D.B. Copeman

High resistance to experimental infection with Fasciola gigantica in Javanese thin-tailed sheep

Innate resistance of Javanese thin-tailed sheep to Fasciola gigantica was investigated in animals infected with single doses of 150 or 500 metacercariae and killed 4, 8, 12 or 16 weeks after infection. Infected and non-infected sheep had similar values for packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin concentration, serum glutamate dehydrogenase, serum gamma glutamyl transferase and serum aspartate transferase throughout the trial, except for one animal infected with 500 metacercariae from which the highest recovery of flukes (55) was made. This animal developed pathologically altered values from 12 weeks post infection, coincident with the period of greatest hepatic haemorrhage and destruction of hepatic tissue by migrating flukes and their entry into bile ducts. However, values were altered much less than those reported in other sheep given as few as 200 metacercaria of F. gigantica. Both susceptibility to infection with F. gigantica, as indicated by percentage take of metacercariae and the severity of pathological changes were low in this study in comparison with reports involving other breeds of sheep infected with this parasite. These findings support the conclusion that Javanese thin-tailed sheep have a high innate resistance to F. gigantica.

A Possible Role for Rusa Deer (Cervus timorensis russa) and Wild Pigs in Spread of Trypanosoma evansi from Indonesia to Papua New Guinea

Movement of transmigrants and livestock from western Indonesia to southeastern areas of Irian Jaya near the border with Papua New Guinea may pose a risk of introducing Trypanosoma evansi into Papua New Guinea via feral Rusa deer (Cervus timorensis russa) and wild pigs which inhabit these areas in large numbers. Pilot experimental studies were conducted to observe infection in pigs and Rusa deer with a strain of T. evansi isolated in Indonesia. Parasitaemia and signs of clinical disease were monitored each second day for 120 days. Trypanosomes were observed in haematocrit tubes at the plasma-buffy coat interface of jugular blood of deer and pigs on 86% and 37% of sampling occasions respectively. Parasitaemia was at a high level in deer for 35% of the time but for only 11.5% of the time in pigs. Results indicate that both Rusa deer and pigs have a high tolerance for infection with T. evansi. The deer suffered mild anaemia evidenced by a 25% reduction in packed cell volume (PCV) 14 days after infection which coincided with the initial peak in parasitaemia. However, PCV had returned to pre infection values by the end of the experiment. The pigs showed no change in PCV. There were no visual indications of disease in either species and appetite was not noticeably affected. It was concluded that both Rusa deer and pigs were capable reservoir hosts for T. evansi but that Rusa deer, with their more persistent higher levels of parasitaemia, have more potential to spread T. evansi into Papua New Guinea from West Irian than pigs.

Evaluation and improvement of parasitological tests for Trypanosoma evansi infection

Research was undertaken to critically evaluate parasitological tests for the detection of Trypanosoma evansi in blood. The relative sensitivity of mouse inoculation (MI), the haematocrit centrifugation technique (HCT) and a modified miniature anion-exchange centrifugation technique (MAECT) were compared using blood and buffy coat. The effect that storage of blood prior to inoculation into mice has on the reliability of the MI test was also evaluated. The tests may be ranked in increasing order of sensitivity: HCT, MAECT with whole blood, MI with whole blood, MAECT with buffy coat and MI with buffy coat. The latter was able to detect 1.25 T. evansi per 4ml of blood. The reliability of the MI test was not reduced with storage of blood containing at least 25 T. evansi per ml for up to 21h prior to inoculation into mice. These results demonstrate that sensitivity of the MI and MAECT are increased approximately 10-fold through the use of buffy coat in place of whole blood. Although, the MI is marginally more sensitive MAECT is better suited to field use.

Evaluation of an antibody-ELISA using five crude antigen preparations for the diagnosis of Trypanosoma evansi infection in cattle

Attempts were made to improve the accuracy of an antibody-detection ELISA for the detection of Trypanosoma evansi infection in cattle by improving the method of preparation of the crude antigen used. An IgG-ELISA was performed with five different antigen preparations: crude soluble antigen, soluble and insoluble fractions of crude antigen treated with 0.1% formalin and whole formalin-fixed trypanosomes treated with either trypsin or 2-mercaptoethanol. An IgM-ELISA using crude soluble antigen was also performed. Each ELISA was evaluated using serum from 44 Indonesian cattle infected with T. evansi and 262 uninfected cattle from Australia. There was no significant difference between the sensitivity or specificity of the IgG-ELISA using each of the five antigens. The IgM-ELISA using a crude untreated lysate was significantly less sensitive (p<0.05) than the IgG-ELISA using the same antigen, trypsin-treated antigen or the 0.1% formalin-treated soluble antigen (68, 64 and 64%, respectively). These results show that these modifications to the method of producing crude antigens for the Ab-ELISA does not improve the accuracy of diagnosis of T. evansi infection in cattle.

The effect of temperature and humidity on longevity of metacercariae of Fasciola gigantica

Studies were undertaken on the viability of the metacercariae of Fasciola gigantica when stored in water at 13˚C for periods up to 23 weeks, exposed to the sunlight for up to 8 h or stored at a range of temperatures and humidities for up to 10 weeks. Excysted metacercariae were catergorized microscopically as viable (motile and undamaged), dubious (not motile and undamaged) or dead (visible necrosis). The infectivity of viable and dubious metacercariae and unselected reference metacercariae held in water at 7˚C for 20 days or longer was assessed by comparing numbers of flukes recovered from infected Merino sheep. Mean recovery rates were 54.6%, 7.2% and 37.2%, respectively, for viable, dubious and unselected metacercariae. Metacercariae immersed in water remained viable longer than those allowed to desiccate. Viability was promoted by decreasing temperature and increasing humidity. Exposure to direct sunlight killed metacercariae within 8 h. Results indicated that in lowland Indonesian irrigated rice paddies, metacercariae immersed in water are likely to survive for less than 5 weeks while those that become desiccated will survive less than 2 weeks. This information, together with the option of exposing fresh rice stalks to direct sunlight before feeding them to livestock, can assist farmers in reducing infection with F. gigantica.

Distribution of Metacercariae of Fasciola gigantica on rice straw

Rice straw and stubble are important sources of fodder for cattle and buffaloes that are kept in areas of Indonesia that produce irrigated rice. Two factors contribute to make use of such fodder a source of infection with F. gigantica: the suitability of irrigated rice fields as a habitat for the aquatic snail intermediate host of F. gigantica, and the practice of using dung from cattle and buffaloes to fertilize rice fields. There are a number of published reports indicating that cercariae of F. gigantica encyst on objects (preferably green plants) just under the surface of the water (Ueno and Yoshihara, 1974; Dumag et al., 1976) and this seems likely to be the case with rice stems. Confirmation that this is so would mean that control could be achieved by feed-

Variations in the survival of Fasciola gigantica eggs in bovine dung stored in the sun as opposed to the shade

Eggs of F. gigantica were placed in dung heaps that were located in the shade or exposed to sun light, and examined at intervals for up to 14 weeks. The rate and extent of decline in viability of eggs was greater in dung exposed to sun light than in shaded dung. This difference was attributed the higher temperature in dung in sun light, owing to the effect of direct sunlight and to a higher rate of fermentation in exposed than in shaded dung. It was concluded that strategies for storing dung that would reduce the risk it poses for infecting L. rubiginosa with F. gigantica when used as fertilizer in rice fields include storing dung in sun light rather than in the shade, preferably in a thin layer to allow sunlight to heat and desiccate it, and mixing a carbohydrate with the stored dung to increase heat through fermentation.

Optimization of the procedure for counting the eggs of Fasciola gigantica in bovine faeces

This paper describes a method for counting eggs of F. gigantica in bovine faeces that optimizes the proportion of eggs recovered and the repeatability of estimates. The method uses 3 g of faeces suspended in 0.05% Tween 20. The suspension is passed through three 6 cm diameter sieves in tandem to remove fibrous debris, with respective apertures of 1 mm, 450 μm, and either 266 or 200 μm. The filtrate is allowed to sediment for 3 min in a conical flask; the sediment is recovered, then resuspended in 200 ml of 0.05% Tween 20 and allowed to sediment. After 3 min the sediment is washed in a sieve with an aperture of 53 μm, which retains the eggs. Eggs suspended in 15 ml of 1% methylene blue are counted using a dissecting microscope. Use of Tween 20 instead of water as the suspending agent for faeces gave a significant threefold increased the proportion of eggs recovered and reduced variability between repeated counts. This method is able to detect about one-third of the eggs present. It was concluded that the high proportion of F. gigantica eggs lost may be due to the presence of hydrophobic and covalent bonds on the eggs that bind them to debris, with which they are discarded.