Canine echinococcosis: genetic diversity of Echinococcus granulosus sensu stricto (s.s.) from definitive hosts.

Canids, particularly dogs, constitute the major source of cystic echinococcosis (CE) infection to humans, with the majority of cases being caused by Echinococcus granulosus (G1 genotype). Canine echinococcosis is an asymptomatic disease caused by adult tapeworms of E. granulosus sensu lato (s.l.). Information on the population structure and genetic variation of adult E. granulosus is limited. Using sequenced data of the mitochondrial cytochrome c oxidase subunit 1 (cox1) we examined the genetic diversity and population structure of adult tapeworms of E. granulosus (G1 genotype) from canid definitive hosts originating from various geographical regions and compared it to that reported for the larval metacestode stage from sheep and human hosts. Echinococcus granulosus (s.s) was identified from adult tapeworm isolates from Kenya, Libya, Tunisia, Australia, China, Kazakhstan, United Kingdom and Peru, including the first known molecular confirmation from Gaza and the Falkland Islands. Haplotype analysis showed a star-shaped network with a centrally positioned common haplotype previously described for the metacestode stage from sheep and humans, and the neutrality indices indicated population expansion. Low Fst values suggested that populations of adult E. granulosus were not genetically differentiated. Haplotype and nucleotide diversities for E. granulosus isolates from sheep and human origin were twice as high as those reported from canid hosts. This may be related to self-fertilization of E. granulosus and/or to the longevity of the parasite in the respective intermediate and definitive hosts. Improved nuclear single loci are required to investigate the discrepancies in genetic variation seen in this study.

Cystic echinococcosis in slaughtered domestic ruminants from Tunisia.

A total of 10,818 domestic ruminants (3913 cattle, 2722 sheep, 3779 goats, 404 dromedaries) slaughtered in various abattoirs in Tunisia between 2003 and 2010 were examined for the presence of Echinococcus granulosus hydatid cysts. The prevalence of cystic echinococcosis (CE) was 16.42% in sheep, 8.56% in cattle, 5.94% in dromedaries and 2.88% in goats. CE prevalence increased with age according to an asymptotic model and there was evidence of variation in infection pressure depending on the region of Tunisia where the animals were slaughtered. Cattle appeared to have the highest infection pressure of the species examined. The mean intensity of hepatic cysts was higher than that of pulmonary cysts in all species. The highest mean intensity of infection with E. granulosus larvae was observed in cattle (18.14) followed by sheep (9.58), goats (2.31) and dromedaries (2.12). The abundance of infection increased in a linear fashion with age in all animal species. Cyst abundance varied with species of animal and district of Tunisia. Cysts from dromedaries were more fertile (44.44%) than those from sheep (30.25%), goats (30.32%) and cattle (0.95%). The viability of the protoscoleces from fertile cysts from cattle (78.45%) was higher than those from sheep (70.71%) and camels (69.57%). The lowest protoscolex viability was recorded for hydatid cysts from goats (20.21%). This epidemiological study confirms the importance of CE in all domestic ruminant species, particularly in sheep, throughout Tunisia and emphasizes the need to interrupt parasite transmission by preventive integrated approaches in a CE control programme.

Cystic echinococcosis in Tunisia: analysis of hydatid cysts that have been surgically removed from patients.

Echinococcosis/hydatidosis caused by Echinococcus granulosus has a widespread distribution in the human population of Tunisia, particularly in the north-west and centre-west of the country. In a recent study, the morphological features, fertility and viability of hydatid cysts that had been excised from patients in Tunis were explored, and the E. granulosus strain or genotype involved in each case was identified from morphology of the protoscolex hooks and the results of molecular genotyping. The hepatic cysts investigated came from 41 patients [31 women and 10 men, with a mean (S.E.) age of 43.41 (14.25) years] who were treated for cystic echinococcosis, by surgery but rarely with chemotherapy, at the La Rabta Hospital in Tunis, in the 12 months ending in June 2008. Most (56%) of these patients originated from rural areas in endemic governorates. Of the 60 hepatic cysts that were studied, 38.3% were located in the right lobe of the liver and 35.0% each involved both hepatic lobes. Almost a third (31.7%) of the excised cysts were degenerating, with the rest considered viable and either multivesicular (38.3%) or univesicular (30.0%). Almost all (93.3%) of the cysts were categorized as fertile, with a mean protoscolex viability of 21.8%. Protoscolex viability was relatively high in the viable univesicular cysts with a visible cyst wall and in the multivesicular and multiseptate cysts with daughter cysts, and lowest in the cysts that appeared to be solid calcified masses. The observed variation in protoscolex viability with cyst type, in cysts excised from patients before any chemotherapy, supports the cyst classification recommended by the World Health Organization but could also be compatible with the imaging-based 'Gharbi' classification. The results of the molecular genotyping showed that all 23 cysts investigated (which came from 20 of the patients) were caused by E. granulosus of the G1 genotype (also known as the 'sheep' or 'sheep-dog' strain).

Use of PCR, IFAT and in vitro culture in the detection of Leishmania infantum infection in dogs and evaluation of the prevalence of canine leishmaniasis in a low endemic area in Tunisia.

The aim of this study was to assess the use of parasitological, serological and molecular methods for the detection of Leishmania infection in blood of 67 dogs and to investigate the prevalence of canine leishmaniasis (CanL) in Kairouan (central Tunisia), an area known to be of reduced endemicity and has not been studied since 1973. Veterinarians clinically examined all dogs, and the titer of anti-Leishmania antibodies was determined by indirect immune-fluorescence antibody test. The presence of Leishmania was performed by PCR and in vitro culture. IFAT was positive in 12% of dogs and promastigote form of the parasite was isolated by in vitro culture from only 4.5% of them. However, DNA of Leishmania was detected by PCR in 20.9% of dogs. PCR was more sensitive than IFAT (p = 0.004) and in vitro culture (p < 10(-5)). A prevalence of 21% was found in Kairouan, which is significant high (p < 10(-3)) when compared to that of thirty years ago. This state is in correlation with the increase in other Mediterranean countries. Furthermore, 50% of positive dogs were asymptomatic. Preventive measures must be taken against these dogs as for symptomatic ones since their role in the transmission of the infection to vectors has been proven.

Ultrasonographic screening for cystic echinococcosis in sheep in Tunisia.

Sheep from the areas of Fondouk-Jeddid, Bir Mchergua and El Fahs, located in the Northeast of Tunisia, were examined by ultrasonography between 2001 and 2004 in order to assess their infection with Echinococcus granulosus, the agent of hydatid disease, and to evaluate this method as an efficient aire for hydatid cysts. A total of 1039 sheep, aged between 1 and 14 years was examined. The highest prevalence was found in sheep aged more than 8 years. The least infected animals were aged between 1 and 2 years. All hydatid cysts detected by ultrasound were located in the liver. In all age-groups, the dead cysts were more numerous than viable cysts. Eighteen positive sheep were autopsied and a comparison between ultrasound and autopsy results was performed. The results showed a prevalence of about 40% for the three areas. Ultrasonography allowed the cysts, deep or superficial to localize in the central or left part in relation to the caudal vena cava of the animals. Consequently, all the cysts were not detected with this technique. This work shows that ultrasonography confirms the importance of ovine hydatid cyst in Tunisia and that its use as a mass screening approach for cystic echinococcosis in sheep could be helpful for the monitoring of this disease in a hydatid control program without great stress for the animals.

Global Distribution of Alveolar and Cystic Echinococcosis.

Alveolar echinococcosis (AE) and cystic echinococcosis (CE) are severe helminthic zoonoses. Echinococcus multilocularis (causative agent of AE) is widely distributed in the northern hemisphere where it is typically maintained in a wild animal cycle including canids as definitive hosts and rodents as intermediate hosts. The species Echinococcus granulosus, Echinococcus ortleppi, Echinococcus canadensis and Echinococcus intermedius are the causative agents of CE with a worldwide distribution and a highly variable human disease burden in the different endemic areas depending upon human behavioural risk factors, the diversity and ecology of animal host assemblages and the genetic diversity within Echinococcus species which differ in their zoonotic potential and pathogenicity. Both AE and CE are regarded as neglected zoonoses, with a higher overall burden of disease for CE due to its global distribution and high regional prevalence, but a higher pathogenicity and case fatality rate for AE, especially in Asia. Over the past two decades, numerous studies have addressed the epidemiology and distribution of these Echinococcus species worldwide, resulting in better-defined boundaries of the endemic areas. This chapter presents the global distribution of Echinococcus species and human AE and CE in maps and summarizes the global data on host assemblages, transmission, prevalence in animal definitive hosts, incidence in people and molecular epidemiology.

Genetic differences between Tunisian camel and sheep strains of the cestode Echinococcus granulosus revealed by SSCP.

Ovine and dromedary Echinococcus granulosus isolates from Tunisia were identified as G1 and G6 strains based on polymorphism of the mitochondrial cytochrome C oxydase CO1. Single strand conformation polymorphism (SSCP) was used in order to examine the genetic variation within and between Tunisian G1 and G6 strains and to estimate the extent of selfing. The dromedary isolates are genetically distinct from sheep isolates (high value of genetic variation between populations: Fst= 0.46). No significant deficiency in heterozygotes was found in sheep isolates, whereas heterozygote deficiency (suggesting selfing) was found in a limited number of camel isolates.

Cystic hydatic disease in sheep: treatment with percutaneous aspiration and injection with dipeptide methyl ester.

An in vitro and in vivo study was conducted to show the effect of dipeptide methyl ester on the protoscolices of Echinococcus granulosus and in naturally infected sheep. Easily punctured cysts were located by ultrasonography. A PAIR and PAI method were performed by the injection of dipeptide methyl ester into these cysts at a final concentration of 110 mmol/L. Follow-up was conducted monthly by ultrasonography. After injection of the compound, the sheep were sacrificed at different times from 6 to 17 weeks. The size and the morphological aspect of treated cysts were noted. Samples were collected for histology and electron microscopy. In conclusion, these studies revealed significant and rapid detachment of the membrane of the treated cyst and alteration of the inner membrane in less than 5 min after injection of the drug, confirming the effect of the compound on the laminated layer of the parasite.

Molecular evidence of ovine (G1) and camel (G6) strains of Echinococcus granulosus in Tunisia and putative role of cattle in human contamination.

Three hundred and seventy-two cysts coming from 50 humans, 166 cattle, 153 sheep and 3 camels were collected in order to establish some epidemiological molecular information in Tunisia for the first time. The analysis by PCR-RFLP of ITS1 sequence showed that all the human, ovine and bovine cysts were due to the common sheep strain of Echinococcus granulosus. The sequencing of the CO1 gene of 37 isolates confirm the G1 genotype of this strain. For seven of these isolates, we found the mutation C56T which is present in the three principal intermediate hosts: human (three cysts), cattle (three cysts) and sheep (one cyst). With regard to the G1 genotype, we identified three other point mutations. The camel strain G6 is uniquely found in the three camels isolates and not in the other intermediate hosts analysed. The fertility of the bovine cyst represents 48% that means that this host is involved in a bovine-dog cycle and consequently represents a reservoir of sheep strain in Tunisia. Our results confirm the importance of the prophylaxis measures in order to disrupt the cycle of transmission sheep-dog in Tunisia. Nevertheless, the supervision of bovine infection should be reinforced because this intermediate host may constitute an important link with the human contamination.

Land snails and periods at high risk for protostrongylid infection on a sheep-grazed pasture of northeast Tunisia.

The population density and level of infection of terrestrial molluscs, and the output by sheep of first stage protostrongylid larvae, were assessed monthly during a 1-year study. Muellerius capillaris was the predominant protostrongylid (approximately 80% of the larval output); Neostrongylus was also found. Eobania vermiculata, Cernuella virgata and Trochoidea elegans were the most common molluscs. Juveniles were less infected than adults. Banded E. vermiculata had a lower intensity of infection than unbanded ones. Nearly 75% of protostrongylid larvae harboured by molluscs on the studied pasture were found in E. vermiculata. The infectivity of pasture was at its lowest in the dry and hot period (June-September). Infection of snails was primarily related to temperature and the importance and frequency of rains, and secondarily to larval excretion in sheep faeces.

Transmission dynamics of the Echinococcus granulosus sheep-dog strain (G1 genotype) in camels in Tunisia.

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.

Echinococcus in the wild carnivores and stray dogs of northern Tunisia: the results of a pilot survey.

Echinococcus granulosus is endemic throughout Tunisia and E. multilocularis has previously been reported as the cause of two cases of human alveolar echinococcosis in the north-west of the country. The aim of the present study was to screen wild carnivores from the north-western Jendouba governorate and semi-stray dogs from the Siliana and Sejnane regions of northern Tunisia for these two zoonotic cestodes. The results of the coproscopy, coproELISA and coproPCR that were undertaken were compared with those of necropsy, where possible. Overall, 111 faecal samples (51 from wild carnivores and 60 from stray dogs) were tested by coproELISA for Echinococcus antigen and by coproPCR for E. granulosus and E. multilocularis species-specific DNA. All 60 dogs and seven of the wild carnivores were necropsied. Eleven (18.4%) of the dogs and one golden jackal (Canis aureus) were found positive for E. granulosus at necropsy. The jackal was found to be carrying 72 E. granulosus tapeworms, which were confirmed to be of the common sheep-dog (G1) genotype. Faecal samples from 10 (19.6%) of the wild carnivores--putatively, four golden jackals, two red foxes (Vulpes vulpes atlantica), one hyaena (Hyaena hyaena) and three genets (Genetta genetta)--gave a positive result in the Echinococcus coproELISA. In the coproPCR-based follow-up, E. granulosus DNA was detected in faecal samples from five jackals, two foxes and six stray dogs. The DNA of E. multilocularis was not, however, detected in any of the faecal samples investigated. This is the first report from Tunisia of (coproPCR-)confirmed E. granulosus infections in golden jackals and red foxes. The possible role of such wild hosts in the transmission of E. granulosus in Tunisia should be investigated further. The possibility of the active transmission of E. multilocularis in Tunisia still remains an open question.

Frequency distributions of Echinococcus granulosus and other helminths in stray dogs in Tunisia.

The intestinal helminths infecting stray dogs were investigated in the Testour and Bouzid regions of Tunisia. Overall, 21% of the dogs necropsied were found to be infected with Echinococcus granulosus. The prevalence among the animals from Testour (27.0%) was significantly higher than that in the dogs from Bouzid (6.9%). Although the prevalence of this species in the dog populations did give a significant fit to the age-prevalence models reported earlier, the best fit was not consistent with the known biological parameters of the parasite. The frequency distribution of E. granulosus was highly aggregated, with a mean abundance of infection of 538 parasites/host and a mean intensity in infected animals of 2534 parasites/host. Several Taenia species were also found in the dogs, but none of these showed evidence of an aggregated distribution and the frequency distribution of each fitted a Poisson distribution, which is unusual for naturally acquired infections. All the other cestode species and the nematode species encountered had aggregated distributions in the dogs. As there was relatively little correlation between the worm burdens of the different parasite species in the dogs, individual dogs may not be generally susceptible or resistant to intestinal helminths of several species.

Echinococcus granulosus larvae in the livers of sheep in Tunisia: the effects of host age.

A baseline survey of hepatic hydatid disease in sheep in Tunisia was undertaken. Prevalence and intensity (i.e. the number of cysts/liver) were both found to increase with age. The age-specific trends in these two parameters indicate that Echinococcus granulosus is in an endemic equilibrium steady-state in Tunisia and therefore amenable to control. The distribution of parasites within the livers of the hosts followed a negative binomial distribution. The negative binomial constant, k, increased with age, indicating that there was a lower propensity for aggregation of parasites in the hosts as the sheep became older.