World association for the advancement of veterinary parasitology (WAAVP): second edition of guidelines for evaluating the efficacy of anthelmintics in swine.

Guidelines are provided for evaluating the efficacy of anthelmintics in swine which, in conjunction with other sets of guidance such as those of the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH GL7 and VICH GL16), should encourage the adoption of uniform registration requirements globally. Testing of efficacy should be carried out according to the principles of "Good Clinical Practice" (VICH GL9, 2000). Data obtained according to these guidelines should be internationally acceptable for the registration of anthelmintics for swine. Further, the use of the guidelines should expedite development, government review, and approval of anthelmintics for swine, as well as contribute towards reducing costs and the number of experimental animals used for drug testing.

Response of two isolates of Fasciola hepatica to treatment with triclabendazole in vivo and in vitro.

In this study, the susceptibility of two isolates of Fasciola hepatica--the Fairhurst and Oberon isolates--to treatment with triclabendazole was investigated, both in vivo and in vitro. The Fairhurst isolate originated in England, but has since been maintained in Australia; the Oberon isolate originated in Australia. Triclabendazole had a very high efficacy against the Fairhurst isolate. In sheep (dose: 10 mg/kg), the efficacy ranged from 78.4% at 2 weeks post-infection to 98.5% at 6 weeks post-infection. In cattle (dose: 12 mg/kg) efficacy was 89% at 2 weeks post-infection and 100% at 12 weeks. In contrast, against the Oberon isolate, triclabendazole had 0% efficacy against 2-week-old flukes in sheep (dose: 10 mg/kg) and 5% against 4-week-old flukes. Surface changes to flukes of the two isolates were assessed by scanning electron microscopy following treatment in vitro for 24 h in triclabendazole sulphoxide (15 and 50 microg/ml). Disruption took the form of blebbing, swelling and furrowing of the tegument and was greater in the Fairhurst than the Oberon isolate. Surface changes generally were more severe in the anterior than posterior region of the fluke and the dorsal surface was also consistently more severely affected than the ventral surface. Disruption was more severe at the higher drug concentration for both isolates. The morphological data is consistent with the efficacy data, which indicates that the Fairhurst isolate of F. hepatica is susceptible to triclabendazole treatment, whilst the Oberon isolate is refractory.

Fasciolicides: efficacy, actions, resistance and its management.

The modes of action of fasciolicides are described. Closantel and other salicylanilides interfere with energy metabolism by uncoupling oxidative phosphorylation in the fluke. Other fasciolicides are believed to have a metabolic action-halogenated phenols (via uncoupling) and clorsulon (via inhibition of glycolysis)-but direct evidence is lacking. Benzimidazoles (in particular, triclabendazole) bind to fluke tubulin and disrupt microtubule-based processes. Diamphenethide inhibits protein synthesis in the fluke. Other potential drug actions may contribute to overall drug efficacy. In particular, a number of fasciolicides-salicylanilides, phenols, diamphenethide-induce a rapid paralysis of the fluke, so their action may have a neuromuscular basis, although the actions remain ill-defined. Resistance to salicylanilides and triclabendazole has been detected in the field, although drug resistance does not appear to be a major problem yet. Strategies to minimize the development of resistance include the use of synergistic drug combinations, together with the design of integrated management programmes and the search for alternatives to drugs, in particular, vaccines.

Fasciola hepatica infection in farmed emus (Dromaius novaehollandiae).

OBJECTIVE: To describe two cases of infection with Fasciola hepatica in young farmed emus, subacute and chronic fasciolosis and a response to treatment of the flock with albendazole. PROCEDURE: Gross lesions were found at necropsy and hepatic lesions in microscopic examination. The parasite recovered from one emu was identified by its morphological characteristics and an egg count reduction test was carried out after treatment of the flock with albendazole. RESULTS: Hepatic lesions resembling subacute and chronic fasciolosis of ruminants were identified. An adult fluke was recovered from the liver of one of the birds and was identified as F hepatica. The eggs of the fluke were irregular in shape and size. No fluke eggs were identifiable in faeces of live emus 10 days after treatment of the flock with albendazole at a dose of 10 mg/kg. CONCLUSIONS: This is the first reported case of infection with F hepatica in farmed emus and the first report of the occurrence of Fasciola infection is the class Aves. The irregular shape and size of the eggs may be attributable to infection of an aberrant host. Treatment with albendazole eliminated eggs from the faeces of the flock.

Zoonotic potential of infection with Fasciola spp. by consumption of freshly prepared raw liver containing immature flukes.

Mice were successfully infected with metacercariae of the Japanese Fasciola sp., resulting in the recovery of a mean number of 110 live immature flukes per mouse 4-5 days after inoculation. Twenty-four mice were then inoculated orally, each with a mean number of 68 freshly recovered immature flukes. The livers of 7 of the 24 recipient mice showed migratory lesions of capsular and subcapsular granulomatous infiltration and 2 of those mice also had haemorrhagic lesions. The lesions were typical of those caused by active migration of early immature flukes. However, no flukes were found in the livers of the recipient mice at necropsy when the flukes were aged 14 weeks. In another experiment, 10 piglets were given fresh livers of mice harbouring approximately 2000 live immature flukes aged 3-7 days. Two additional piglets were inoculated with 2000 metacercariae of Fasciola. All pigs were killed when the flukes were 14 days old. Granulomatous lesions were present in all pigs, except in those that were given livers containing flukes aged 7 days. The lesions were localized, forming well-defined foci, different from the typical migratory lesions normally observed in mouse or sheep liver at the early stage of fluke migration. From the 10 pigs given livers, 65 live flukes were recovered at necropsy, 0.29% of the estimated number of immature flukes given. From the 2 pigs which received 2000 metacercariae each, a total of 198 flukes were recovered (5%). The results of the experiments suggest that humans consuming raw liver dishes prepared from fresh livers infected with immature Fasciola spp. could become infected with liver fluke.

Fasciola hepatica infection in sheep: changes in liver metabolism.

Several aspects of liver function during infection with Fasciola hepatica were examined in sheep four weeks after infection and compared with the changes observed in infected rats. Previously reported respiratory abnormalities in mitochondria isolated from the left lobe of the liver of infected sheep were characterised further. Evidence is presented that the respiratory lesion is located in the mitochondrial electron transport chain and that the aberrant respiratory behaviour is not associated with an increase in nonesterified fatty acids and the depletion of mitochondrial phospholipids, as is the case in the rat. Microsomal membranes, which have also been shown to be depleted of phospholipids in the fluke-infected rat liver, showed no such changes in the sheep. However, in common with the rat, a substantial loss of cytochrome P450 was recorded in microsomes prepared from the left lobe, and the glycogen content of the left lobe was found to be less than 50 per cent of control values. No change was observed in glucose 6-phosphatase activity. All these changes were localised effects, confined to areas of fluke infiltration.

Pyrethroid resistance in Australian field populations of the sheep body louse, Bovicola (Damalinia) ovis.

Synthetic pyrethroid (SP) resistance has developed in Australian field populations of the sheep body louse, Bovicola (Damalinia) ovis. Laboratory bioassays were used to measure the susceptibility of lice to cypermethrin and the other registered SPs. Results of these bioassays indicated resistance to cypermethrin, deltamethrin, cyhalothrin and alphacypermethrin. So far, high-level resistance has been diagnosed in only a few strains. The toxicological responses of these strains were clearly separated from those of the majority of louse strains tested. Furthermore, these strains had survived immersion in commercial SP dips. The level of resistance described in some strains was sufficient to cause pour-on products to fail despite the fact that the LC50s of these strains fell within the normal range of field responses.

Pathology of infection with Paramphistomum ichikawai in sheep.

Lambs were given 40,000 or 50,000 metacercariae of Paramphistomum ichikawai by injection into the rumen and necropsied at 21, 42 and 84 days after infection. Pathological changes were observed grossly and confirmed histologically in the small intestine and rumen. The numbers of flukes and their location in the gastrointestinal tract were recorded and the populations of eosinophils, mast cells and globule leucocytes estimated. Changes varied, according to the numbers of flukes present, from a localised enteritis and villous atrophy in the duodenum in light infections to severe destruction of the mucosa extending into most of the jejunum in heavy infections. As the infection progressed changes were characterised by extensive thickening and fibroplasia in the mucosa and submucosa. Severe damage to the mucosa of the rumen was also observed in heavy infections. Heavy infections were associated with increased infiltration with eosinophils. Mast cells were generally depleted and globule leucocytes only appeared after the flukes had left the small intestine. Migration of the flukes from the small intestine was delayed in heavy infections exacerbating the effect of the infection. It is suggested that the presence of 20,000 to 25,000 flukes would result in clinical disease; smaller numbers would cause significant subclinical disease.

Glutathione S-transferases as markers of salicylanilide resistance in isolates of Fasciola hepatica.

A possible link between the level of glutathione S-transferase (GST, E.C. 2.5.1.18) activity and the development of salicylanilide resistance in Fasciola hepatica was investigated. Various isolates of F. hepatica with varying susceptibilities to salicylanilides were isolated and maintained in the laboratory. Individual flukes of these isolates were surveyed for their level of GST activity and a correlation between the level of GST activity and drug efficacy was found. In contrast to most other studies, a decrease in GST activity was associated with an increase in drug resistance. Evidence was collected to show that this may be a selective process since flukes which had survived exposure to rafoxanide and closantel in vivo (in sheep) had lower activity levels of GST than flukes from untreated sheep. Treatment with other flukicides (oxyclozanide, luxabendazole and triclabendazole) did not have this effect. Furthermore, in vivo treatment with closantel induced selection of particular isoenzymes in different isolates of F. hepatica having different degrees of susceptibility to closantel. However, no single isoenzyme or isoenzyme profile was associated with resistance and, in total, up to 8 different isoenzymes could be present in a single isolate. Thus, GST has some potential as a marker enzyme for salicylanilide resistance in F. hepatica. However, the precise role of GST in resistance is unclear and the extensive inter- and intra-isolate variation in activity levels and isoenzyme characteristics of this enzyme indicate the need for considerably more study before application in field situations.

Host effects on glutathione S-transferase activity in Fasciola hepatica.

Glutathione S-transferases (GST, E.C. 2.5.1.18) in Fasciola hepatica from sheep were previously found to be extremely variable with regard to specific GST activity and isoenzyme profile within and between parasite isolates. The effect of the host on GST activity and isoenzyme profile was examined by infecting mice, rats and cattle as well as sheep with one or the other of two isolates--either salicylanilide-resistant or salicylanilide-susceptible F. hepatica. In the case of both isolates, GST activity in hosts relatively resistant to reinfection--rats and cattle--was lower and more restricted in range compared with hosts susceptible to multiple infection--mice and sheep. In the case of the rat flukes, there was little variation in isozyme profiles whereas cattle flukes appeared to exhibit more variation than sheep flukes. In mice, despite the apparent variability in GST activity, only one GST band was found in the isoenzyme profiles. Therefore, the host appears to exert a pronounced effect on the activity and expression of GSTs in F. hepatica which may be related to variation in the immune responses of the different hosts during infection.

Prevalence of the causes of fleece derangement among sheep flocks in New South Wales.

An investigation of sheep flocks with unknown histories of fleece derangement, selected at random from the main sheep raising areas of New South Wales, showed that Psorergates ovis was present in 53 of the 177 flocks surveyed and in 162 of the 3495 sheep examined. Of the single causes of fleece derangement, the most prevalent was grass seed contamination (15.7%) followed by infestation with sheep body lice, Bovicola ovis, (14.7%) and infestation with itch mite (7.8%). The prevalence of fleece derangement was significantly higher on sheep in which lice were the only ectoparasites found than on those in which itch mites were the only ectoparasites present. The greatest prevalence of P ovis was in flocks in the Young, Yass, Mudgee and Tenterfield districts. In most affected flocks, more than one potential cause of fleece derangement was present. Itch mite infestation was more prevalent in flocks that received no treatment for ectoparasites than in those that had been treated in the year preceding the survey. Our results did not support a commonly held belief among farmers that arsenical compounds control infestations of itch mites more effectively than other types of pesticides. There was no significant difference in prevalence of fleece derangement between flocks treated with different pesticide groups and those that received no treatment, but mean fleece derangement score was significantly lower in sheep treated with organophosphates than in untreated sheep. Infestations of lice were significantly more prevalent in sheep from flocks treated with synthetic pyrethroids than from those treated with organophosphates or those that received no treatment.

Antipyrine, erythromycin and oxytetracycline disposition in experimental fasciolosis.

The effects of fasciolosis on drug disposition were studied by administration of antipyrine, erythromycin and oxytetracycline to sheep and cattle. Fasciolosis was produced by administration of 200 or 400 metacercariae (MC) of Fasciola hepatica to sheep and 500 MC to cattle. The disease was subsequently confirmed by determination of plasma glutamate dehydrogenase and gamma-glutamyl transferase and identification and quantitation of mature flukes in the liver at necropsy. Acute or subacute fasciolosis in sheep was accompanied by a significant decrease in the elimination rate constant (beta) and increase in the elimination half-time (t 1/2) for antipyrine and erythromycin when compared with controls or infected sheep which had been treated with the anthelmintic luxabendazole. An increase in apparent volume of distribution (Vd) was seen only for erythromycin in sheep given 400 MC. There were no changes in the disposition of oxytetracycline in sheep with either acute or subacute infection and no effects on disposition of the three test drugs in chronically infected sheep. With early chronic disease in calves, only the disposition of oxytetracycline was affected; not that of antipyrine or erythromycin.

Resistance to synthetic pyrethroid pour-on insecticides in strains of the sheep body louse Bovicola (Damalinia) ovis.

Groups of sheep infested with strains of Bovicola (Damalinia) ovis were obtained from flocks either with a history of failure to control lice with synthetic pyrethroid (SP) pour-on insecticides, or from farms where SP compounds were not used. The sheep were treated according to the manufacturer's recommendations with registered "off-shears" SP formulations. All treatments were applied under ideal conditions with doses calculated on an individual body weight basis and applied to the dorsal mid-line from the base of the neck to the butt of the tail. Treated sheep were kept in pens and maintained in separate groups. The pour-on SP treatments significantly reduced the lice population but failed to eliminate the infestation in 7 of 13 experiments in sheep carrying strains of lice with resistance factors of greater than 4 to at least one of the SP compounds. Failures occurred with all three of the SP pour-ons currently registered for lice control in NSW and with both water-based and organic solvent-based formulations.

Fasciola hepatica: free radical generation by peritoneal leukocytes in challenged rodents.

Free radical generation by peritoneal leukocytes from hosts able to develop resistance to reinfection with Fasciola hepatica (rats) was compared with that of hosts unable to develop resistance (mice). Free radical generation by rat leukocytes was 3.5 times higher per cell and 30 times higher per animal than radical production by mouse leukocytes. The capacity of peritoneal leukocytes to produce free radicals in response to adult fluke crude antigen was increased by the presence of host plasma and was quantitatively greater in challenged rats than in naive or primary infected rats. This was not the case for mice, in which cells from primary infected animals were equally as responsive as cells from challenged mice. Further experiments revealed that challenge infection in rats apparently caused the in vivo activation of peritoneal leukocytes and increased levels of unidentified factors in plasma and that both of these responses were involved in the initiation of free radical generation in response to F. hepatica. Dramatic increases in the number of eosinophils present in the peritoneal cavities of primary infected and challenged rats (but not mice) were observed but the role of eosinophils in the production of free radicals in response to F. hepatica remains to be determined.

Human fascioliasis successfully treated with triclabendazole.

Fascioliasis, caused by the common liver fluke Fasciola hepatica, is an endemic infection in sheep and cattle in many parts of Australia. Infections in humans in Australia have been reported infrequently and the most appropriate therapy for human fascioliasis remains to be determined. This case report describes a patient with the infection unsuccessfully treated with multiple high doses of praziquantel, mebendazole or albendazole. The infection was successfully eliminated by the administration of triclabendazole in two single doses of 900 mg (12 mg/kg) two days apart. No side effects were observed after the treatment. Based on our experience and that recently reported in the literature, triclabendazole may be regarded as the drug of choice for the treatment of human fascioliasis.

Epidemiology of paramphistomosis in cattle.

The epidemiology of paramphistomosis in cattle was studied using tracer calves in a subtropical location in eastern Australia. Two species of paramphistomes were present; Calicophoron calicophorum and Paramphistomum ichikawai. The former species was the most abundant. Gyraulus scottianus and Helicorbis australiensis acted as intermediate hosts, respectively. Paramphistome burdens varied seasonally and were dependent upon the number of infected host snails. Peak fluke burdens and clinical paramphistomosis occurred in late summer in year 1 and early winter in year 2. The peak fluke burdens coincided with prolonged inundation of the grazing areas resulting in rapid multiplication and infection of host snails, and the period after the inundated areas dried out. The prevalence of infection in snails was high in both years, peaking at 98% in year 1 and 58% in year 2. The main host snail, G. scottianus, aestivated and retained infection for at least 24 weeks in soil, and in vegetable debris on the surface of the soil, resulting in rapid reappearance of host snails and infective metacercariae after the onset of seasonal rain. Metacercariae survived on herbage for up to 12 weeks, depending on the environmental conditions. Paramphistome burdens in calves could be predicted from the prevalence of infection in the host snail, the water levels and an index of surface water on the grazing site. Control of paramphistomosis during and after flooding may be achieved by removal of susceptible cattle from pasture or regular treatment during these periods. Strategic treatment during the dry season may reduce contamination of snail habitats and infectivity of the pasture in the following wet season.

Resistance of internal and external parasites to antiparasitic drugs.

Resistance to drugs and pesticides among internal and external parasites is an increasingly widespread problem both geographically and taxonomically. The development of highly effective pesticides by the chemical industry has led to an overreliance on chemotherapy aimed at maximum animal production or public health improvement through parasite control. Resistance to antiparasitic agents has developed in protozoa, helminths and insects. Considerable research efforts are being made around the world in an attempt to formulate sustainable programs of parasite control and resistance management. More than 120 delegates attended a spirited discussion at a Round table on resistance of parasites to antiparasitic drugs at ICOPA VII. Herein we attempt to summarise the main points made at the meeting. The complete texts of the papers submitted for discussion are being published: Resistance of Parasite to Antiparasitic Drugs (Boray, Martin and Roush, editors), MSD AGVET Division of Merck and Co. Inc. Rahway, NJ.

The prevalence of itchmite, Psorergates ovis, among sheep flocks with a history of fleece derangement.

An investigation of sheep flocks in the main sheep raising areas of New South Wales showed that the itchmite Psorergates ovis was frequently associated with fleece derangement. In 26 of the 41 flocks examined, P. ovis was the only ectoparasite detected. P. ovis and the sheep body louse Damalinia ovis, were found in 5 flocks. No external parasites were found on sheep examined from the 10 remaining flocks. The type of fleece derangement most frequently recorded was rubbing which in some cases was combined with areas of chewed fleece. Among flocks, there were positive relations between the prevalence of fleece derangement and prevalence of itchmite or scurf and between itchmite count and mean scurf score. Within flocks, itchmite infested sheep or sheep with scurf had higher prevalences of fleece derangement than sheep on which no mites or no scurf were found. Itchmite infested sheep had a higher prevalence of scurf than those with no detectable mite infestation. There were no significant differences in itchmite populations or fleece derangement between untreated flocks and flocks treated with synthetic pyrethroids, organophosphates or arsenic and rotenone.