Antibodies to Fasciola hepatica antigens during experimental infections in cattle measured by ELISA

Chromatography of a soluble PBS extract of adult Fasciola hepatica on Sephadex G200 produced 7 fractions, F₁?F₇. The antigenicity of these fractions varied with F₁ being the most antigenic. Antibody responses to these fractions showed different time courses. Antibodies to F₁, F₂ and F₃ appeared early on in infection and, for the most part, persisted throughout infection, whereas antibodies to F₄ and F₅ increased gradually during infection.A standardised assay was developed to measure anti-F₁ antibodies. The assay was reproducible and able to detect antibodies by 3 weeks post-infection in experimentally-infected calves.     

Depression of productivity in sheep infected with Fasciola hepatica

The productivity of 24 Corriedale weaner wethers in eight groups of 3, artificially infected with a range of fluke burdens, was studied by measuring body weight change, midside patch wool growth, fleece weights and feed digestibility over a period of 24 weeks.Data from bodyweight and patch wool measurement and feed digestibility were analysed by regression procedures. All parasitised groups exhibited a reduction in growth rate, patch wool growth and fleece weight, when compared with the control groups. Reduction in feed conversion efficiency was detected in groups having mean burdens of 45, 67 and 117 flukes. Because groups with mean burdens of 230 or more flukes had reduced feed intake, and sheep in these groups died, the feed conversion information from these groups cannot be considered unequivocal.The group mean liveweight changes, patch wool growth and fleece weights were closely related to the mean number of flukes in the respective group. Prediction equations for wool growth rate and body weight gain in fluke infected sheep are presented.     

Maintenance in vitro of adult Fasciola hepatica in a continuous-flow system

A continuous-flow apparatus is described for the maintenance of groups of adult Fasciola hepatica. The ammonia produced by the flukes was used as a measure of their metabolic activity and it was shown that this is best maintained in culture fluids containing a large proportion of bovine serum. More ammonia per fluke was consistently produced when the flow rate of the medium was increased but including small pieces of clotted sheep blood or increasing the number of flukes per culture had no significant effects.     

Anthelmintic activity of artesunate against Fasciola hepatica in naturally infected sheep

In light of rapidly spreading triclabendazole resistance alternative fasciocidal drugs are urgently needed. Following up on promising results obtained with artemether in Fasciola hepatica infected sheep, we here report the efficacy and safety of artesunate in sheep with a natural F. hepatica infection. Artesunate was administered intravenously and intramuscularly, adverse events were monitored and drug efficacy was elucidated by means of faecal egg and worm burden reductions. A single 40 mg/kg intravenous dose of artesunate induced an egg count reduction of 68.9% and a worm burden reduction of 77.4%. Intramuscular artesunate at 40 mg/kg reduced faecal egg count and worm burden by 97.6% and 91.9%, respectively; whereas at 60 mg/kg it caused 93.2% and 87.1% reduction in faecal egg count and worm burden, respectively. Three sheep died 24-72 h post-treatment with a double dose of 40 mg/kg intramuscular artesunate, showing lethargy, sialorrhoea, reduced rumination and tremors. Egg and worm burden reductions of 93.3% and 83.9%, respectively, were calculated in the three surviving sheep. In conclusion, the interesting fasciocidal properties of artesunate in sheep warrant further investigations with an emphasis on toxicity studies.     

Precipitating antibodies against excretory/secretory antigens of Fasciola hepatica in sheep serum

Gel diffusion techniques were used to study antigen-antibody reactions in precipitates forming around juvenile, semi-mature and adult Fasciola hepatica cultured in serum from infected sheep. The number of reactions was analysed in both primary and challenge infections. The number of antigens shared by the various developmental stages of the fluke was also examined.At least 2 antigens were involved in precipitate formation around juvenile flukes. These antigens were also produced by the later stages of development. Two additional antigens were produced by semi-mature flukes and these, together with two others, were produced by adult flukes.Challenge infections had little effect on the number of antigens or antibodies in the precipitate.     

Immunological features of LPS from Ochrobactrum intermedium on sheep experimentally infected with Fasciola hepatica

The effects of the lipopolysaccharide (LPS) from Ochrobactrum intermedium in sheep with fasciolosis was reported previously, resulting in lower fecal egg counts and fluke burden. In the current study, we analyzed its immunological effects in two groups of sheep, treated (T) and controls (C). Fasciolosis induces a T helper (Th) type-2 response, characterized by IL-4 and IL-10 production; however, at the beginning of the infection, the IFN-γ production predominates (Th type-1 response). Although we did not find differences in IL-4 production or in the expression level of this gene in the hepatic lymph nodes, the expression level of IL-10 was higher (P < 0.05) in the T group at 4 wpi. The IFN-γ production was higher (P < 0.01) at 12 wpi as well as its level of expression at 4 wpi (P < 0.05) in the T group. We found a higher expression level of TGF-β at 4 wpi in the T group (P < 0.05), associated with the previous report of thicker fibrous tracks in a treated group. Immunoglobulin G1, related with a Th type-2 response, was higher (P < 0.01) in the T group at 4 and 12 wpi. In conclusion, the effects of LPS from O. intermedium could have resulted from a predominant Th type-2 immune response.     

The laboratory maintenance of field-collected Lymnaea tomentosa for the production of Fasciola hepatica metacercariae

A culture method is described for the production of Fasciola hepatica metacercariae in Lymnaea tomentosa collected from the field habitat. Two-litre glass jars, filled with artificial spring (A.S.) water and having a substratum of soil-bentonite (S.B.) agar to provide habitat and food, maintain groups of 40 to 100 snails graded according to size. Small quantities of washed lettuce are fed as a supplement. Compressed air, pre-warmed to 25°C, is piped to each jar to maintain thermoregulation, aeration, filtration and circulation of the water. Infection is achieved by reducing the A.S. water level to 3–4 mm and exposing the snails to miracidia for 6 h. During the aquatic developmental phase of 5–6 weeks, twice-weekly changes of A.S. water and the filter minimise proliferation of protozoans and Chaetogaster spp. Accumulation of mature cercariae is achieved by sustaining the infected snails on moist S.B. agar while humidification within the jar is maintained. Alternatively, groups of five to ten snails may be held in disposable petri dishes lined with S.B. agar. Maturation of cercariae may be delayed by refrigeration at 4–5°C. Upon maximum accumulation, the cercariae are harvested by dissection of the snails and metacercariae are stored in plastic containers at 4°C.     

The role of the gut in acquired resistance to Fasciola hepatica in the rat

The role of the gut in acquired resistance to Fasciola hepatica in the rat was assessed by comparison of the number of flukes recovered 4 and 10 weeks after oral or intraperitoneal challenge infection of male outbred Wistar rats previously infected by the oral or intraperitoneal routes.Previous infection given by either route generated signigicant protection against both oral and intraperitoneal challenge. The rats were resistant to Fasciola challenge in the presence of the primary infection or after its removal by anthelmintic treatment.It was concluded that passage of juvenile flukes through the gut was not essential for either the acquisition or the expression of acquired resistance to F. hepatica in the rat.     

A comparison of two laboratory methods of maintaining field-collected Lymnaea tomentosa for the production of Fasciola hepatica metacercariae

Four replicates of 50 field-collected Lymnaea tomentosa were maintained in two laboratory culture systems and their efficiency and practicability for the production of Fasciola hepatica metacercariae were assessed. There was no significant difference in the total number of metacercariae produced (56 552 from 64 snails in the closed container system and 54 073 from 82 snails in the shallow aquarium system).There was no significant difference between culture systems as assessed by snail survival, the number of infected snails or the number of infected snails harvested for the recovery of metacercariae. Within both systems there was a significant effect of snail size on the number of metacercariae produced. Optimum survival and production of metacercariae was achieved in young adult snails of 5–8 mm shell length at the time of infection.The collection of L. tomentosa from the field habitat and their maintenance in either culture system was shown to be a practical alternative to the breeding of laboratory adapted strains of L. tomentosa for the regular production of large numbers of F. hepatica metacercariae.     

Fasciola hepatica: Attempts to immunise rats using fluke eggs and in vitro culture products

Rats were sensitised on days 0 and 12 by subcutaneous injection of Fasciola hepatica ova and/or the excretion/ secretion products obtained from in vitro maintained adult flukes and then challenged orally with 30 F. hepatica metacercariae on day 14. Worm burdens were determined 3 and 8 weeks after infection. No resistance to challenge infection was detected, but those fluke recovered from rats sensitised with excretion/secretion products were smaller than those from control groups.     

Failure to induce homologous immunity to Fasciola hepatica in sheep vaccinated with irradiated metacercariae

Vaccination of sheep with either 100 or 1000 γ-irradiated (2.5 krad) metacercariae of Fasciola hepatica, on two occasions six weeks apart, did not generate significant protection against intraruminal challenge with F. hepatica six weeks after the second vaccinating dose as measured by recovery of flukes from liver and bile ducts, twenty weeks after challenge. There was, however, a significant increase in the proportion of flukes retarded in the parenchyma of both vaccinated groups. The percentage of retarded flukes was positively correlated with the degree of liver damage and increased weight of the hepatic lymph nodes. It was not possible to determine if the retarded flukes were derived from the vaccine or challenge infections or both.Challenge infection of both vaccinated and unvaccinated sheep significantly increased the numbers of eosinophils and globule leucocytes in the parenchymal bile duct and the numbers of mast cells and globule leucocytes in the abdominal bile duct. In addition the numbers of eosinophils and globule leucocytes in the parenchymal bile duct were significantly correlated with the percentage of retarded flukes in both vaccinated groups. In the abdominal bile duct, only the numbers of eosinophils in the low level vaccination group were significantly correlated with fluke retardation.Vaccination did not protect against the pathogenic effects of challenge infection as measured by reduced packed cell volumes and weight gain.     

Effect of dietary supplementation with flaxseed oil or vitamin E on sheep experimentally infected with Fasciola hepatica

The effects of two diets supplemented with flaxseed oil (FO) or vitamin E (VE) were studied in sheep infected with Fasciola hepatica. The control group (CO), not supplemented, had higher weight gain and faecal egg count (P < 0.05) at the end of the study. At necropsy, the number (71.2 ± 26.5) and size of flukes were lower in the FO group (P < 0.01), probably due to the higher levels of white blood cells and lymphocytes (P < 0.01). This group also had higher red blood cell and haematocrit values (P < 0.01). The CO group had more severe hypoproteinaemia and hypoalbuminaemia, due to having the highest fluke burden (123.0 ± 35.2), and also the highest IgG1 titre (P < 0.01). The diet did not affect production or gene expression levels of IFN-γ and IL-4. On the other hand, the supplementation with VE led to a reduction in adult fluke burden (97.7 ± 39.9) and lower lipid oxidation in the liver (P < 0.001).     

Comparative efficacy of albendazole against Fasciola hepatica in sheep and calves: Relationship to serum drug metabolites levels

Calves were given albendazole (ABZ) daily in feed at levels of 0,3 or 5 mg kg^?1 day^?1. None of the ABZ treatment levels was significantly effective in reducing Fasciola hepatica burdens. A dose rate of 5 mg kg^?1 day^?1 did significantly reduce the fecal egg count. Measurements of serum druf levels from calves following a single dose of ABZ showed ABZ levels to be low, but sulfoxide and sulfone metabolites of ABZ were present in (significant) larger quantities. The total available sulfoxide present in calves, however, was much lower than in sheep receiving the same dose of ABZ. Measurements of serum metabolite levels from sheep and calves which were given daily low-level doses of ABZ also indicated that the seum sulfoxide levels of calves were much lower than those of sheep receiving tha same ABZ dose. The results indicated that ABZ is not an effective prophylactic treatment for bovine fascioliasis and the differences in efficacy between sheep and cattle correlated with the differences in serum metabolites levels.     

MM3-ELISA evaluation of coproantigen release and serum antibody production in sheep experimentally infected with Fasciola hepatica and F. gigantica

During an experimental infection of sheep with Fasciola hepatica or F. gigantica, MM3-SERO and MM3-COPRO ELISA tests were applied to compare the kinetics of antibody production and coproantigen release between the 2nd and 32nd week post-infection (wpi). The Kato-Katz technique was used to measure the kinetics of egg shedding by both Fasciola species (eggs per gram of feces, epg). The kinetics of IgG antibodies for all sheep infected with F. hepatica and F. gigantica followed a similar pattern. Optical density (OD) increased rapidly between the 4th until the 12th wpi, when the highest values were reached and then decreased slowly until the 32nd wpi. Coproantigen levels increased above the cut-off value between 6 and 9 wpi in the F. hepatica group, and between 9 and 11 wpi in the F. gigantica group. The comparison between coproantigen levels and epg indicated that F. hepatica-infected sheep had detectable amounts of coproantigens 4–7 weeks before patency (egg shedding), while F. gigantica-infected sheep had detectable amounts of coproantigens 3–6 weeks before patency. When comparing the kinetics of coproantigen release vs the kinetics of epg, a similar pattern emerged, but with a two-week time-lag in epg, for both F. hepatica and F. gigantica infections. The amount of coproantigen release by each adult was not burden dependent for F. hepatica infection (burden of 33–66 adults), while it was for F. gigantica infection (burden of 17–69 adults). The results demonstrate the usefulness of the MM3-SERO and MM3-COPRO ELISAs as tools for the diagnosis of early as well as long-term fascioliasis infections, and suggest that they can potentially be applied to human fascioliasis even in countries where F. hepatica and F. gigantica co-exist. These tests can be employed not only in the diagnosis, but also in studies on epidemiology as well as pathogenesis and treatment in animals and humans since they allow post-treatment infection monitoring.     

The effect of age of host in resistance expressed at the gut level in rats infected with Fasciola hepatica

The development of oral and intraperitoneal infections with Fasciola hepatica in young and old rats showed that the gut was involved in the expression of age resistance. The role of the gut was assessed by comparison of the number of flukes recovered 4 and 10 weeks after oral or intraperitoneal infection of 2-, 6-, 15-, and 35-week-old male Wistar rats and 6- and 15-week-old male DA rats with encysted metacarcariae of F. hepatica.Rats of both strains behaved similarly in their response to F. hepatica infection. For both routes of infection the number of flukes recovered decreased as host age increased. In 22- and 6-week-old rats equal numbers of flukes were recovered at 4 and 10 weeks after oral or intraperitoneal infection. In 15-week-old rats, fluke burdens 4 weeks after infection were significantly greater following intraperitoneal infection than oral infection. A significant loss of flukes from the intraperitoneal infection of 15-week-old Wistar rats occured between 4 and 10 weeks after infection. In 35-week-old Wistar rats there was no significant effect for route or age of infection.As intraperitoneal infection (to by-pass rejection at the gut level) only partially eliminates the age response, additional age related mechanism(s), able to reject flukes at some time after they have entered the peritoneal cavity, must be operative in the peritoneal cavity and/or the liver.     

Pathophysiology of ovine fascioliasis: The influence of dietary protein and iron on the erythrokinetics of sheep experimentally infected with Fasciola hepatica

Worm-free sheep were fed diets with a high or low protein and iron content and infected with 1,000 F. hepatica metacercariae. The adult fluke burdens of the sheep on each diet were similar, but animals on the poorer plane of nutrition developed an earlier and ultimately more severe anaemia and as a result had to be necropsied earlier. By measuring the attendant changes in blood volume and erythrokinetics, it was found that the underlying cause of the anaemia in all animals was a combination of haemodilution, intra-hepatic and biliary haemorrhage, and its earlier appearance and faster development in the poorer-fed group was a refelection of the earlier onset of these changes in association with a faster rate of fluke migration. The ultimate degree of anaemia was not related to the severity of biliary haemorrhage, but rather to the animals' erythropoietic capacity. This was substantially lower in the sheep maintained on the poorer diet, which unlike their better-fed counterparts, became iron deficient by virtue of their inability to replace from the diet iron lost in the faeces as a result of haemorrhage. It was concluded that this difference was primarily a reflection of the better appetite and hence iron intake of the supplemented sheep, but differences in the efficiency of dietary iron utilization, possibly related to protein intake, may also have been involved.In a further experiment involving sheep infected with 600 metacercariae, there was no evidence of haemodilution. When provided with a diet of high protein and iron content, these animals were able to maintain constant, albeit reduced PCV values in the face of substantial biliary haemorrhage. However, when switched to an inferior diet, the same degree of haemorrhage produced a sharp fall in PCV, suggesting that erythropoiesis was adversely affected by reduced protein and iron intake.     

A novel method for the administration of metacercariae of Fasciola hepatica to adult cattle

A gelatin bolus containing the infective stage of Fasciola hepatica was prepared and administered to adult cattle. The bolus was formed to fit into the nozzle of a standard size bolus-type dosing gun. Administration proved easy and effective causing minimal stress to the animal, yet ensuring that experimental infection took place.     

Molecular detection of Theileria and Babesia infections in cattle

This study was carried out to determine the presence and distribution of tick-borne haemoprotozoan parasites (Theileria and Babesia) in apparently healthy cattle in the East Black Sea Region of Turkey. A total of 389 blood samples were collected from the animals of various ages in six provinces in the region. Prevalence of infection was determined by reverse line blot (RLB) assay. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified with a set of primers for members of the genera Theileria and Babesia. Amplified PCR products were hybridized onto a membrane to which generic- and species-specific oligonucleotide probes were covalently linked. RLB hybridization identified infection in 16.19% of the samples. Blood smears were also examined microscopically for Theileria and/or Babesia spp. and 5.14% were positive. All samples shown to be positive by microscopy also tested positive with RLB assay. Two Theileria (T. annulata and T. buffeli/orientalis) and three Babesia (B. bigemina, B. major and Babesia sp.) species or genotypes were identified in the region. Babesia sp. genotype shared 99% similarity with the previously reported sequences of Babesia sp. Kashi 1, Babesia sp. Kashi 2 and Babesia sp. Kayseri 1. The most frequently found species was T. buffeli/orientalis, present in 11.56% of the samples. T. annulata was identified in five samples (1.28%). Babesia infections were less frequently detected: B. bigemina was found in three samples (0.77%), B. major in two samples (0.51%) and Babesia sp. in five samples (1.28%). A single animal infected with T. buffeli/orientalis was also infected with B. bigemina.     

Detection of Fasciola hepatica antigen in cattle faeces by a monoclonal antibody-based sandwich immunoassay

A monoclonal antibody-based sandwich immunoassay (mAb sandwich ELISA) was developed for the detection of Fasciola hepatica antigen in the faeces of cattle. The assay was applied to samples from 100 cattle infected with F hepatica, 56 animals with parasitologically proven infections of other parasites and 100 uninfected animals. F hepatica antigen was detected in all the faecal samples from animals with fasciolosis, but none of the samples from the uninfected animals or from those with other parasitic infections had significant levels of F hepatica antigens. The results indicate that the mAb sandwich ELISA is a rapid, simple and useful method for the diagnosis of active F hepatica infection in cattle.