Use of two polysaccharide antigens in ELISA for the detection of antibodies to Echinococcus granulosus in sheep sera

Polysaccharide antigens were obtained from either the secretions produced during in vitro cultivation of Echinococcus granulosus protoscoleces or from mouse hydatid cyst membranes by phenol extraction. When either of these antigens was used in an enzyme-linked immunosorbent assay antibody activities were detected in sera from sheep infected 27 or more weeks earlier with at least 100 E granulosus eggs. These antibody responses were significantly higher (P less than 0.05) than those of sheep infected with Taenia hydatigena or T ovis and tested with the E granulosus antigens. Very high cross-reacting antibody responses in sera from sheep recently infected with T hydatigena were only detected with the protoscoleces secretions antigen. Neither antigen was sufficiently sensitive or specific for serodiagnostic use. However, when sera were first tested with one antigen and then with the other, and only sera that were positive in both tests were regarded as positive, the overall sensitivity and specificity of this two antigen method increased to about 80 per cent.     

THE ROLE OF THE TASMANIAN DEVIL IN THE EPIZOOTIOLOGY OF OVINE CYSTICERCOSIS AND ECHINOCOCCOSIS

Tasmanian devils were fed infective cystic material of Taenia ovis, T. hydatigena and Echinococcus granulosus and later killed and examined for adult tapeworms. No infections with T. ovis were found after feeding 23 cysts to 8 devils. T. hydatigena were recovered from 5 to 41 devils and 10 worms established after feeding 417 cysticerci. No E. granulosus were recovered from any of 3 devils. Specimens of T. hydatigena appeared to develop as well in devils as in dogs. From these and previous results devils could be successful hosts to T. hydatigena and T. ovis. A survey of 320 devils failed to detect any natural infections with any of these 3 species.     

Control of Echinococcus granulosus in Uruguay: evaluation of different treatment intervals for dogs.

This study attempted control of transmission of Echinococcus granulosus from dogs to sheep in different areas in the Department of Florida, Uruguay, by treating dogs with praziquantel at intervals of 6, 12 and 16 weeks. The 6-week interval was based on the prepatent period of infection with E. granulosus, the 12- and 16-week intervals were based on the rate of reinfection with tapeworms in dogs in the area. Dogs had become reinfected with E. granulosus between 2 and 4 months after treatment, whereas they became reinfected with the Taenia spp. tapeworms within 2 months of treatment. One year after the start of treatments sentinel lambs were born and grazed the farms in the three treatment areas. Approximately, 15 months later when the sentinel lambs were killed and examined for parasites the six weekly treatments had stopped the transmission of E. granulosus to the sentinel lambs. Treatment of dogs at 12- and 16-week intervals failed to stop transmission of E. granulosus but both the numbers of farms and the numbers of sheep infected with E. granulosus were lower where dogs received 12 weekly treatments compared with dogs receiving 16 weekly treatments and a fourth area where dogs had received no treatments (chi(2)P=0.002). Lambs continued to become infected with the Taenia spp. tapeworms in all the areas. Control was complicated by large changes in the dog population. From a starting population of 1164 dogs in the three treated areas, 832 new dogs, most of these adult hunting dogs, entered the population and 793 dogs were lost from the population.     

The effect of nitroscanate tablets of Echinococcus granulosus and Taenia hydatigena infections in dogs

Tablets of micronised nitroscanate (nominal particle size 2--3 microns) were given to a total of 190 dogs that had been experimentally infected with either Echinococcus granulosus or Taenia hydatigena. The efficiency of the drug in tablet form in freeing dogs from tapeworms, was investigated. The dose rate at which 50 per cent of normally fed dogs can be expected to be freed from E granulosus was found to be 89 mg/kg (95 per cent confidence limits 55 mg/kg to 140 mg/kg). The 90 per cent effective dose rate was not determined within the range 32 mg/kg to 250 mg/kg. The dose rate at which 90 per cent of normally fed dogs can be expected to be freed from T hydatigena was 37 mg/kg (95 per cent confidence limits 23 mg/kg to 60 mg/kg).     

Efficacy of praziquantel against ovine cysticercosis caused by Taenia hydatigena

Praziquantel was evaluated for its larvicidal activity against naturally or experimentally induced Taenia hydatigena infections in sheep. The major criterion used for assessing efficacy was an estimate of the dose rate required to free 90 per cent of the sheep from infection (ED90) together with a 95 per cent confidence interval. With both light and heavy infections of three-month-old T hydatigena in lambs up to 12 months old, many larvae were killed. The ED90 and 95 per cent confidence limits for six-month-old and aged organisms was estimated to be 13.1 (5.5 to 31.4) mg/kg and 6.1 (2.4 to 15.5) mg/kg, respectively. Equivalent data for the ED50 were 3.4 (1.4 to 8.3) mg/kg and 1.3 (0.3 to 5.4) mg/kg, respectively. It was established that one of the factors involved in the efficacy of praziquantel was associated with the age of the larvae at the time of treatment, the drug being markedly more effective against older than younger organisms.     

Comparative immunoelectrophoretic analysis of Echinococcus granulosus, Taenia hydatigena and Taenia pisiformis cyst fluid antigens by hyperimmune rabbit sera.

Cyst fluid antigens of Echinococcus granulosus, Taenia hydatigena and T pisiformis were examined by electrophoresis using homologous and heterologous hyperimmune rabbit sera to these antigens. While arc 5 forming antibodies were identified in sera from rabbits immunised with E granulosus and T hydatigena cyst fluids, antibodies responsible for forming precipitating antigen B band were detected in rabbit antisera to E granulosus, T hydatigena and T pisiformis antigens. T hydatigena cyst fluid appears to contain antigen similar to E granulosus antigen 5 and probably antigen B while T pisiformis cyst fluid has mainly an antigen close to hydatid antigen B.     

Frequency distribution of Echinococcus granulosus hydatid cysts in sheep populations in the Xinjiang Uygur Autonomous Region, China.

Age-prevalence and age-intensity data of Echinococcus granulosus hydatid cysts in sheep populations were collected in an abattoir in the Xinjiang Uygur Autonomous Region, People's Republic of China. The frequency distribution of the larval cysts per sheep was empirically described by the negative binomial model, with parameter k being 0.5273. A mathematical model for the life cycle of E. granulosus was applied to the collected data and the results show that the infection pressure on sheep was 0.4362 (female) or 0.4119 (male) infections per year, the mean number of cysts increased linearly by 0.8824 (female) or 0.9971 (male) cysts every year and acquired immunity was too low to depress this rate of increase. According to certain definitions of steady states for taeniid populations, it was concluded that at least in some parts of Xinjiang, the life cycle of E. granulosus was and may still be in an endemic steady state. Consequently, the regular dog-dosing program would readily drive the infection from an endemic state towards extinction.     

Detection of Echinococcus granulosus coproantigens in faeces from naturally infected rural domestic dogs in south eastern Australia

OBJECTIVE: To investigate the occurrence of Echinococcus granulosus in rural domestic dogs in farming areas around Yass, New South Wales, and Mansfield and Whitfield, Victoria. DESIGN: Faeces were collected per-rectally from farm dogs voluntarily presented by their owners in four farming districts in New South Wales and two in Victoria. PROCEDURE: Faeces were collected in the field, an extract prepared from each sample and E granulosus coproantigens detected in an ELISA. Farmers were also questioned about their dog feeding and worming practices. RESULTS: Echinococcus granulosus coproantigens were detected in 99 of 344 dogs (29%) from 95 farms in south eastern New South Wales and 38 of 217 dogs (17.5%) from 43 farms in Victoria. Cross-reactions between E granulosus coproantigen trapping antibody and coproantigens in faeces from dogs monospecifically infected with other species of intestinal helminthes (Taenia ovis, T hydatigena, T pisiformis, Spirometra ericacei, Dipylidium caninum, hookworm, Toxocara canis, Trichuris vulpis) were not evident. Dietary and worming data revealed many owners fed raw meat and occasionally offal from domestic livestock and wildlife to their dogs and few owners wormed their dogs frequently enough to preclude the chance of patent E granulosus being present in their dogs. CONCLUSION: Echinococcus granulosus occurs commonly in rural dogs in south eastern Australia and an education program promoting the public health importance of responsible management of rural dogs is urgently needed.     

Trials to induce protective immunity in mice and sheep by application of protoscolex and hydatid fluid antigen or whole body antigen of Echinococcus granulosus

In the current study, soluble proteins prepared from 200 mature Echinococcus granulosus and protoscolices of sheep hydatid cysts were applied to immunize sheep and mice respectively. The samples were mechanically homogenized in a blender, sonicated and the final yield was maintained at -20 degrees C until analysis. Hydatid fluid was isolated from liver or lung of sheep under sterile conditions. In the first experiment, 15 mice were randomly allocated to three groups of five mice each. Each mouse in groups 1 and 2 was immunized with 100 microg of hydatid fluid and protoscolex proteins in 100 microl of phosphate-buffered saline (PBS) and emulsified with an equal volume of Freund's complete adjuvant (FCA) respectively. The mice of group 3 were immunized with adjuvant in PBS. The mice were boosted 4 weeks after the first vaccination with the same preparation except that FCA was replaced by Freund's incomplete adjuvant (FIA). In the second experiment, eight male or female lambs 4-6 months of age, were allocated to two groups of four lambs each. Each lamb in the test group was vaccinated subcutaneously in the neck with a 2-ml dose of vaccine (1 mg of whole body protein of E. granulosus dissolved in 1 ml of PBS plus 1 ml of FCA). Control lambs were vaccinated with adjuvant in PBS. Lambs were boosted the same way as in the first experiment. Three weeks after the second vaccination, each mouse and lamb received a challenge infection with 2000 protoscolices intraperitoneally and each lamb additionally received 10 gravid E. granulosus. All mice and sheep were killed after 7 months and examined for hydatid cysts. In these studies, protective immunity was induced in mice with protoscolex protein and with hydatid fluid, and in sheep with whole-body homogenate of E. granulosus and the levels of protection afforded were found to be 72.1, 82.6 and 90.9% respectively.     

Evaluation of three enzyme-linked immunosorbent assays (ELISAs) for the detection of serum antibodies in sheep infected with Echinococcus granulosus

The aim of this study was to develop an immunological method for the identification of sheep infected with Echinococcus granulosus which would allow the monitoring of animals imported into countries free from hydatidosis and as an aid to countries where control schemes for the disease are in operation. Three enzyme-linked immunosorbent assays (ELISAs) were developed and validated, using as antigen either a purified 8 kDa hydatid cyst fluid protein (8kDaELISA), a recombinant EG95 oncosphere protein (OncELISA) or a crude protoscolex preparation (ProtELISA). Sera used for the assay validations were obtained from 249 sheep infected either naturally or experimentally with E. granulosus and from 1012 non-infected sheep. The highest diagnostic sensitivity was obtained using the ProtELISA at 62.7 and 51.4%, depending on the cut-off. Assay sensitivities were lower for the 8kDaELISA and the OncELISA. Diagnostic specificities were high, ranging from 95.8 to 99.5%, depending on the ELISA type and cut-off level chosen. A few sera from 39 sheep infected with T. hydatigena and from 19 sheep infected with T. ovis were recorded as positive. Western immunoblot analysis revealed that the dominant antigenic components in the crude protoscolex antigen preparation were macromolecules of about 70-150 kDa, most likely representing polysaccharides. This study demonstrated that the ProtELISA was the most effective immunological method of those assessed for detection of infection with E. granulosus in sheep. Because of its limited diagnostic sensitivity of about 50-60%, it should be useful for the detection of the presence of infected sheep on a flock basis and cannot be used for reliable identification of individual animals infected with E. granulosus.     

Prevalence of chlamydia, toxoplasma, toxocara and ringworm in farm cats in south-west England

The prevalence of infection with Chlamydia psittaci, Toxoplasma gondii, Toxocara cati and Microsporum canis was examined in 51 cats on 22 sheep farms in the Bristol area. Serum antibody to C psittaci and T gondii was present in 45 per cent and 47 per cent of cats, respectively. At the time of sampling C psittaci was isolated from 6 per cent of the cats, T cati was identified in 63 per cent of faecal samples but neither T gondii nor M canis was isolated. When examined according to the farm of origin, 22.7 per cent of farms had cat populations with no evidence of infection with C psittaci or T gondii. Of the remainder, 45.5 per cent supported cat populations with evidence of both infections and 31.8 per cent had evidence of T gondii infection alone. None of the farms had cat populations with evidence of C psittaci infection alone. Two of the cats infected with C psittaci were excreting viable organisms in the faeces. The possible significance of this to the epidemiology of ovine enzootic abortion is discussed.     

Detection of Taenia hydatigena copro-antigens by ELISA in dogs

A sandwich-ELISA was developed for the detection of soluble Taenia hydatigena antigens in fecal samples of dogs. Affinity-purified polyclonal catching antibodies and alkaline phosphatase-conjugated detecting antibodies were employed, which had been obtained from rabbits hyperimmunized with excretory/secretory antigens derived from in vitro maintained adult Taenia hydatigena. The assay allowed the detection of 800 ng T. hydatigena antigen g-1 of feces as a lower limit. Six helminth-free dogs were each infected with 10 T. hydatigena cysticerci isolated from Swiss sheep. After prepatent periods ranging from 57 to 71 days, the dogs started to excrete Taenia eggs and/or proglottids. The ELISA detected Taenia antigens in all six dogs during the prepatent period starting individually between Day 18 and 45 post-infection (p.i.). Anthelmintic treatment of three dogs at Day 95 p.i. resulted in elimination of the cestodes and within the 5 following days in the disappearance of Taenia antigens from feces. The specificity of the assay was evaluated by testing crude antigens derived from helminths or bacteria. Four Taenia species showed cross-reactivity at concentrations of 5 micrograms protein ml-1. Conversely, no cross-reactions occurred with various antigen batches derived from Echinococcus granulosus, E. multilocularis, Dipylidium caninum, Mesocestoides corti, Diphyllobothrium sp., Toxocara canis and bacterial antigens (Salmonella and Escherichia). Moreover, fecal samples from dogs naturally infected with T. canis (n: 13), hookworms (n: 2), Trichuris vulpis (n: 13) and of 10 dogs with mixed infections with these three nematode groups were tested, and results confirmed the high degree of specificity. The Taenia antigens detectable by this ELISA remained immunologically stable in native feces stored at +25 degrees, +4 degrees or at -20 degrees C for at least 5 days.     

A survey of gastro intestinal helminth parasites of stray dogs in Zaria, Nigeria.

A survey of helminth parasites in stray dogs was carried out in the Zaria area. Out of 180 dogs examined 65.6 per cent (118/180) were found positive for Ancylostoma caninum, while Toxocara canis and Trichuris vulpis were detected in 41.1 per cent (74/180) and 13.9 per cent (25/180) respectively. Other helminth parasites found Taenia ovis 35 per cent (63/180) Taenia hydatigena 67.8 per cent (122/180), Echinococcus granulosus 0.006 per cent (1/180) and Dipylidium caninum 97.8 per cent (176/180).     

The prevalence of cysticercosis and hydatidosis in Ethiopian sheep

Five hundred and sixty sheep, slaughtered at an Addis Ababa abattoir, were examined for cysticercosis and hydatidosis. The prevalence of the metacestodes of Taenia hydatigena was 37.1% and that of Echinococcus granulosus 16.4%. In addition, the age and sex prevalences of hydatid cysts, their organ distribution and the ratio of fertile cysts were determined.     

Fox predation as a cause of lamb mortality on hill farms

Data collected between 1993 and 1996 from two Scottish hill farms were used to assess the potential importance of predation by foxes as a cause of lamb mortality. Farm 1 was in Midlothian and farm 2 in West Perthshire. Overall lamb mortality rates from birth onwards were 10.2 per cent for farm 1 and 9.3 per cent for farm 2. Sixteen lambs were identified as killed by foxes on the two farms during the four-year period. The minimum rates of fox predation were 0.6 per cent for farm 1 and 0.2 per cent for farm 2. All confirmed cases of fox predation were recorded during the period until the lambs were marked at about six weeks of age, and during this period another 53 lambs went missing from the two farms. Both groups of missing lambs were characterised by having significantly more siblings than the remaining lambs. A logistic regression model was able to classify the lambs killed by foxes and the missing lambs with 75 per cent accuracy on the basis of their birth rank alone. Of the total potential revenue from lamb production, the maximum losses due to confirmed fox predation in any one year were equivalent to 1.5 per cent on farm 1 and 0.6 per cent on farm 2.     

The efficacy of praziquantel against cestodes in cats, dogs and sheep

Praziquantel is a new type of acylated isoquinoline-pyrazine. A single, low oral or subcutaneous dose of the compound is reliably effective against all tested juvenile and adult cestodes in cats, dogs and sheep. Praziquantel is the first cestodicide which is also effective on bile duct cestodes. In cats and dogs, 5 mg praziquantel per kg is completely effective on all stages of Taenia hydatigena, T pisiformis, T ovis, T taeniaeformis, Dipylidium caninum, Mesocestoides corti, Echinococcus multilocularis and E granulosus. Because of its very wide therapeutic index praziquantel is thus particularly suited for eradication programmes, eg, echinococcosis.     

Efficacy, safety and palatability of a new broad-spectrum anthelmintic formulation in dogs

The efficacy, safety and palatability of a new flavoured chewable anthelmintic tablet were investigated in dogs. The efficacy, based on worm counts, of a single recommended therapeutic dose (RTD) of 5 mg pyrantel + 20 mg oxantel + 5 mg praziquantel/kg bodyweight was assessed in experimental infections (EI) and natural infections (NI) with Trichuris vulpis, Echinococcus granulosus and Toxocara canis. For T vulpis, the efficacy of the treatment was 99.3 per cent in EI (comparing groups of six treated and six control dogs) and 100 per cent in NI (nine treated and nine control dogs). For E granulosus, the efficacy was more than 99.9 per cent in EI (11 treated and 11 control dogs). For T canis, the efficacy was 94.3 per cent in EI (10 treated and 10 control dogs) and 100 per cent in NI (12 treated and 13 control dogs). In a field study, Ancylostoma caninum (11 dogs) and T canis (11 dogs) faecal egg counts were reduced by more than 99 per cent, and in eight dogs with Dipylidium caninum proglotides in the faeces the efficacy was 100 per cent. The tablets were readily consumed by 56 of 64 (87.5 per cent) privately owned dogs. Safety was assessed in groups of six dogs treated either once with twice the RTD, once with six times the RTD, with twice the RTD on three consecutive days, or untreated. There were no significant differences in blood parameters between the groups, and no abnormal clinical findings. Two dogs treated with six times the RTD vomited, but no vomiting was observed when administration was repeated two days later.     

Specific antibody responses to Taenia hydatigena, Taenia pisiformis and Echinococcus granulosus infection in dogs

Groups of dogs reared free of both nematodes and cestodes were infected with Taenia hydatigena, Taenia pisiformis or Echinococcus granulosus. After infections with the Taenia spp became patent, dogs were purged to remove the worms. They were later reinfected and the second infections again removed by purging after patency. A group of 3 uninfected worm free dogs was kept as age-matched controls. The dogs were bled at intervals of 5 days and their serums tested for antibodies using the enzyme-linked immunosorbent assay (ELISA) with excretory/secretory (ES) antigens collected during in vitro incubation of evaginated scoleces (scolex ES antigen) and oncosphere antigens. Antibodies to scolex ES antigen were detected by 3 weeks after infection with each cestode species whereas antibodies to oncosphere antigen were not detected until about one week after eggs were found in the faeces of the infected dogs. Antibody responses to both oncosphere and scolex ES antigens decreased rapidly following removal of the worms by purging. Uninfected control dogs were invariably negative to both oncospheral and scolex ES antigens. There were cross-reactions between the serums from dogs infected with T. pisiformis and T. hydatigena when tested with scolex ES antigens, but oncospheral antigens showed a high degree of species specificity. Scolex ES antigens from E. granulosus were compared with those prepared from T. hydatigena and T. pisiformis for their ability to discriminate between antibodies in serums collected from dogs 31 and 32 days after infection with 100,000 protoscoleces of E. granulosus or dogs infected with Taenia spp.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of Taenia hydatigena infection on existing and concurrent infections of Fasciola hepatica in sheep

Sheep given a primary infection of Fasciola hepatica were challenged 18 weeks later with Taenia hydatigena or F hepatica, or both parasites together, or were not challenged. At the same time, control sheep were infected with T hydatigena and/or F hepatica separately or concurrently. All sheep were killed seven weeks after challenge and the number of cysts and flukes counted. Challenge infection with T hydatigena did not affect the numbers of flukes recovered from either primary or challenge F hepatica infections. On the other hand, the numbers of cysticerci were reduced in sheep previously infected with F hepatica but not in those given T hydatigena and F hepatica concurrently.     

Abattoir survey of acquired reproductive abnormalities in ewes.

Abnormalities of the reproductive tract of female sheep were studied by examining 9970 reproductive tracts from cull ewes and 23,536 tracts from nulliparous sheep (prime lambs) over a period of 12 months in abattoirs in south-west England. Overall, 3.37 per cent of the tracts were pregnant (8.11 per cent of cull ewes, and 1.36 per cent of nulliparous sheep), with a peak incidence between September and December. A total of 655 ewes (6.57 per cent) and 459 nulliparous sheep (1.95 per cent) had acquired abnormalities of the reproductive tract. Within these totals, abnormalities of the ovaries accounted for 3.51 per cent (for the ewes) and 10.68 per cent (for the nulliparous sheep) of all the abnormalities, and abnormalities of the ovarian bursa and uterine tube accounted for 42.1 per cent (for the ewes) and 5.23 per cent (for the nulliparous sheep). In addition, uterine lesions (hydrometra and metritis) accounted for 9.92 per cent (for the ewes) and 13.51 per cent (for the nulliparous sheep); lesions of the cervix and vagina (total of 1.44 per cent) and Cysticercus tenuicollis cysts associated with the reproductive tract (total of 3.05 per cent) were less common. Among the ewes the most common ovarian lesions were ovulation tags, and follicular cysts were the most common in nulliparous animals. Lesions such as bursitis, parametritis and abscesses of the reproductive tract were much more common in cull ewes than in nulliparous sheep, probably having arisen from peripartum infections. Hydrosalpinx and hydrometra, in which the intraluminal fluid was clear, were present at relatively high incidence in nulliparous animals, but not in cull ewes. The proportion of tracts containing macerated fetal remnants (2.14 per cent of all abnormalities in cull ewes) was lower than expected. It was considered that the functional significance of many of the lesions, such as ovulation tags and C tenuicollis cysts, was likely to be low, although in some cases of the latter calcification of the cyst had occluded the uterine tubes. Other lesions, notably hydrosalpinx, bursitis and metritis were likely to have made the affected animals sterile. The acquired abnormalities were therefore more significant in terms of individual animal infertility than as a major cause of infertility in flocks.