The metabolic profile of adult Fasciola hepatica obtained from rafoxanide-treated sheep.

Sheep infected with adult Fasciola hepatica were drenched with rafoxanide. At 4, 8, 16 and 24 h after drenching the sheep were killed and the flukes removed, washed and rapidly frozen in liquid nitrogen. The content of key metabolites in the fermentation pathway were determined and compared with those in control F. hepatica, whose hosts were not treated with rafoxanide. Rafoxanide decreased glycogen, malate, NADH and ATP levels. The level of other metabolites in the pathway increased for the first 8-16 h after rafoxanide treatment. The marked decrease in ATP and glycogen, and the increase in total [NAD+]/[NADH] and [oxaloacetate]/[malate], together with the changed content of other metabolites, led to the conclusion that the mode of action of rafoxanide against F. hepatica in vivo is by uncoupling oxidative phosphorylation.     

The metabolic integrity of Fasciola hepatica during in vitro maintenance

Levels of metabolic intermediates and end products in F. hepatica after 24 and 48 h in Hédon-Fleig salt solution with added glucose were compared with levels obtained immediately on removal from the host. Glycogen levels dropped initially, probably due to the expulsion of eggs; thereafter they remained constant. Internal glucose concentrations increased as the parasites equilibrated with the medium. Other changes in internal pool sizes were consistent with regulation to the in vitro conditions. ATP levels increased; ATP/ADP ratios were maintained. Comparisons of mass action ratios and equilibrium constants suggest that hexokinase, pyruvate kinase and phosphofructokinase are regulatory. Output of excretory products approached linearity; from the calculated regressions the proportions of lactate, acetate and propionate were 1: 2: 4. The implications for metabolic regulation in F. hepatica are briefly discussed, and it is concluded that, for at least 48 h in vitro, energy metabolism is not adversely affected.     

Development of the large American liver fluke, Fascioloides magna, in white-tailed deer, cattle, and sheep.

The comparative development of Fascioloides magna in white-tailed deer, cattle and sheep has been studied. Flukes were recovered from 72% of 32 deer administered 40 to 500 metacercariae, from 82% of 11 cattle administered 10 to 500 metacercariae, and from 53% of 15 sheep administered 8 to 200 metacercariae. The percentage recovery of the flukes administered as metacercariae was 4.1% of 6,130 in deer, 5.7% of 2,510 in cattle, and 4.7% of 1,213 in sheep. Flukes were recovered only from livers of infected deer, while in cattle, 1 fluke was also found in the lungs of each of 2 animals. In sheep, all but 10 flukes were recovered from the livers; 6 were found in the lungs and 4 in the abdominal cavities. The black iron porphyrin pigment associated with F. magna infection was found to be most widespread in cattle and sheep, but was also a pathognomonic feature in deer. Growth of the fluke was similar in all 3 host species tested, but eggs were passed only from deer, the normal definitive host. In cattle, the eggs were retained in the liver, and F. magna was lethal to sheep before its own maturity was attained. In cattle and deer, flukes matured approximately 7 months after exposure, but immature migrating flukes were found 12 months after infection and apparently can remain in this retarded state for an undetermined period of time.     

Changes in energy metabolism due to anthelmintics in Fasciola hepatica maintained in vitro

The effects of rafoxanide (RFX), nitroscanate (NSC) and mebendazole (MBZ) on oxidative pathways in whole F. hepatica maintained in a simple salt solution have been examined. The anthelmintics did not alter glucose uptake or glycogen mobilization. NSC and RFX depressed ATP and increased AMP levels. MBZ behaved similarly at first, but later depressed the total adenine nucleotides. All three drugs influenced end product formation, increasing it initially, although by different mechanisms. With NSC, early increases in lactate and acetate excretion were later abolished. With RFX, there was an initial increased production of acetate and propionate. Later, excretion of propionate was reduced and that of succinate was increased. MBZ also increased succinate excretion, but to a much greater extent. In addition, it inhibited lactate production. A number of effects of the drugs on the internal concentrations of metabolic intermediates are described. The mechanisms of action of the drugs are discussed.     

Fasciola hepatica: Surface tegumental responses to in vitro and in vivo treatment with the experimental fasciolicide OZ78

The tegumental alterations in adult Fasciola hepatica induced by the experimental fasciolide OZ78 were investigated utilizing scanning electron microscopy (SEM). Twelve weeks post-infection with F. hepatica, rats were treated with a single 100mg/kg oral dose of OZ78 and flukes were recovered from the bile ducts after 24-72 h. In vitro F. hepatica were incubated with OZ78 for 48 h at a concentration of 10 microg/ml in the absence or presence of haemin. Twenty-four and 48 h post-treatment of rats disruption of the tegument of F. hepatica as blebbing, swelling and furrowing was evident. The recovery of flukes 72 h post-treatment was low. Flukes examined at this time point showed an increasing severity of tegumental damage as sloughing and absence of spines, in particular in the tail region. SEM analysis of F. hepatica incubated in the presence of OZ78 without haemin showed only minor and localized damage of the tegument. In the presence of haemin extensive tegumental damage, including sloughing or blebbing, in particular in the anterior part, was observed. In conclusion, our experiments confirm the interesting fasciocidal properties of OZ78. The tegument of adult F. hepatica might play a role in the action of this drug.     

Scanning electron microscopic study of Opisthorchis viverrini tegument and its alterations induced by amoscanate

Scanning electron microscopic study of Opisthorchis viverrini tegument and its alterations induced by amoscanate. International Journal for Parasitology16: 19–26. When examined by scanning electron microscopy, the surface of adult Opisthorchis viverrini is covered with short microvilli that are closely packed together. Microvilli are more numerous and are taller on the ventral surface. Distributed among microvilli are two types of papillae, each one with a dome-shaped base (approx. 3 μm in diameter) with a projecting cilium in one case but not the other. Papillae are scattered in groups over the surface but are especially numerous around the suckers and laterally.When the flukes were treated with a potent schistosomicidal agent, amoscanate (C 9333-Go/CGP 4540), the tegument was damaged. Lesions that occurred on the flukes recovered from infected hamsters 1, 9, 30 and 90 days after treatment were compared with those which occurred when the flukes were exposed to the agent in vitro. Total disruption of the basic structure of the tegument was noted within 2 h of in vitro incubation with 1% amoscanate; the damage was more severe after 24 h of treatment. Flukes obtained from hamsters 24 h after treatment for 4 consecutive days with a total of 40 mg also showed tegumental lesions, including pronounced swellings into large bulbs that eventually ruptured and sloughed. However, complete regeneration of the tegument was noted within 30 days after treatment.     

In vitro cultivation of Fasciola hepatica metacercariae and of partially developed flukes recovered from mice

Attempts were made to culture the metacercariae of Fasciola hepatica under a wide variety of conditions. Of the media tested, the most successful was NCTC 135 plus 50% heat inactivated chick serum and sheep red blood cells at 37°–38°C. In this medium, somatic development of newly excysted juveniles was similar to that of flukes recovered from the liver of a mouse 11 days post-infection. There was, however, no corresponding development of the genital rudiment. Various supplements, such as liver extract, bile, yeast extract, embryo extract, egg products, monolayer cells and diphasic media were tested, but none enhanced development. The effects of various physical parameters on growth and development in vitro were examined. Cultured metacercariae appeared to be in a state of ‘suspended animation’; when injected intraperitoneally into mice they developed into egg-producing adults. Flukes recovered from the abdomen and liver of mice continued their somatic growth in vitro but their genitalia failed to develop further.     

The stimulation of resistance in sheep to Fasciola hepatica by infection with Cysticercus tenuicollis

Infection of sheep with Cysticerus tenuicollis for 12 weeks generated a high level of protection (> 95%) against intra-ruminal challenge with metacercariae of Fasciola hepatica as measured by recovery of flukes from liver and bile ducts and counts of fluke eggs in faeces. The animals were resistant to Fasciola whether challenge was superimposed upon the cestode infection or after removal of the cestode with mebendazole.Previous infection with C. tenuicollis also protected against the pathogenic effects of challenge infection with F. hepatica. Liver fibrosis was much less extensive in resistant sheep than controls and PCV's were not affected although these were reduced during fluke infection in the control animals.     

Haemonchus contortus: The in vitro effects of dl-tetramisole and rafoxanide on glycolytic enzymes

Kaur R. and Sood M. L. 1982. Haemonchus contortus: the in vitro effects of dl-tetramisole and rafoxanide on glycolytic enzymes. International Journal for Parasitology 12: 585–588. Various enzymes of glycolysis (hexokinase, phosphoglucomutase, phosphoglucoisomerase, adolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglyceromutase-enolase-pyruvate kinase and lactate dehydrogenase) have been detected in adult Haemonchus contortus. Low pyruvate kinase and lactate dehydrogenase activities suggested an alternate pathway from phosphoenolpyruvate. In vitro incubation had no significant effects on these enzymes and the worm was able to maintain normal metabolism for 12 h. Varying degrees of inhibition of glycolytic enzymes were observed with 50 μg/ml of dl-tetramisole and rafoxanide. The enzymes were inhibited to a greater extent by dl-tetramisole. These effects may block the glycolytic pathway and deprive the parasite of its ATP production.     

Evidence for two distinct mechanisms of resistance in the rat to reinfection with Fasciola hepatica

Doy T. G. and Hughes D. L. 1982. Evidence for two distinct mechanisms of resistance in the rat to reinfection with Fasciola hepatica. International Journal for Parasitology12: 357–361. Congenitally athymic nude (rnu/rnu) and heterozygous hairy (rnu/ + ) rats were found to be equally highly resistant to oral reinfection with Fasciola hepatica. Accompanying the development of this resistance was a marked increase in intestinal eosinophil numbers. The sensitised rnu/ + rats showed a similar marked resistance to intraperitoneal challenge with newly excysted juvenile (NEJ) flukes. This was much less effective in the rnu/rnu rats, although there was some evidence of reduced numbers and stunting of parasites. Serum from infected rnu/rnu rats, unlike that from the infected rnu/+ rats, failed to induce the adherence of eosinophils to NEJ flukes in vitro. Flukes recovered from rnu/rnu rats were in general considerably larger than comparable flukes from their rnu/ + counterparts.There thus appears to be two distinct mechanisms of resistance to reinfection with F. hepatica operating in the rat. The first a T-independent system effective at the gut wall and the second, effective after penetration of the gut and requiring a T-dependent response for full expression. If eosinophils are involved in protection they can apparently function in the gut wall in the absence of an adherence promoting antibody in the serum.     

A 31P-NMR study of the intact liver fluke Fasciola hepatica

³¹P-NMR techniques offer a useful method of studying changes in the metabolism of intact parasitic worms. The liver flukes, Fasciola hepatica, provide good quality ³¹P high resolution NMR spectra for at least 6 h under anaerobic conditions. The levels of ATP remain constant throughout this period. There is no signal for phosphocreatine or phosphoarginine. In contrast to the findings in mammalian tissues, there is a distinct peak for the terminal phosphate of ADP. A number of signals are observed in the phosphodiester region of the spectrum the largest of which is identified as l-α-glycerophosphoryl choline. Serotonin (5-hydroxytryptamine) causes an appreciable increase in the levels of sugar phosphates when the flukes are incubated in the absence of glucose. The addition of glucose also causes a marked increase in the signals for the hexose phosphate.     

The impact of refugia on the development of thiabendazole resistance in Haemonchus contortus

Martin P. J., Le Jambre L. F. and Claxton J. H. 1981. The impact of refugia on the development of thiabendazole resistance in Haemonchus contortus. International Journal for Parasitology11: 35–41. Beginning with a mixture of 5% thiabendazole-resistant and 95% non thiabendazole-resistant Haemonchus contortus larvae, worm free sheep were artificially infected with either 10,000, 9000, 7000, 2500 or zero larvae. Four weeks later, the infected sheep were treated with 44 mg/kg of thiabendazole. Seven days after anthelmintic treatment the same sheep were infected with zero, 1000, 3000, 7500 or 10,000 larvae respectively so that each sheep was given a total of 10,000 larvae. The larvae given after thiabendazole treatment are referred to as being in refugia (defined as an area where individual members of a population can escape exposure to a drug). This was repeated for six generations of parasites, maintaining the same proportion of larvae in refugia in each generation. The egg hatch assay for resistance indicated that refugia delayed the development of resistance in Haemonchus contortus. Where none or a small proportion of larvae were in refugia, a rapid increase in resistance occurred. With an increased proportion of larvae in refugia, resistance was slower to develop. Population size, as estimated by faecal egg counts done after anthelmintic treatment and corrected for the number of larvae given in each treatment, was lower at the higher levels of refugia. This was due to a lower level of resistance in the higher refugia lines and consequently a greater effect of the drug. However, the estimate of population size, from egg counts done after the larvae introduced from refugia had matured, were similar in all lines.     

Fasciola hepatica: An immunofluorescent study of antigenic changes in the tegument during development in the rat and the sheep

Serum from sheep was collected throughout a 30-week period of infection with Fasciola hepatica and specificity for the tissues of flukes of various ages was tested by an indirect fluorescent antibody labeling technique, using as antigen JB4 plastic-embedded sections of flukes up to 30-weeks old grown in rats. Quantitative estimates of host antibody concentration and fluke tissue antigenicity were determined by titration using serially diluted serum. Serum from early infections (pre-7 weeks) gave strong labeling over the tegument of young flukes, but the reaction became progressively weaker with older fluke tissue. This was associated with a decline in the number of T1 bodies in the tegument as revealed by electron microscopy. T1 bodies contain glycocalyx precursor substances and during development they replace the antigenically similar T0 secretory bodies characteristic of early juvenile flukes. Glycocalyx turnover may help protect the pre-bile duct flukes against immunological attack. Serum from sheep with F. hepatica infections older than 7 weeks gave moderate reaction with T2 bodies which accumulated in the tegument during the early stages of infection but only expressed their antigens on the surface about the time of entry into the host's bile ducts. The antigenicity of the gut and excretory system of flukes seemed to remain unchanged throughout adult life. Levels of host antibody specific for juvenile tegument, gut, and excretory system peaked at 3–5 weeks postinfection, and declined once the flukes entered the bile ducts. Anti-T2 antibody appeared 6 weeks postinfection and began to decline 5–6 weeks later.     

Fasciola gigantica: Anthelmintic effect of the aqueous extract of Artocarpus lakoocha

The effect of the crude extract of Artocarpus lakoocha (70% composition is 2,4,3′,5′- tetrahydroxystilbene -THS) on adult Fasciola gigantica was evaluated after incubating the parasites in M-199 medium containing 250, 500, 750 and 1000 μg/ml of the crude extract, or triclabendazole (TCZ) at the concentrations of 80 and 175 μg/ml as the positive control, for 3, 6, 12 and 24 h, using relative motility (RM) assay and observation by scanning electron microscope (SEM). Decreased contraction and motility were first observed after 3 h incubation with TCZ at the concentration 80 and 175 μg/ml. TCZ markedly reduced the parasite’s motility at the concentration of 175 μg/ml at 6 h, and killed the worms after 12 h exposure. The crude extract of A. lakoocha at all concentrations reduced the parasite’s motility similar to TCZ at 3 h incubation. In 250 and 500 μg/ml of the crude extract, the values were decreased from 3 to 12 h, then they were stable between 12 and 24 h and reduced to the level approximately 30-40% of the control. At 750 and 1000 μg/ml concentrations the crude extract rapidly reduced the RM values from the start to 12 h and killed the parasites between 12 and 24 h incubation. The crude extract also inhibited the larval migration by 75% and 100% at the concentrations of 250-500 and 750-1000 μg/ml, respectively. TCZ and the crude extract caused sequentially changes in the tegument including swelling, followed by blebbings that later ruptured, leading to the erosion and desquamation of the tegument syncytium. As the result, lesion was formed which exposed the basal lamina. The damage appeared more severe on the dorsal than the ventral surface, and earlier on the anterior part and lateral margins when compared to the posterior part. The severity and rapidity of the damages were enhanced with increasing concentration of the crude extract. Hence, the crude extract of A. lakoocha, may exert its fasciolicidal effect against adult F. gigantica by initially causing the tegumental damage.     

Studies on substrate specificity and activity regulating factors of trehalose-6-phosphate synthase of Saccharomyces cerevisiae

Purified trehalose-6-phosphate synthase (TPS) of Saccharomyces cerevisiae was effective over a wide range of substrates, although differing with regard to their relative activity. Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity, particularly when a pyrimidine glucose nucleotide like UDPG was used, rather than a purine glucose nucleotide like GDPG. A high V_max and a low K_m value of UDPG show its greater affinity with TPS than GDPG or TDPG. Among the glucosyl acceptors TPS showed maximum activity with G-6-P which was followed by M-6-P and F-6-P. Effect of heparin was also extended to the purification of TPS activity, as it helped to retain both stability and activity of the final purified enzyme. Metal co-factors, specifically MnCl₂ and ZnCl₂ acted as stimulators, while enzyme inhibitors had very little effect on TPS activity. Metal chelators like CDTA, EGTA stimulated enzyme activity by chelation of metal inhibitors. Temperature and pH optima of the purified enzyme were determined to be 40 °C and pH 8.5 respectively. Enzyme activity was stable at 0–40 °C and at alkaline pH.     

Inhibitory effect of essential oils of Allium sativum and Piper longum on spontaneous muscular activity of liver fluke, Fasciola gigantica

Effects of essential oil of Allium sativum (garlic) and Piper longum (Indian long pepper) were evaluated on muscular activity of whole Fasciola gigantica and its strip preparation. The whole flukes and longitudinal strip preparations of the flukes were isometrically mounted to record the spontaneous muscular activity (SMA) and to evaluate effects of cumulative doses (0.1, 0.3, 1.0 and 3.0 mg/ml) of the plant essential oils. Whole flukes and the strip preparations exhibited continuous SMA without any significant difference in its baseline tension, frequency and amplitude for 2 h. Essential oil of A. sativum produced significant reduction in the frequency and the amplitude of the SMA of whole fluke at 1 and 3 mg/ml concentrations. It caused complete paralysis of the fluke after 15 min of administration of 3 mg/ml concentration. Similar to whole fluke, essential oil of A. sativum (3 mg/ml) also produced flaccid paralysis in the strip preparations of the flukes. Essential oil of P. longum firstly induced marked excitatory effect and then there was flaccid paralysis of the whole fluke following 15 min exposure at 3 mg/ml concentration. Complete flaccid paralysis of the strip preparation was also ensued after 15 min of administration of 3 mg/ml concentration of P. longum. In both the essential oils, the whole fluke and strip preparations did not recover from paralysis following 2-3 washes. In conclusion, the observations demonstrated irreversible paralytic effect of essential oils of A. sativum and P. longum on F. giganticain vitro which might possibly help to developing herbal-based anthelmintic.     

Kinetics of primary and secondary infections with Strongyloides ratti in mice

Dawkins H. J. S. and Grove D. I. 1981 Kinetics of primary and secondary infections with Strongyloides ratti in mice. International journal for Parasitology11: 89–96. The kinetics of infection with S. ratti were quantitated in normal and previously exposed C57B1 /6 mice. In primary infections, larvae penetrated the skin rapidly and were seen in peak numbers 12 h after infection. By 24 h after infection, larval numbers had declined appreciably and there was a slow decrease in numbers thereafter. Larvae were first observed in the lungs at 24 h and maximal recovery occurred at 48 h. It is thought that larval migration through the lungs is rapid. Worms were first seen in the intestines two days after infection. Maximum numbers were seen on the fifth day and worm expulsion was complete by day 10. Two moults took place in the small intestine during days 3 and 4 after infection. Rhabditiform larvae were first noted on the fourth day after infection. Mice exposed to S. ratti four weeks previously had significantly less larvae in the skin 4 and 12 h after infection but by 24 h there was no difference when compared with mice with primary infections. Peak recovery of larvae from the lungs occurred 24 h after infection; significantly less larvae were recovered on days 2 and 3 when compared with normal mice. There was a marked reduction in the adult worm burden in the gut; the number of worms recovered was less than one fifth of that seen in primary infections. Those worms which did mature were less fecund and were expelled from the intestines within 7 days of infection. It is suggested that in previously exposed animals, the migration of larvae from the skin is hastened, many of these larvae are destroyed in the lungs and that expulsion of worms which do mature in the intestines is accelerated.     

Ordering of C-terminal loop and glutaminase domains of glucosamine-6-phosphate synthase promotes sugar ring opening and formation of the ammonia channel

Glucosamine-6-phosphate synthase (GlmS) channels ammonia from glutamine at the glutaminase site to fructose 6-phosphate (Fru6P) at the synthase site. Escherichia coli GlmS is composed of two C-terminal synthase domains that form the dimer interface and two N-terminal glutaminase domains at its periphery. We report the crystal structures of GlmS alone and in complex with the glucosamine-6-phosphate product at 2.95 Å and 2.9 Å resolution, respectively. Surprisingly, although the whole protein is present in this crystal form, no electron density for the glutaminase domain was observed, indicating its mobility. Comparison of the two structures with that of the previously reported GlmS-Fru6P complex shows that, upon sugar binding, the C-terminal loop, which forms the major part of the channel walls, becomes ordered and covers the synthase site. The ordering of the glutaminase domains likely follows Fru6P binding by the anchoring of Trp74, which acts as the gate of the channel, on the closed C-terminal loop. This is accompanied by a major conformational change of the side chain of Lys503^# of the neighboring synthase domain that strengthens the interactions of the synthase domain with the C-terminal loop and completely shields the synthase site. The concomitant conformational change of the Lys503^#-Gly505^# tripeptide places catalytic His504^# in the proper position to open the sugar and buries the linear sugar, which is now in the vicinity of the catalytic groups involved in the sugar isomerization reaction. Together with the previously reported structures of GlmS in complex with Fru6P or glucose 6-phosphate and a glutamine analogue, the new structures reveal the structural changes occurring during the whole catalytic cycle.     

Experimental Fascioloides magna infections of mule deer (Odocoileus hemionus hemionus).

Six mule deer (Odocoileus hemionus hemionus) and one white-tailed deer (Odocoileus virginianus), approximately 5-mo-old, each were inoculated orally with 500 metacercariae of Fascioloides magna. All mule deer died from liver fluke infection between 69 and 134 days (mean = 114, SE = 9.9) after inoculation. Between 38 and 326 immature F. magna (mean = 102, SE = 45.5) were recovered from each deer at necropsy. Flukes were present in livers, lungs, and free in pleural and peritoneal spaces. Infection was characterized by necrotizing hepatitis, fibrosing peritonitis and pleuritis, and hematin pigment accumulation in liver, lung, and many other internal organs. Eggs of F. magna first were detected in feces of the white-tailed deer 28 wk after inoculation, and weekly thereafter until the healthy deer was euthanized at 31 wk. At necropsy, 205 F. magna, including 12 encapsulated mature and 193 nonencapsulated immature flukes were recovered from liver, lungs, and free in abdominal and thoracic spaces of the white-tailed deer. Based on these results, F. magna may be fatal to mule deer within 5 mo of infection. Like domestic sheep and goats, mule deer may be highly susceptible to infection, and it is unlikely mule deer can survive infection with large numbers of F. magna.     

Trypanosoma brucei: Biochemical and morphological changes during in vitro transformation of bloodstreamto procyclic-trypomastigotes

Trypanosoma brucei brucei in whole rat blood inoculated into a semidefined medium undergoes complete morphological transformation (observed by light microscopy) in 72 hr. This reproducible system permits the biochemical and physiological study of transformation from bloodstream to procyclic trypomastigotes and mitochondrial biogenesis in these organisms. Infectivity for mice is lost after 6 days. Proline stimulates cell growth after transformation. High levels of glucose adversely affect the growth of transforming cells. Respiration during transformation is by an α-glycerophosphate oxidase although a cyanide-sensitive pathway is present after 24–48 hr but does not become fully functional with respect to procyclic trypomastigotes until 20–24 days in culture. The success of this system will permit the biochemical characterization of African trypanosomes as the development of the cytochrome system occurs.