannWhitney U-test; site 1, U[24,24] = 871, P < 0.0001; site 2, U[29,29] = 456, P < 0.0001;

control, U[25,25] = 494, P = 0.15). The observation of numerous dead rats at the parasitetreated sites between days 20 to 30 (Fig. 1) conrmed rodent mortality (Table 1). No dead rats were noticed at the control site. Intriguingly, most moribund rats left their burrows shortly before death in search for food or water; thus, the majority of individuals could be recovered at the treated sites. Application of a speciesspecic mAb, SS11D5/H3, on impression smears of lung tissue conrmed that merozoites of S. singaporensis were present in all out of 20 samples taken from dead rats at the parasitetreated sites (Fig. 2). There were >20 to several hundreds of merozoites per sample. Twenty samples from rats trapped at the control site were negative. There are no statistically signicant dierences between: (i) parasite-treated sites and the control site with regard to proportions of males and females that succumbed to infection or were recovered from the site by trapping and treatment with coumatetralyl, respectively (statistical analysis refers to numbers of recovered rats in Table 1, excluding some animals of which sex could not be determined unequivocally due to

Table 1 Comparison of footprint cover of tracking plates and bait take before and after treatment of brown rats with Sarcocystis singaporensis Location Censusa Control Site 1 Site 2
a

Mean amount No. No. Bait Total No. No. rats Percentage Footprint cover eaten tray 1 rats/tray tracking taken no. rats trays recoveredc mortalityd (mean %2 S.E.M.) b plates (g) (g2S.E.M.) (estimate) (estimate) 82.325.5 73.425.7 92.523.2 13.824.2 82.324.5 23.525.2 25 25 24 24 29 29 879.9 931.2 1256.4 212.9 2163.8 458.1 17 17 20 23 23 23 51.828.2 54.8211.2 62.8218.7 9.323.2 94.1214.6 19.925.5 3.54 3.74 4.23 0.63 5.27 1.11 60 64 85 14 121 26 38 70 17 93 17

Pre-treatment Post-treatment Pre-treatment Post-treatment Pre-treatment Post-treatment

83, 86 68, 80

Refers to Fig. 1; days of reference are 13 (footprint cover) and 6 (food consumption) for the pre-treatment census, and day 28 for the post-treatment census. b It was assumed that each rat consumes 5% of its body mass day 1 [24]. Mean body masses of rats were 293 g (n = 28; control), 297 g (n = 62; site 1), 357 g (n = 83; site 2). c After the experiment, remaining rats were recovered from all sites by kill-trapping and treatment with coumatetralyl. Post-treatment values include these animals, and pre-treatment values are the sum of the latter plus animals that were collected dead after treatment. d The rst value refers to mean footprint cover, the second value to mean bait take.

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Fig. 2. Detection of a merozoite (arrowhead) of Sarcocystis singaporensis in an imprint of the lungs of a brown rat which was recovered dead in the study area. Indirect immunouorescence with monoclonal antibodies of the mouse hybridoma cell line SS11D5/H3. Host cells (hc) were counterstained with Evans Blue.

an advanced stage of decay; 17 males/21 females at control site, 25/24 site 1, 29/36 site 2; w 2 = 0.54, df = 2, P = 0.76), (ii) proportions of males and females that survived within the treated sites (site 1; eight males out of 33 males and nine females out of 33 females, w 2 = 0.003, df = 1, P = 0.96, site 2; eight out of 37 and nine out of 45, w 2 = 0.015, df = 1, P = 0.90). This indicates that the eect of the parasite on the rats was not related to sex. When rats that had died inside the treated plots were grouped according to their body mass (Hage) and the chronology of their appearance in the eld, it became apparent that adult females with more than 300 g body mass succumbed rst to infection followed by males and juveniles of the recent breeding season (weight group 0 100 g) (Fig. 3). Necropsy revealed that most of these females were pregnant or lactating (Fig. 3).

Fig. 3. Demography of the population decline of Rattus norvegicus. Shown are the numbers of dead rats recovered from the two parasite-treated sites of experiment 1 (upper and lower panel, respectively) grouped according to sex, body mass, and the chronology of appearance in the eld. Numbers adjacent to bars indicate numbers of pregnant or lactating females.

T. Ja kel et al. / International Journal for Parasitology 29 (1999) 13211330 Table 2 Activity of bandicoot rats before and after treatmenta with Sarcocystis singaporensis No. active burrowsb No. footprints

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Percentage Location February 35 February 2426 March 2022 February 35 February 2426 March 2022 mortalityc (before treatment) (after 1st treatment) (after 2nd treatment) Control Site 1 Site 2
a

93 98 112

87 50 71

125 42 24

489 548 1092

298 481 250

640 233 121

65, 68 84, 92

Dissemination of 3.5107 and 3.8107 sporocysts ha 1 on two 4-ha experimental plots, respectively, at February 57 and March 57, 1997, each. A bait pellet contained 1105 sporocysts. Bandicoot rats accepted 65.3 and 50.3% of the parasite bait, respectively, during the rst treatment round, and 15.2 and 28.2% in the second round. Placebos were oered to rats in the control eld. b Approximately one entrance per burrow system was closed. It was reported about a closely related bandicoot species that on average 1.5 adults (range 04) inhabitate a burrow system [25]. c The data of February 35 and March 2022 were compared; the rst value refers to the burrow data, the second to footprint data.

3.2. Field experiment with bandicoot rats Parasite-bait was oered to bandicoot rats, B. indica, on two occasions inside two 4-ha riceelds (Table 2). After the second round, activity indices were signicantly decreased in both parasite-treated elds when compared with the control (Table 2; burrows, w 2 = 24.5, df = 1, P < 0.0001 [control/site 1], w 2 = 52.5, df = 1, P < 0.0001 [control/site 2]; footprints, w 2 = 133.1, df = 1, P < 0.0001 [control/site 1], w 2 = 579.6, df = 1, P < 0.0001 [control/site 2]). Additionally, the

data show a signicant dierence between the two parasite-treated sites (burrows, w 2 = 5.1, df = 1, P = 0.024; footprints, w 2 = 127.0, df = 1, P < 0.0001) indicating that there was variation in the course of treatments at the dierent sites. In rice elds where parasites had been released, a smell of decay conrmed the presence of dead rodents 1219 days after the rst round. Due to the dense vegetation and high ambient temperatures recovery of dead rats was minimal (20 adult bandicoot rats from both treated sites). All out of six lung samples obtained from these rats were

Table 3 Numbers of Malaysian wood rats and their activity before and after treatmenta with Sarcocystis singaporensis No. rats capturedb Location Footprint cover (mean %2 S.E.M., n = 150)

2831 January 2528 March 57 February 12 March 2425 March Percentage (before treatment) (after 2nd treatment) (before treatment) (after 1st treatment) (after 2nd treatment) mortalityc 72 58 21 23 51.122.5 38.422.3 52.922.8 59.022.7 25.122.1 33.822.2 9.621.5 9.821.3 31.1 21.7 51.3 22.2 17.2 21.9 23.0 21.8

Control 1 31 Control 2 51 Site 1 45 Site 2 50

71, 65 71, 58

a Rodents inside the parasite-treated core areas accepted 94 and 100%, respectively, out of 300 bait-pellets during the rst round at February 15, 1998, and 100% out of 200 bait-pellets on both plots during a second application at March 3. Each bait-pellet contained 4105 sporocysts. Placebos were applied in the control plots. b Numbers captured in 300 trap-nights. b The rst value refers to rat numbers, the second value to footprint data. Values of sites 1 and 2 were compared with the mean of the controls.

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positive for S. singaporensis when probed with mAb SS11D5/H3. In the control eld, no smell of decay was noticed nor were dead rats seen.

4. Discussion The results from three independent experiments indicated that an endemic protozoon, S. singaporensis, signicantly increased mortality of eld populations of rodents when oered at articially high doses. Rodents in treated populations had a level of mortality that is comparable to chemical rodenticides tested under similar environmental conditions [26]. It was previously suggested that control of eruptive pests may require tactically released control agents [27]. Our data indicate the feasibility of such a concept: the parasite's eect is amplied by manipulation [15] and can be terminated if not required. Sarcocystis singaporensis was eective against three dierent rat species in dierent agricultural habitats proving that the observations on virulence obtained in the laboratory [4, 15] are also valid in the wild. Non-susceptible hosts [4, 13, 14] appeared not to be aected by this parasite. Although, the experimental sites had been selected to be largely inhabitated by single target species only, some other rodents such as Mus spp. were present but no dead specimens of these were found. With respect to the ethical questions involved in killing of vertebrates, it is noteworthy that rats do not appear to be ill until shortly before death because food consumption is unaltered most of the time during acute infection [4]. The present results conform with this; food consumption remained stable until a sharp decline occurred (Fig. 1). To measure changes in population size, we used various indices of abundance or activity. All proved to be useful because estimates of mortality within single sites were quite similar in most cases. Occasionally, indices obtained with tracking plates showed considerable variation and did not conform with results of robust techniques like live-trapping (e.g. experiment 3). This indicates that tracking plates should not serve as a sole source of information in the eld. Besides that, dierential hostparasite interactions may also account for the variation observed among parasite-treated elds (e.g. experiment 2). Within the endemic area of S. singaporensis, dierent

3.3. Field experiment with Malaysian wood rats As in the previous experiment, parasite-bait was oered to rats twice. Compared with the situation before treatment, signicantly less wood rats were captured in the parasite-treated elds after the second round, whereas numbers in the control elds had increased (Table 3). There is no signicant dierence between parasite-treated sites regarding rodent numbers before and after application of parasites (w 2 = 0.02, df = 1, P = 0.89), in contrast, data are signicantly dierent when compared to the controls (e.g. control 2/site 1, w 2 = 6.8, df = 1, P < 0.01; control 2/site 2, w 2 = 7.5, df = 1, P < 0.01). A similar trend is apparent for the footprint data, although there was considerable variation among the controls with control eld 1 showing a signicant drop of rodent activity compared with pre-treatment values (MannWhitney Utest, U[150,150] = 26975, P < 0.0001, after second treatment round) despite a marked increase in rodent numbers (Table 3). Footprint cover in control eld 2 was similar to pre-treatment values after the rst baiting round (U[150,150] = 23571, P = 0.19), but was signicantly increased after the second round (U[150,150] = 19495, P < 0.0001). Inside the parasite-treated elds, a signicant decrease of rodent activity after the rst baiting round (site 1, U[150,150] = 30870, P < 0.0001; site 2, U[150,150] = 31432, P < 0.0001) translates into mortality rates of 72 and 74%, respectively. This was paralleled by the observation of 54 and 34 dead wood rats, respectively, 11 to 17 days after dissemination of sporocysts. After the second treatment, another 10 and 12 rats, respectively, were found dead at these sites. At that time, footprint frequency also was signicantly lower than pre-treatment values (site 1, P < 0.0001; site 2, U[150,150] = 29388, U[150,150] = 29297, P < 0.0001). No dead rodents were seen at the control sites.

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populations of a particular rodent species may express genetical dierences with regard to susceptibility to infection (role of immunity, see below). We have shown that brown rats, R. norvegicus, living outside the endemic area are more susceptible to infection with this parasite than those living within; additionally, there also exist dierences between rodent species [28]. The experiment with brown rats focused on the temporal aspects of the disease. Because activity declined 1013 days after the rst application of parasite-bait (Fig. 1) and most of the dead rats were seen in the eld after this interval, it can be concluded that the lethal dose had been already consumed within the rst 2 days. The above interval is exactly the time required for merozoites to appear in lung tissue [4]. The two other rodent species tested also accepted the parasite-bait immediately. This underlines the high degree of palatability of the bait material. It also indicates that aversion towards novel food, or neophobia (which is assumed to be generally true for brown rats, [29]), may not always apply to rodents in particular geographic locations or control situations. The rst experiment further demonstrates that the eect of the parasite is not related to the sex of the host. This is an important result because if males were predominantly aected then the population consequences would be less pronounced [30]. Interestingly, our data show that pregnant or lactating females were the rst to be infected with the parasite. This is probably due to increased energy requirements of females during the reproductive period; thus, they probably accept novel foods faster. The experiments with bandicoot rats and wood rats diered from the previous one in that the study areas were considerably larger, no pre-baiting was used before application of parasite-bait (therewith avoiding a possible selection of rats that liked the food), and two treatment rounds were performed instead of one only. The rst two points outline that the experimental procedures conformed with current control practices. The latter is important because the data show that rodents were also aected by a second treatment round. Dead wood rats were found after

the second application of parasite-bait; similarly, activity of bandicoot rats decreased after the second treatment. In a previous study, we have shown that consumption of a sublethal amount of sporocysts can immunise rats, and argued that protective immunity could be an obstacle to consecutive treatments [4]. Obviously, this appears not to be a problem in the short term. Intriguingly, articial infection with S. singaporensis is highly pathogenic for rats in Thailand despite the fact that this protozoon is highly prevalent in the wild [12]. We regularly observed rats with natural infections of S. singaporensis that do not resist challenge infection in the laboratory (unpublished observation). The mechanisms of immunity are still poorly understood and, therefore, the subject of our current research. There is a promising prospect for an application of S. singaporensis within the endemic area in Southeast Asia. Its application elsewhere may be considered, provided its high degree of host specicity can be further conrmed.

Acknowledgements We thank B. J. Wood and two anonymous referees for comments on an earlier version of the manuscript.

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