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.     

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.     

Observation of mucosal pathology after praziquantel treatment in experimental Fibricola seoulensis infection in rats

It is well known that the duodenum of mice or rats infected with Fibricola seoulensis shows atrophy of villi (shortening, blunting, widening, fusion) and hyperplasia of crypts. This study was performed to observe healing process of these pathological changes after deworming with anthelmintic treatment. Albino rats infected each with 1,000 metacercariae of F. seoulensis were treated with single dose of 10 mg/kg praziquantel on day 15 post-infection. On day 1, 3, 5, 7, 15, 21 and 28 after the treatment, they were sacrificed and their duodenums were histopathologically studied. Control (uninfected) rats showed their normal finger-like projections of duodenal villi and well arranged crypts. In comparison, untreated (infected) controls revealed severe mucosal changes characteristic of villous atrophy and crypt hyperplasia in their duodenum. The damaged duodenal mucosa was found to restore its normal morphology after praziquantel treatment; until day 3 post-treatment the mucosa was severely atrophied; on day 5 long and slender villi sometimes appeared among the fused and stout ones; after day 15 the villi were in their normalizing process. From this experiment, it was shown that the mucosal changes in the duodenum of rats caused by F. seoulensis infection were completely reversible in 21-28 days after anthelmintic treatment.     

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.     

Alveolar echinococcosis in humans: the current situation in Central Europe and the need for countermeasures.

Alveolar echinococcosis (AE) in humans is caused by a larval stage (metacestode) of Echinococcus multilocularis, which exhibits a tumor-like growth, initially in the liver, with the potential to induce serious disease. At the end of the 1980s, E. multilocularis was known to occur in four countries of Central Europe, but has now been identified in ten countries. Red foxes are the principal definitive hosts of E. multilocularis and sources of human infection, but dogs and cats can also be infected. Growing populations of foxes and their increasing immigration to urban areas are new risk factors. Human AE is rare but its potential high fatality rate, considerable costs of treatment and the persisting infection risk should be reasons for health authorities in European countries to establish coordinated systems of surveillance and risk assessment in combination with measures to reduce morbidity and mortality of AE in the human population. Here, J. Eckert and P. Deplazes outline the current epidemiological situation in Central Europe, and discuss options for surveillance, prevention and control.     

Observations on Echinococcus multilocularis in the definitive host

Six dogs were found to be susceptible to experimental infections with a European isolate of Echinococcus multilocularis from southern Germany. Two cats were only poorly susceptible. Adult worms were not evenly distributed throughout the small intestine and the majority of parasites were found in the posterior region. The mode of attachment of E. multilocularis in the dog was similar to that for E. granulosus with the adult worm extending its rostellum deep within a crypt of Lieberkühn. In cats only few worms were found to have penetrated deeply between the villi. E. multilocularis was found to possess a modified group of rostellar tegumental cells, morphologically and functionally identical to those described in E. granulosus and previously referred to as the "rostellar gland". By studying development in vivo and in vitro, the time required for the production of shelled eggs was demonstrated to be only 28 days. Concurrent experimental infections in dogs with E. multilocularis and E. granulosus revealed that both species will develop together in the same host. Their development was not retarded in any way by the presence of the other and both species were able to coexist in the same area of the intestine.     

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.     

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.     

New distribution records of Echinococcus multilocularis in the brown lemming from Barrow, Alaska, USA

We identified Echinococcus multilocularis for the first time in brown lemmings (Lemmus trimucronatus) from Barrow, Alaska, USA. Of 467 brown lemmings trapped between 1995 and 2000, two males and two females (0.9%; 95% confidence interval=0.9+/-0.9%) were found to be infected with metacestodes of E. multilocularis. No metacestodes were found in 17 collared lemmings (Dicrostonyx rubricatus) also trapped at Barrow. In humans, E. multilocularis causes alveolar echinococcosis, which is potentially fatal. Knowledge of the distribution of this parasite is important to protect the public health.     

Licking behaviour induces partial anthelmintic efficacy of ivermectin pour-on formulation in untreated cattle

Licking behaviour in cattle has been reported to account for the disposition of topically administered macrocyclic lactones. However, its impact on anthelmintic efficacy remains to be established. Therefore, we evaluated the impact of ivermectin exchange between cattle on the reduction in the faecal egg count (FEC) after pour-on administration in a group of 10 heifers experimentally infected with Ostertagia ostertagi and Cooperia oncophora. Four treated (500 μg/kg, pour-on) and six untreated animals were put together after treatment and plasma and faecal exposure to ivermectin as well as the FECs were evaluated before and over 40 days after treatment. Ivermectin was detected in plasma and faeces of the six untreated heifers, with maximal exposures two- to three-fold lower than the minimal exposures in treated animals. The interindividual variability of exposure was very high in untreated animals, with a ten-fold difference between the upper and lower limits compared with treated heifers, where there was only a two-fold difference. Anthelmintic efficacy, expressed as an average reduction of the FECs over the experimental period, was maximal in the treated group. In untreated heifers, anthelmintic efficacies ranged from zero to maximal efficacy, with intermediary values between 30% and 80%. The use of a classical pharmacodynamic model demonstrated a clear relationship between exposure and efficacy and enabled us to define the critical plasma or faecal ivermectin concentrations delimiting an exposure window associated with partial anthelmintic efficacy. This range of ivermectin plasma concentrations (0.1-1 ng/mL) could be considered as a potential selection window for anthelmintic resistance. Finally, our results show that macrocyclic lactone exchange between cattle after pour-on administration, resulting from natural grooming behaviour, can significantly impact on anthelmintic efficacy. This raises several issues such as the design of comparative clinical trials and the occurrence of partial efficacy which is considered a risk factor for the development of anthelmintic resistance.     

Oxamniquine, praziquantel and lovastatin association in the experimental Schistosomiasis mansoni

The activity of lovastatin associated with oxamniquine or praziquantel against schistosomiasis mansoni was evaluated in mice infected with Schistosoma mansoni. Forty days after infection, mice were treated with lovastatin, 400 mg/kg for five consecutive days by oral route, and on the last day of this sequence with 50 mg/kg oxamniquine or with 200 mg/kg praziquantel, both by oral route, single dose. Fifteen days later, the animals were perfused in parallel with an untreated control group. Studies were carried out in vitro, using lovastatin in culture medium containing S. mansoni worms proceeding from experimentally infected mice. In the in vivo trials, the association of lovastatin with oxamniquine or praziquantel did not show any additive action, but there were oogram changes when lovastatin was associated with oxamniquine. In vitro lovastatin was able to interrupt the maturation of S. mansoni eggs, which remained at the 1st or 2nd stages, depending on the dose used. The total number of morphologically dead eggs found in culture of worms exposed to 2 microg/ml or 4 microg/ml concentrations of lovastatin was significantly higher than the number of viable eggs. Using the probe Hoescht 33258 it was observed that 70% of the eggs considered morphologically viable in the treated groups (against 16% in the control group) were labeled, indicating that the majority of the viable eggs had membrane permeability increased due to lovastatin action.     

Ancylostoma caninum: Adult worm removal,corticosteroid treatment,and resumed development of arrested larvae in dogs

Beagles, 2 months old and helminth naive, were infected with chilled arrest-prone larvae of Ancylostoma caninum. Eighteen days after infection, the pups were treated with an adulticidal anthelmintic (disophenol or dichlorvos) to remove adult worms while allowing dormant larvae to survive. Examinations of treated pups at necropsy demonstrated that the remaining hypobiotic larvae could develop and mature in the same dogs within which their development was arrested. Removal of adult worms was not a stimulus for resumption of larval development. Indeed, larvae resumed development in untreated control dogs harboring substantial populations of adult worms. Prednisolone treatment of dogs, although apparently producing some degree of immunodepression as judged by lymphocyte transformation assays, did not release larvae from dormancy. In fact, the dogs treated with the corticosteroid harbored significantly greater populations of hypobiotic larvae at 100 days after infection than did their untreated controls. Some hypobiotic larvae appeared to resume development spontaneously and idiosyncratically during the 2 to 3 month duration of these experiments. Whether a synchronous resumption of development would occur given other stimuli or spontaneously after a longer period of dormancy remains to be determined.     

Use of a Taqman PCR to determine the response of Mycoplasma haemofelis infection to antibiotic treatment

A quantitative Taqman polymerase chain reaction (PCR) assay was used to evaluate the response of Mycoplasma haemofelis experimentally infected cats to three antibiotic treatment regimes. Sixteen cats were intravenously inoculated with M. haemofelis from a chronically infected donor. The cats were randomly assigned to one of four treatment groups each containing four cats: oral doxycycline at 10 mg/kg/day for 14 days, oral enrofloxacin at 5 mg/kg/day for 14 days, oral enrofloxacin at 10 mg/kg/day for 14 days, and an untreated control group. DNA, extracted from blood samples collected on days 0, 7, 14, 21, 25, 28, 32, 35, 42 and 54 post-inoculation (PI), was subjected to quantitative Taqman PCR. The M. haemofelis copy number was significantly lower in the doxycycline group (P=0.008), the 5 mg/kg/day enrofloxacin group (P=0.006) and the 10 mg/kg/day enrofloxacin group (P=0.005) compared to the untreated control group. No significant differences were found between any of the three antibiotic treated treatment groups. All three antibiotic treatment regimes evaluated in this study were effective at reducing M. haemofelis copy number.     

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.     

Natural Echinococcus multilocularis infection in a Norway rat, Rattus norvegicus, in southern Hokkaido, Japan.

Forty-two rats, Rattus norvegicus, captured at a garbage dump in southern Hokkaido, Japan, were examined, and one was found to be infected with Echinococcus multilocularis. The lesions were found in the liver, lung, mesenteric lymph nodes, greater omentum and also free in the abdominal cavity. No necrosis was observed in any of the lesions, and inflammatory reactions were mild. Protoscoleces were observed in the large liver cysts. A homogenate of these cysts, when transplanted into the abdominal cavity of three Mongolian gerbils and a rat, yielded numerous fully developed protoscoleces at 4-7 months post-inoculation. Judging from this, it is postulated that the rat could become a natural intermediate host for E. multilocularis in this area.     

Effects of cyclosporin A on the course of murine alveolar echinococcosis and on specific cellular and humoral immune responses against Echinococcus multilocularis.

The effects of cyclosporin A (CsA) on Echinococcus multilocularis (E. multilocularis) metacestode growth, and on the specific immune responses of the hosts, were examined in AKR mice. Mice were intra-peritoneally infected with a metacestode homogenate. CsA (40 mg kg(-1) day(-1)) was injected subcutaneously from the 45th day after infection (Group 1), and from the day before infection (Group 2) until the day of autopsy (days 125 and 80, respectively). Results showed that unlike ths situation with some other helminthiases, CsA had no antiparasitic effect, although it lengthened the maturation time of protoscoleces in Group 1. The parasitic burden, unmodified in Group 1, was significantly enhanced in Group 2. This enhancement was associated with a decrease in antibody levels, whereas the delayed-type hypersensitivity was decreased in the two groups. These results confirm the role of cellular immunity in controlling the first stages of the larval development of E. multilocularis and indicate the necessity for a careful follow-up of any recurrence of alveolar echinococcosis in patients treated with CsA after liver transplantation.     

Complete life cycle of the canid tapeworm, Echinococcus multilocularis, in laboratory rodents

Mongolian gerbils, Meriones unguiculatus, when treated at intervals of 2-6 days with prednisolone tertiary butyl acetate, sustained infection with adult Echinococcus multilocularis in the small intestine, with the tapeworm exhibiting normal strobilation and egg production as in the natural canid host. Host age is critical for the survival of the tapeworm in normal gerbils; parasites survive for only 2 days in 20-wk-old animals, 4 days in 4-wk-old animals, but at least 7 days in 3-wk-old animals. The host age dependence in parasite recovery between days 28-37 postinfection was not affected by treatment from around the day of infection. Starting the treatment before infection (on day -17 relative to infection) remarkably improved the tapeworm's survival within the intestine of older animals. Eggs produced in this rodent model system 28 days postinfection were infective to rodents such as Mongolian gerbils and gray red-backed voles, Clethrionomys rufocanus bedfordiae, by oral or intraperitoneal inoculation. The E. multilocularis/Mongolian gerbil system can replace the natural canid hosts as a new way to obtain infective eggs and to analyze host-parasite interactions. The development of an alternative definitive host for zoonotic tapeworms may accelerate experimentation in this field.     

Failure to identify alveolar echinococcosis in trappers from South Dakota in spite of high prevalence of Echinococcus multilocularis in wild canids

Echinococcus multilocularis causes a rare but potentially lethal zoonotic disease in humans. This tapeworm has been known to be endemic in foxes (Vulpes vulpes) and coyotes (Canis latrans) within the northern United States since the 1960s. One purpose of this study was to provide recent data on the prevalence of E. multilocularis in foxes and coyotes from eastern South Dakota. In a survey conducted from 1987 to 1991 and involving 137 foxes and 9 coyotes from this area, 74.5% of the foxes and 4 of the coyotes were infected. To assess the possible prevalence of alveolar echinococcosis in a group at presumptive high risk, we also conducted a serological survey of members of the South Dakota Trappers Association in 1990 and 1991. Serum samples from 115 trappers were evaluated for the presence of E. multilocularis antibodies using enzyme-linked immunosorbent assay tests involving a purified antigen called Em2, a crude E. multilocularis antigen, and a recombinant E. multilocularis antigen called II/3-10. None of the trappers showed antibody evidence for the presence of E. multilocularis. Roughly half of the surveyed individuals had trapped more than 50 foxes during their life, and almost one-fourth had trapped more than 1,000 foxes.     

Humoral and cellular response to infection with Echinostoma revolutum in the golden hamster, Mesocricetus auratus

Laboratory hamsters (Mesocricetus auratus) were infected with Echinostoma revolutum (Trematoda). Immunoelectrophoretic studies of hamster serum showed no demonstrable antibody response to E. revolutum. Histopathologic examination of intestinal tissue of infected hamsters showed erosion of intestinal villi and lymphocytic infiltration as the primary host response. Spleens from infected hamsters were hyperplastic during the first 3 weeks of infection and atrophic from 4 to 8 weeks postinfection. Hamsters were unable to acquire a resistance to E. revolutum infection. Lack of resistance was demonstrated in hamsters where the parasite infection was no longer detected based on the absence of eggs in the faeces; these hamsters were then reinfected. Hamsters treated with the anthelmintic oxyclozanide were also reinfected with E. revolutum.     

Recovery from immunesuppression in mice infected with Schistosoma japonicum by treatment of praziquantel

To learn the possible alteration of immune response, hemolytic plaque-forming cells (PFC) produced in the spleen of Schistosoma japonicum infected mice treated with Praziquantel and untreated group were counted. There was no significant difference in the immunesuppression percentage between the treated and untreated groups 1 and 3 weeks after treatment. In 5 weeks after treatment, however, the immunesuppression percentage in the treated mice was markedly reduced in comparison with that of the untreated group. Recovery from immunesuppression appears to be associated with elimination or impairment of adult worms.