in camels in Tunisia

Veterinary Parasitology 121 (2004) 151–156

Short communication

Transmission dynamics of the Echinococcus granulosus sheep–dog strain (G1 genotype) in camels in Tunisia S. Lahmar a , H. Debbek a , L.H. Zhang b , D.P. McManus b , A. Souissi c , S. Chelly c , P.R. Torgerson d,∗ b

a Service de Parasitologie, Ecole Nationale de Médecine Vétérinaire, 2020 Sidi Thabet, Tunisia Molecular Parasitology Laboratory, Australian Centre for International and Tropical Health and Nutrition, The Queensland Institute of Medical Research, University of Queensland, 300 Herston Road, Brisbane, Qld. Q4029, Australia c Services Vétérinaires, C.R.D.A., Benguerden, Tunisia d Institute of Parasitology, Winterthurerstrasse 266a, CH-8057 Zürich, Switzerland

Received 10 September 2003; received in revised form 29 January 2004; accepted 9 February 2004

Abstract Cystic echinococcosis, caused by Echinococcus granulosus, is highly endemic in North Africa and the Middle East. This paper examines the abundance and prevalence of infection of E. granulosus in camels in Tunisia. No cysts were found in 103 camels from Kébili, whilst 19 of 188 camels from Benguerden (10.1%) were infected. Of the cysts found 95% were considered fertile with the presence of protoscolices and 80% of protoscolices were considered viable by their ability to exclude aqueous eosin. Molecular techniques were used on cyst material from camels and this demonstrated that the study animals were infected with the G1 sheep strain of E. granulosus. Observed data were fitted to a mathematical model by maximum likelihood techniques to define the parameters and their confidence limits and the negative binomial distribution was used to define the error variance in the observed data. The infection pressure to camels was somewhat lower in comparison to sheep reported in an earlier study. However, because camels are much longer-lived animals, the results of the model fit suggested that older camels have a relatively high prevalence rate, reaching a most likely value of 32% at age 15 years. This could represent an important source of transmission to dogs and hence indirectly to man of this zonotic strain. In common with similar studies on other species, there was no evidence of parasite-induced immunity in camels. © 2004 Elsevier B.V. All rights reserved. Keywords: Echinococcus granulosus; Camels; Mathematical modelling; Strain variation; Epidemiology; Maximum likelihood ∗ Corresponding author. Fax: +41-1-63-58907. E-mail address: [email protected] (P.R. Torgerson).

0304-4017/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2004.02.016

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S. Lahmar et al. / Veterinary Parasitology 121 (2004) 151–156

1. Introduction Many countries in North Africa and the Middle East are highly endemic for Echinococcus granulosus (Torgerson and Budke, 2003). In Tunisia the annual human incidence of surgical cases of cystic echinococcosis is approximately 15 cases per 100,000 (Anon, 1993). The importance of small ruminants, particularly sheep as an intermediate host in this region has been demonstrated in previous studies (e.g. Torgerson et al., 1998; Lahmar et al., 1999; Khan et al., 2001). Less is known about the importance of the camel for transmission, although the camel strain (G6 genotype) has been isolated from camels, cattle, humans and goats in sub Saharan Africa, China, Argentina and Nepal (McManus, 2002). Infection of camels is widespread in the Middle East (Ibrahem and Craig, 1998; Tashani et al., 2002). Therefore, it is important to define the role of this species in the transmission of E. granulosus. Models describing the transmission dynamics of E. granulosus have been developed (Roberts et al., 1986, 1987; Torgerson, 2003; Torgerson and Heath, 2003). These models, together with data from Australia, China, South America, the Middle East and central Asia (Roberts et al., 1986; Ming et al., 1992; Torgerson et al., 1998, 2003; Cabrera et al., 1996; Mukbel et al., 2000; Dueger and Gilman, 2001), suggest that there is no regulation of the parasite population by intermediate host immunity and have demonstrated that E. granulosus is normally aggregated in the intermediate host. Although this hypothesis has been tested and supported by field data from sheep, goats, cattle and horses (reviewed by Torgerson and Heath, 2003), no such analysis has been undertaken in camels. This paper confirms that echinococcosis of camels in Tunisia does not appear to be regulated by intermediate host immunity, that the oldest animals are most intensely infected, and that camels in this region are infected by the common sheep strain (G1) of E. granulosus. 2. Materials and methods 2.1. Study area and animals A total of 291 camels (Camelus dromedarius) were examined at necropsy in abattoirs in Kébili and Benguerden regions of southern Tunisia for the presence of E. granulosus cysts. The age of each animal was ascertained by careful examination of its dentition, together with any supplementary data by questioning the animal’s owner. Viscera were examined for the presence of hydatid cysts. Lungs and livers were palpated, examined visually and drawn individually. Large cysts were dissected free of the surrounding tissue. The organs were then carefully cut into thin strips (2 mm thick) to identify smaller cysts, which were again carefully dissected free of the tissue when identified. Cysts found from animals were also examined in more detail for the presence of protoscolices. For each cyst, fluid was extracted and the sediment observed under a microscope for fertility and the viability of protoscolices: 100 protoscolices from each cyst were tested for their ability to exclude 0.2% (v/v) aqueous eosin. In addition, the sizes of the large and small hooks from the protoscolices were measured.


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2.2. Analysis The variation in the numbers of cysts with age and the variation in the prevalence with age were analysed using the model proposed by Roberts et al. (1986, 1987). Because of the aggregated distribution of the parasites within the camels, maximum likelihood techniques using the negative binomial function were used to demonstrate the lack of evidence of parasite-induced host immunity and consequently the most likely value of the parameters of infection pressure (infections per year and cysts per year) and their 95% confidence limits. The numbers of cysts per infection were then calculated from these parameters. The method was exactly the same as that described in detail by Torgerson et al. (2003). 2.3. Strain typing of cyst material Protoscoleces were collected and preserved in 80% ethyl alcohol in small glass containers. These isolates were sent to the Brisbane laboratory where isolation and amplification of DNA was performed by standard PCR technology. A portion (391 bp) of the mitochondrial coding gene, cytochrome c oxidase subunit I (cox1), was examined for each isolate and compared with the profile of existing genotypes of the species E. granulosus as described by Zhang et al. (2000).

3. Results and discussion A total number of 103 camels from Kébili and 188 from Benguerden regions were examined. Of these, there were no cysts found in animals from Kébili, whilst 19 animals from Benguerden were infected. Only the camels from Benguerden were included for subsequent analysis. Attempts to fit the data to the full non-linear, age-abundance model failed to demonstrate evidence of parasite-induced immunity, with parameters for immunity not significantly different from 0. When the data fitted to the linear model, this suggested that the camels were acquiring a mean of 0.056 (95% CI 0.034–0.089) cysts per year (Table 1). Likewise, when the data was fitted to the model that described the variations of prevalence with age, again all parameters for parasite-induced immunity were not significantly different from 0. The prevalence model suggested that the camels were being exposed to a mean of 0.025 (95% CI 0.015–0.039) infections per year and hence 2.21 (95% CI 1.05–4.42) cysts per infection. Despite the limited data available from just 19 positive animals, the model Table 1 Maximum likelihood values and 95% confidence limits of parameters and negative binomial constant for E. granulosus in camels in Benguerden regions of southern Tunisia Parameter

Most likely

Lower 95%

Upper 95%

Infection pressure (cysts per year) Infection pressure (infections per year) Cysts per infection Negative binomial constant k

0.0559 0.0253 2.21 0.222

0.0340 0.0153 1.05 0.105

0.0890 0.0390 4.42 0.476

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Table 2 Morphology of hooks of protoscolices isolated from fertile hydatid cysts obtained from camels in southern Tunisia

Total number of hooks Length of hook (␮m) Handle length (mm) Blade length (mm) Length of garde: width

Large hooks

Small hooks

19 (17–21) 27.92 (24.7–30.1) 8.58 (6.09–9.15) 8.97 (7.37–9.98) 2.1 (2.07–4.17)

11 (9–12) 19.19 (15.84–22.05) 5.98 (3.75–7.21) 4.67 (3.1–5.81) 1.9 (1.7–2.43)

suggests that by the time that camels are 15 years of age 32% of animals will be infected (95% CI 21–44%). These old animals represent a significant threat of infection to dogs particularly since they are likely to be infected with fertile cysts. Cyst numbers were aggregated within the camel population, but the negative binomial constant, k, did not vary significantly with age, and was a value of 0.22 (95% CI 0.11–0.48) for the whole population. A total of 65 cysts were found in these animals. Of these, 60 cysts were found in the lungs whilst only five were found in the liver. Of these cysts, 95% were considered fertile with a mean of 80% viability of protoscoloces. Examination of hook morphology suggested that the parasites were of sheep origin rather then camel origin. Eckert et al. (1989) reported that the mean size (±S.D.) of large hooks from protoscolices of camel, cattle and horse origin are 35.4 ␮m (1.2), 39.1 ␮m (0.8) and 40.2 ␮m (1.6), respectively. Likewise the size (±S.D.) of small hooks from cyst material from these species was reported as 28.2 ␮m (2.0), 32.7 ␮m (1.1) and 31.7 ␮m (1.4). Each of these reported sizes is significantly larger (P < 0.05) then the mean size of the large and small hooks found in the camel cyst material in this study (Table 2). The mean sizes of large and small hooks from cyst material of sheep origin reported by Eckert et al. (1989) was 31.8 ␮m (2.5) and 24.6 ␮m (2.9), which is not significantly different from the hook size reported in this study. Molecular analysis of 13 cysts confirmed that camels in southern Tunisia are infected with E. granulosus of sheep origin. The sequences obtained were identical to the published cox1 sequence for the common sheep strain or G1 genotype of E. granulosus. The results of the research described in this paper have a number of important implications. Firstly, it confirms that camels in Tunisia are infected with the sheep strain (G1) of E. granulosus. This is in agreement with results reported from neighbouring Libya (Tashani et al., 2002) where the cox1 sequence examined for each of 28 protoscolex isolates excised from cysts from several intermediate hosts (sheep, cattle, humans), including camels, were identical to that of the published G1 genotype (Tashani et al., 2002). There is no evidence of transmission of the camel strain (G6) in this part of Africa although this strain is transmitted in a number of other countries including Somalia, Sudan, Mauritania and Kenya (McManus and Thompson, 2003). In the current study, camels were infected with fertile cysts, which suggests that camels are suitable hosts for the sheep strain. In agreement with other studies (Roberts et al., 1986; Ming et al., 1992; Cabrera et al., 1996; Torgerson et al., 1998, 2003; Dueger and Gilman, 2001) there was little or no evidence of parasite-induced host immunity due to natural infection despite the small number of infected animals. The infection pressure was somewhat lower than that of sheep in Tunisia (Lahmar et al., 1999) and Jordan (Torgerson et al., 1998). This may reflect either grazing behaviour of camels resulting in the ingestion of fewer eggs from the environment than


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sheep, or camels being kept in more arid areas where the viability of eggs could be lower. The number of cysts per infection from various studies in sheep has been reported as between 2.02 and 6.03. The 2.21 cysts per infection (95% CI 1.05–4.42) in camels is within this range, which would suggest that the aggregation of viable eggs in the environment is similar to the situation of grazing sheep. This could therefore be interpreted that it is grazing behaviour, leading to the ingestion of fewer clusters of eggs that results in the lowered infection pressure rather than lower viability of eggs. The negative binomial constant of 0.22 (95% CI 0.105–0.476) was of a similar order of magnitude as other studies in sheep (Torgerson et al., 1998, 2003; Lahmar et al., 1999) but greater than that found in cattle (Torgerson et al., 2003) or donkeys (Mukbel et al., 2000). The reason for this extreme distribution is due, partly at least, to the aggregation of the infectious stages. Eggs are likely to be concentrated around areas where dogs have defecated, thus even if the infection process is random, an infectious insult may contain a large number of eggs thus resulting in considerable aggregation of cysts in camels. The results reported provide valuable information with regard to the base line infection pressure in livestock. Such information can be used to simulate a control programme (Torgerson, 2003), which is potentially useful for planning such a programme. Although the overall prevalence rate for camels was considerably lower then in sheep, they could still act as an important reservoir of the parasite. Old camels in particular have a high prevalence rate and the cysts are highly fertile. Thus, uncontrolled slaughtering of these animals will provide dogs with a ready source of infection with this parasite. Using the models of Roberts et al. (1986, 1987) this report suggests, despite the relatively small sample size, that camels are less likely to acquire immunity on natural expose to Echinococcus eggs then become resistant to reinfection. Furthermore, camels in Tunisia appear to be infected with the common sheep (G1) strain of E. granulosus. References Anon, 1993. The surgical incidence rate of hydatidosis in Tunisia (1988–1992). Report of the D.S.S.B. (Direction de la Santé et des Soins de base), Ministry Public Health, Tunis. Cabrera, P.A., Haran, G., Benavidez, U., Valledor, S., Perera, G., Lloyd, S., Gemmell, M.A., Baraibar, M., Morana, A., Maissonave, J., Carballo, M., 1996. Transmission dynamics of Echinococcus granulosus, Taenia hydatidgena and Taenia ovis in sheep in Uruguay. Int. J. Parasitol. 25, 807–813. Dueger, E.L., Gilman, R.H., 2001. Prevalence, intensity, and fertility of ovine cystic echinococcosis in the central Peruvian Andes. Trans. R. Soc. Trop. Med. Hyg. 95, 379–383. Eckert, J., Thompson, R.C.A., Michael, S.A., Kumaratilake, L.M., El-Sawah, H.M., 1989. Echinococcus granulosus of camel origin: development in dogs and parasite morphology. Parasitol. Res. 75, 536–544. Ibrahem, M.M., Craig, P.S., 1998. Prevalence of cystic echinococcosis in camels (Camelus dromedarius) in Libya. J. Helminthol. 72, 27–31. Khan, A.H., El-Buni, A.A., Ali, M.Y., 2001. Fertility of the cysts of Echinococcus granulosus in domestic herbivores from Benghazi, Libya, and the reactivity of antigens produced from them. Ann. Trop. Med. Parasitol. 95, 337–342. Lahmar, S., Kilani, M., Torgerson, P.R., Gemmell, M.A., 1999. Echinococus granulosus larvae in the livers of sheep in Tunisia: the effects of host age. Ann. Trop. Med. Parasitol. 93, 75–81. McManus, D.P., 2002. The molecular epidemiology of Echinococcus granulosus and cystic hydatid disease. Trans. R. Soc. Trop. Med. Hyg. 96, S1/151–S1/157. McManus, D.P., Thompson, R.C.A., 2003. Molecular epidemiology of cystic echinococcosis. Parasitology 127, S37–S51.

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