Observations on the development of Echinococcus multilocularis in cats

The development of a European isolate of Echinococcus multilocularis was compared in cats and dogs at the end of the prepatent period. Echinococcus multilocularis established in all dogs and cats, but worm recovery was significantly greater from dogs than from cats. Overall, worms in cats were not as advanced as those in dogs in terms of development and maturation, but there was no evidence of retarded development or stunted forms. These results confirm that dogs are highly susceptible to E. multilocularis, whereas cats have lower and more variable recovery rates. However, because cats produce thick-shelled eggs of E. multilocularis after experimental and natural infections, they have to be regarded as potential sources of infection both for intermediate and accidental hosts, including humans. However, their general role in the epidemiology of the infection has yet to be determined.     

Efficacy of Droncit Spot-on (praziquantel) 4% w/v against immature and mature Echinococcus multilocularis in cats

The causative agent of alveolar hydatidosis in humans, the fox tapeworm Echinococcus multilocularis, is extending its geographical range in Europe and has been found in domestic cats in some areas. A dermally applied cestocidal treatment for domestic cats has been developed and the efficacy of this treatment is reported. Thirty purpose-bred cats were experimentally infected each with 10000 protoscoleces of Echinococcus multilocularis. Ten days later one group of ten cats was treated with Droncit(R) Spot-on (Praziquantel) 4% w/v dermally in one place on the dorsal aspect of the neck at a dose of 8 mg/kg. Eleven days later (21 days p.i.) a second group of ten cats was also treated with Droncit(R) Spot-on the same way. One group of ten cats was left untreated as controls. Twenty three days after infection the cats were examined for the presence of E. multilocularis tapeworms. No E. multilocularis were recovered from any of the cats in either of the treated groups. Echinococcus multilocularis were recovered from eight of the ten cats left untreated as controls. The worm burdens in the untreated cats were 0, 0, 5, 15, 75, 110, 220, 815, 2635, and 3045 worms per cat. The worms ranged in development from the three to four segment stage. Many of the E. multilocularis with four segments contained unshelled eggs in the terminal segment. This study indicates that Droncit(R) Spot-on (Praziquantel) 4% w/v applied dermally at 8 mg/kg is highly effective in removing E. multilocularis from the small intestine of cats infected with immature and mature (prepatent) infections of E. multilocularis. In the cats with the mature infections all tapeworms were absent from the small intestine within 2 days of treatment.     

Meriones meridianus and Lagurus lagurus as alternative definitive hosts of Echinococcus multilocularis and E. granulosus.

The utilization of Meriones meridianus and Lagurus lagurus as alternative definitive hosts for Echinococcus multilocularis and E. granulosus was investingated. Tapeworm stage development of E. multilocularis was observed and their recovery rate was determined in the small intestine of M. meridianus and L. lagurus. These were compared with those in golden hamsters, which are known as alternative definitive hosts. The animals were treated with PTBA (prednisolone tertiary butylacetate) and PA (prednisolone acetate), after which M. meridianus showed the highest recovery rate, whereas L. lagurus had few or no worms. The recovery rate of worms from golden hamsters was between those of M. meridianus and L. lagurus. On day 20 post-infection, developing tapeworms with mature segments were collected from M. meridianus treated with PA. The worms were mostly from the proximal and medial small intestine. The three species of animals infected orally with E. granulosus were divided into two groups, PTBA-treated and non-treated control groups. PTBA promoted/enhanced the recovery rate of the worms until 5 days, but none or only a few worms were found in PTBA treated animals thereafter. The highest recovery rate was obtained from M. meridianus treated with PTBA on day 5 post-infection. Some worm developments were observed on days 5 and 10 post-infection. These results demonstrate that M. meridianus could be useful as an alternative definitive host of Echinococcus.     

Echinococcus multilocularis coproantigen detection by enzyme-linked immunosorbent assay in fox, dog, and cat populations

A sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Echinococcus multilocularis coproantigens (EM-ELISA) was developed with polyclonal rabbit (solid phase) and chicken egg (catching) antibodies that were directed against E. multilocularis coproantigens and somatic worm antigens, respectively. In experimentally infected dogs and cats, coproantigens were first detectable 6-17 days postinfection (PI) in samples of 8 dogs (worm burdens at necropsy: 6,330-43,200) and from 11 days PI onward in samples of 5 cats infected with 20-6,833 worms. After anthelmintic treatment of 4 dogs and 5 cats at day 20 PI, coproantigen excretion disappeared within 3-5 days. The sensitivity of the ELISA was 83.6% in 55 foxes infected with 4-60,000 E. multilocularis, but reached 93.3% in the 45 foxes harboring more than 20 worms. The EM-ELISA was used in surveys of "normal" dog and cat populations in Switzerland. Among 660 dogs and 263 cats, 5 dogs and 2 cats exhibited a positive reaction. In 2 of these dogs (0.30%) and 1 cat (0.38%), intestinal E. multilocularis infections were confirmed by necropsy, polymerase chain reaction PCR, or both. The specificites of the ELISA in these groups were found to be 99.5% and 99.6%, respectively, if positive ELISA results that could not be confirmed by other methods were classified as "false positive" reactions.     

Canine echinococcosis in Kyrgyzstan: using prevalence data adjusted for measurement error to develop transmission dynamics models

Echinococcosis is a major emerging zoonosis in central Asia. A cross-sectional study of dogs in four villages in rural Kyrgyzstan was undertaken to investigate the epidemiology and transmission of Echinococcus spp. A total of 466 dogs were examined by arecoline purgation for the presence of Echinococcus granulosus and E. multilocularis. In addition, a faecal sample from each dog was examined for taeniid eggs. Any taeniid eggs found were investigated using PCR techniques (multiplex and single target PCR) to improve the diagnostic sensitivity by confirming the presence of Echinococcus spp. and to identify E. granulosus strains. A total of 83 (18%) dogs had either E. granulosus adults in purge material and/or E. granulosus eggs in their faeces as confirmed by PCR. Three genotypes of E. granulosus: G1, G4 and the G6/7 complex were shown to be present in these dogs through subsequent sequence analysis. Purge analysis combined with PCR identified 50 dogs that were infected with adult E. multilocularis and/or had E. multilocularis eggs in their faeces (11%). Bayesian techniques were employed to estimate the true prevalence, the diagnostic sensitivity and specificity of the procedures used and the transmission parameters. The sensitivity of arecoline purgation for the detection of echinococcosis in dogs was rather low, with a value of 38% (credible intervals (CIs) 27-50%) for E. granulosus and 21% (CIs 11-34%) for E. multilocularis. The specificity of arecoline purgation was assumed to be 100%. The sensitivity of coproscopy followed by PCR of the isolated eggs was calculated as 78% (CIs 57-87%) for E. granulosus and 50% (CIs 29-72%) for E. multilocularis with specificity of 93% (CIs 88-96%) and 100% (CIs 97-100%), respectively. The 93% specificity of the coprological-PCR for E. granulosus could suggest coprophagia rather than true infections. After adjusting for the sensitivity of the diagnostic procedures, the estimated true prevalence of infection of E. granulosus was 19% (CIs 15-25%) and the infection pressure in the dog population was estimated to be 0.29 infections per year (CIs 0.014-0.75). Logistic regression analysis failed to identify any significant risk factors for infections for E. granulosus. After adjusting for the sensitivity of the test procedures, the estimated true prevalence for E. multilocularis was 18% (CIs 12-30%). Dogs that were restrained had a significantly lower prevalence of E. multilocularis of 11% (CIs 6-29%) compared with 26% in free-roaming dogs (CIs 17-44%) and independently within these groups hunting dogs were more likely to be infected than non-hunting dogs.     

Reproductive potential of Echinococcus multilocularis in experimentally infected foxes, dogs, raccoon dogs and cats

A total of 15 red foxes, 15 raccoon dogs, 15 domestic dogs and 15 domestic cats were each infected with 20,000 protoscolices of Echinococcus multilocularis. At 35, 63, and 90 days post inoculation (dpi), five animals from each group were necropsied and the worm burdens determined. The highest worm burdens in foxes (mean of 16,792) and raccoon dogs (mean of 7930) were found at 35 dpi. These declined to a mean of just 331 worms in foxes and 3213 worms in raccoon dogs by day 63 with a further decline to 134 worms in foxes and 67 worms in raccoon dogs by day 90. In dogs, there was no significant difference between worm burdens recovered at days 35 (mean of 2466) and day 90 (mean of 1563), although reduced numbers were recovered on day 63 (mean of 899). In cats, worms were found in four animals 35 dpi (mean of 642), in three at 63 dpi (mean of 28) and in two at 90 dpi (mean of 57). Faecal egg counts were determined at 3 day intervals from 25 dpi. A mathematical model of egg excretion dynamics suggested that the mean biotic potential per infected animal was high in foxes (346,473 eggs); raccoon dogs (335,361 eggs) and dogs (279,910 eggs) but very low for cats (573 eggs). It also indicated that approximately 114, 42 and 27 eggs per worm were excreted in the faeces of dogs, raccoon dogs and foxes, respectively. The fecundity of worms in cats was low with an average of less than one egg per worm. The peak levels of coproantigen were detected earlier in foxes and raccoon dogs than in dogs. Eggs recovered from foxes, raccoon dogs and dogs resulted in massive infections in experimental mice. However, metacestodes did not develop from eggs originating from infected cats. It is concluded that foxes, raccoon dogs and dogs are good hosts of E. multilocularis. In contrast, the low worm establishment, the very few excreted eggs and the lack of infectivity of eggs strongly indicate that cats play an insignificant role in parasite transmission.     

Modeling the transmission of Echinococcus granulosus and Echinococcus multilocularis in dogs for a high endemic region of the Tibetan plateau

Echinococcus granulosus and Echinococcus multilocularis abundance and prevalence data, for domestic dogs of Shiqu County, Sichuan Province, People's Republic of China, were fitted to mathematical models to evaluate transmission parameters. Abundance models, assuming the presence and absence of immunity, were fit for both E. granulosus and E. multilocularis using Bayesian priors, maximum likelihood, and Monte Carlo sampling techniques. When the models were compared, using the likelihood ratio test for nested models, the model assuming the presence of immunity was the best fit for E. granulosus infection, with a purgation based prevalence of 8% (true prevalence interval of 8-19% based on the sensitivity of purgation) and a mean abundance of 80 parasites per dog, with an average infection pressure of 560 parasites per year. In contrast, the model assuming the absence of immunity was the best fit for E. multilocularis infection, with a purgation based prevalence of 12% (true prevalence interval of 13-33% based on the sensitivity of purgation) and a mean abundance of 131 parasites per dog, with an average infection pressure of 334 or 533 parasites per year assuming a 5 or 3 month parasite life expectancy, respectively. The prevalence data for both parasites was then fit to a set of differential equations modeling the transition between infection states in order to determine number of infectious insults per year. Infection pressure was 0.21, with a 95% credibility interval of 0.12 to 0.41, infections per year for E. granulosus and 0.52, with a 95% credibility interval of 0.29-0.77, infections per year for E. multilocularis assuming a 5 month parasite lifespan or 0.85, with a 95% credibility interval of 0.47-1.25 infections per year, assuming a 3 month E. multilocularis lifespan in dogs.     

Milbemycin oxime in a new formulation, combined with praziquantel, does not reduce the efficacy of praziquantel against Echinococcus multilocularis in cats

Twenty European domestic cats were each infected with 15,000 protoscoleces of Echinococcus multilocularis extracted from metacestodes grown in experimentally infected common voles (Microtus arvalis). Sixteen days after infection, ten cats were treated with a broad-spectrum anthelmintic and acaricide comprising praziquantel and milbemycin oxime. Five days later treated and untreated cats were euthanized and the intestine examined for E. multilocularis. Five of ten untreated cats were infected with E. multilocularis with worm burdens ranging from 235 to 1920 worms per cat. No E. multilocularis were recovered from any of the treated cats. This study has demonstrated that this new combination broad spectrum anthelmintic and acaricide for cats is highly efficacious against E. multilocularis and the relevance of this is discussed.     

Erroneous reports of the neotropical Echinococcus oligarthrus as a cause of echinococcosis in India

Two cases of submandibular echinococcosis have been reported in adult residents of India (Kini et al., 1997; Sahni et al., 2000). It was stated in both publications that the cestode was E. oligarthrus, "a rare variety of E. granulosus", based on morphogical characteristics. It was also mentioned that 3 cases of E. granulosus with similar submandibular metacestode had been previously diagnosed in India. The metacestode is readily distinguishable from that of E. oligarthrus. The final hosts of E. oligarthrus are several neotropical species of wild cats and metacestode occurs in hystricognath, all of them absent in India. The larval stage of all 4 species of Echinococcus can be differentiated on the basis of the form and dimensions of rostellar hooks of protoescoleces. With the information provided in the two papers we concluded that the 2 cases of echinococcosis described in India were due to E. granulosus.     

An oral recombinant vaccine in dogs against Echinococcus granulosus, the causative agent of human hydatid disease: a pilot study

Dogs are the main source of human cystic echinococcosis. An oral vaccine would be an important contribution to control programs in endemic countries. We conducted two parallel experimental trials in Morocco and Tunisia of a new oral vaccine candidate against Echinococcus granulosus in 28 dogs. The vaccine was prepared using two recombinant proteins from adult worms, a tropomyosin (EgTrp) and a fibrillar protein similar to paramyosin (EgA31), cloned and expressed in a live attenuated strain of Salmonella enterica serovar typhimurium.In each country, five dogs were vaccinated with the associated EgA31 and EgTrp; three dogs received only the vector Salmonella; and six dogs were used as different controls. The vaccinated dogs received two oral doses of the vaccine 21 d apart, and were challenged 20 d later with 75,000 living protoscoleces. The controls were challenged under the same conditions. All dogs were sacrificed 26-29 d postchallenge, before the appearance of eggs, for safety reasons.We studied the histological responses to both the vaccine and control at the level of the duodenum, the natural localization of the cestode. Here we show a significant decrease of parasite burden in vaccinated dogs (70% to 80%) and a slower development rate in all remaining worms. The Salmonella vaccine EgA31-EgTrp demonstrated a high efficacy against E. granulosus promoting its potential role in reducing transmission to humans and animals.     

Diagnosis of canine echinococcosis: comparison of coproantigen detection with necropsy in stray dogs and red foxes from northern Jordan

The sandwich enzyme linked immunosorbent assay (ELISA) was used as a diagnostic test for Echinococcus granulosus infection by detecting coproantigens in 94 stray dogs Canis familiaris and eight red foxes (Vulpes vulpes) from northern Jordan. The results were analyzed in relation to actual helminth infection as revealed by necropsy. The infection rate of dogs with E. granulosus was 13.8% with a worm load ranging between 3-> 10,000 per infected dog. In contrast, eight of 13 E. granulosus infected dogs were coproantigen positive (overall sensitivity 61.5%). The sensitivity increased to 87.5% and 100% in dogs harboring > 20 and > 100 worms/dog, respectively. The specificity of coproantigen-ELISA was 91%. The greatest cross-reactivity was found in dogs infected with Dipylidium caninum. The positive and negative predictive values for the coproantigen-ELISA test were 50% and 94.2%, respectively. Thus, a coproantigen negative dog is most probably truly negative for E. granulosus. In contrast, a coproantigen positive dog may not be truly positive for E. granulosus, except if it has a high worm burden of > 100 worms/animal.     

ITS-1 ribosomal DNA sequence variants are maintained in different species and strains of Echinococcus

This study investigated sequence heterogeneity in the first internal transcribed spacer (ITS-1) of ribosomal DNA within and among species and strains of Echinococcus. Different ITS-1 sequence variants exist in Echinococcus granulosus and Echinococcus multilocularis, which represent at least four evolutionary lineages: (1) a sheep strain-lineage of E. granulosus, (2) a sister lineage of a cervid and camel E. granulosus ITS-1 variants, (3) a lineage including the ITS-1 variants representing horse, bovine and camel strains of E. granulosus, as well as variants from E. multilocularis, Echinococcus oligarthrus and Echinococcus vogeli and (4) a distinct lineage of ITS-1 variants including E. granulosus strains from sheep and cervid, and E. multilocularis. At least two of the species (E. granulosus and E. multilocularis) were paraphyletic for ITS-1. Divergent ITS-1 variants from these two species shared distinct evolutionary lineages. The sequence data provided evidence that at least two turnover mechanisms, namely slippage and unequal crossing over/transposition, have led to the divergence and maintenance of sequence variants in Echinococcus species and strains.     

Mating behaviour of Echinostoma trivolvis and E. paraensei in concurrent infections in hamsters

Young adults of Echinostoma trivolvis and E. paraensei were recovered from hamsters previously infected with metacercarial cysts. Some worms of each species were exposed for 1 h to 3-H-tyrosine to label sperm and transplanted singly to uninfected hamsters with several unlabelled worms of the same or opposite species or both species. After 5 days, recovered worms were processed for paraffin sectioning and autoradiography. The resulting slides were observed for the location of radioactive sperm in the seminal receptacles of donor (labelled) and recipient (unlabelled) worms. When E. trivolvis was the donor with the recipient E. paraensei, self-insemination took place, but only one interspecies mating occurred out of 72 possible recipient worms. When E. paraensei served as the donor, self-insemination again occurred, but no cross-insemination was observed among the 59 E. trivolvis recipient worms. When single donor worms had a choice of either species of recipient worms, no interspecies mating took place, but both self- and cross-insemination occurred in the normal, unrestricted behaviour found in single species mating studies. Rates of both self- and cross-insemination were higher in concurrent infections of both recipient species than in single species mating studies. After transplant, both species localized in their natural habitat within the small intestine, with 1/3 overlapping in the duodenum, making interspecies mating a possibility. The correlation between mating and electrophoretic studies on the genetic relationship between 37-collar-spined echinostomes is discussed.     

Echinococcus multilocularis: developmental stage-specific expression of Antigen B 8-kDa-subunits

Antigen B (AgB) initially found in hydatid cyst fluid of Echinococcus granulosus is a polymeric lipoprotein of 160 kDa, and is an aggregate of several different but homologous small proteins with approximately 8 kDa. Four genes encoding these 8-kDa-subunits have been identified from E. granulosus. In this study we isolated five genes encoding 8-kDa-subunits of AgB from Echinococcus multilocularis. Sequence comparison of isolated cDNA clones demonstrated that one of these five clones was completely identical to EmAgB8/1 which had been isolated previously by our group, and three of them were 94.5, 90.8, and 91.9% homologous to E. granulosus antigen B 8-kDa subunit genes, EgAgB8/2, EgAgB8/3, and EgAgB8/4, respectively. The remaining clone shared 51-58% homology with the nucleotide sequences of AgB genes. Gene-specific RT-PCR and Western blot analyses revealed that these genes were expressed in a developmentally regulated manner in E. multilocularis vesicles, protoscoleces, and immature adult worms. Possible functions of different expression manners are also discussed.     

Echinococcus multilocularis laminated-layer components and the E14t 14-3-3 recombinant protein decrease NO production by activated rat macrophages in vitro.

Echinococcus multilocularis and Echinococcus granulosus cause alveolar and cystic (unilocular) echinococcosis, respectively, in humans and animals. It is known that these parasites can affect, among other molecules, nitric oxide (NO) production by periparasitic host cells. Nevertheless, detailed dissection of parasite components specifically affecting cell NO production has not been done to date. We compare the effect of E. granulosus and E. multilocularis defined metacestode structural (laminated-layer associated) and metabolic (14-3-3 protein, potentially related with E. multilocularis metacestode tumor-like growth) components on the NO production by rat alveolar macrophages in vitro. Our results showed that none of these antigens could stimulate macrophage NO production in vitro. However, a reversed effect of some Echinococcus antigens on NO in vitro production was found when cells were previously exposed to LPS stimulation. This inhibitory effect was found when E. multilocularis laminated-layer (LL) or cyst wall (CW) soluble components from both species were used. Pre-stimulation of cells with LPS also resulted in a strong, dose-dependent reduction of NO and iNOS mRNA production after incubation of cells with the E14t protein. Thus, the E. multilocularis 14-3-3 protein appears to be one of the components accounting for the suppressive effect of the CW and LL metacestode extracts.     

Detection of Echinococcus granulosus coproantigens in Australian canids with natural or experimental infection

Coproparasitological and purging methods for diagnosing canids infected with the intestinal helminth Echinococcus granulosus, an important zoonotic parasite, are unreliable. Detection of coproantigens in feces of infected dogs by enzyme-linked immunosorbent assay (ELISA) is suitable for detecting patent and prepatent infections with a high degree of sensitivity and specificity. In the present study, natural and experimental infections in domestic and wild Australian canids were investigated using a coproantigen capture ELISA. Experimental infection of dogs with E. granulosus was detected at between 14 and 22 days postinfection (PI), and optical density (OD) values remained high until termination of experiments 35 days PI. After chemotherapy, coproantigen levels in infected dogs dropped rapidly, becoming negative 2-4 days after treatment. In experimentally infected red foxes (Vulpes vulpes), the coproantigen excretion profile was different, with ELISA OD levels peaking 15-17 days PI, then falling to low or undetectable levels by 30 days PI. Coproantigens were detected in the feces of naturally infected Australian wild dogs (dingoes, dingo/domestic dog hybrids) with infection levels ranging between 2 worms and 42,600. Preliminary data on the stability of coproantigen in dog feces exposed to environmental conditions indicated that there was no change in antigenicity over 6 days. The results suggest the coproantigen ELISA could be successfully used to monitor E. granulosus prevalence rates in Australian domestic dogs, foxes, and wild dogs.     

Serological differentiation between Echinococcus granulosus and E. multilocularis infections in man

An enzyme-linked immunosorbent assay (ELISA) was adapted for the serological differential diagnosis of cystic or alveolar echinococcosis in man caused by Echinococcus granulosus or E. multilocularis respectively. By affinity chromatography using rabbit anti hydatid fluid IgG coupled covalently to CNBr-Sepharose 4B a protein fraction (Em 1) containing shared antigens of both parasites could be isolated from an extract of E. multilocularis metacestode tissue. From the same source another antigen fraction (Em 2) with a high degree of specificity for E. multilocularis was prepared by immunosorption. Antigen Em 1 was equally sensitive for the detection of antibodies against E. granulosus and E. multilocularis, whereas antigen fraction Em 2 appeared to be more specific for E. multilocularis. A correct serological differential diagnosis was achieved in 95% of 57 confirmed cases of human cystic or alveolar echinococcosis by the simultaneous use of both antigen fractions in the ELISA and by comparison of their reactivities.     

The dispersion of Echinococcus granulosus in the intestine of dogs

We studied the dispersion of adult Echinococcus granulosus in the intestine of experimentally infected dogs at 2 scales of habitat use. On a coarse scale, worms were found most frequently in the anterior third of the small intestine. On a fine scale, clumps or aggregations, typically of 4-5 worms in an area of 12 mm2, occurred throughout the anterior two-thirds of the intestine. The most likely proximate cause of aggregative behavior is attraction between individual worms. There are at least 2 equally plausible ultimate causes of the behavior: to enhance cross-fertilization and to improve the quality of the environment. Restriction of worms to the anterior small intestine may be a consequence of aggregative behavior on a finer scale or a response to different proximate and ultimate factors.     

Ecology and epidemiology of Echinococcus multilocularis in Europe

Human alveolar echinococcosis (AE), caused by the larval stage of Echinococcus multilocularis has a high mortality rate in untreated patients. The life-cycle of E. multilocularis in Europe predominantly involves foxes as definitive hosts. However, experimental studies demonstrated a comparable biotic potential of E. multilocularis in dogs and raccoon dogs but an insignificant potential in cats. AE occurs in central and eastern Europe at low incidence rates. Recent studies in foxes have shown that E. multilocularis has a wider geographic range (including Italy) than previously thought. In recent years, increases in fox populations have been observed in many European countries, especially in urban areas. As a result, the E. multilocularis cycle is now established in the urban environment. This presents an increased risk of infection for a large human population. Based on these facts and new epidemiological data, possible intervention strategies are presented.     

Echinococcus shiquicus n. sp., a taeniid cestode from Tibetan fox and plateau pika in China

The taeniid cestode Echinococcus shiquicus n. sp. was found from the Tibetan fox Vulpes ferrilata and the plateau pika Ochotona curzoniae in the Qinghai-Tibet plateau region of China. In the adult stage, E. shiquicus from the foxes is morphologically similar to Echinococcus multilocularis. However, the new species is differentiated by its smaller rostellar hooks, fewer segments, distinct position of genital pore in the mature segment and fewer eggs in the gravid segment. Hydatid cysts of E. shiquicus found in the livers from the pikas were essentially unilocular but an oligovesicular cyst was also found. The data of mitochondrial and nuclear DNA sequences proved E. shiquicus to be a valid taxon.