DPIRD approved fluke egg sedimentation test (FEST) procedure

Appendix 1: Validation report - validation of the faecal sedimentation method (PAM-26) for the detection of Fasciola hepatica eggs in faeces from cattle and horses, by Jeff Mitchell and Dieter Palmer

Final report

Introduction

Egg counts for the detection of liver fluke infection are routinely conducted in many laboratories. Because liver fluke infection is common in most parts of the world, methods for the detection of liver fluke eggs in faeces have concentrated mainly on the quantitative aspect of the assessment. Surprisingly, very few studies have validated the technique and to our knowledge only one study has attempted to determine the detection limit of the test.

The most commonly used technique is a sedimentation method described by Boray (1960) and later modified by Happich and Boray (1969). They found that the technique was able to detect eggs in all samples with more than 10 eggs/gram using 3g of sheep faeces. The percentage of recovered eggs was approximately 40%. Faeces with less than 10 eggs/g were not tested.

Breza and Corba (1973) compared a flotation-sedimentation method with the sedimentation method of Happich and Boray (1969) and found that in an analysis of 120 naturally infected animals, the flotation-sedimentation method had a sensitivity of 100% compared to 92% for the sedimentation method. The flotation-sedimentation method recovered up to 10 times more eggs in the samples than the sedimentation methods. However, the actual number of eggs present in the samples was unknown.

More recently a glass bead sieving method has been described for the detection of Fasciola hepatica eggs in faeces (Taira, 1985). The test was able to detect eggs in all samples that contained at least eight eggs/g faeces. The test examines only one gram of faeces. At a faecal egg concentration of one egg/g only 55% of all samples were positive.

The parasitology laboratories at the Department of Agriculture and Food, Western Australia (now DPIRD) use an in-house modification of the sedimentation method described by Boray (1960) and Happich and Boray (1969). A validation of our test method is important because we rely on the test to prevent liver fluke infected animals from entering WA.

Methods

Sheep faeces containing large number of Fasciola hepatica eggs were obtained from NSW Agriculture (courtesy of Dr Joan Lloyd and Dr Gareth Hutchinson). Eggs were purified using large-scale sieving followed by sedimentation.

Faeces from cattle and horses free of liver fluke infection were obtained and 4g or 10g of faecal samples were spiked with a known number of F. hepatica eggs. The samples were examined following our standard sedimentation technique (PAM-26), except that in all but one series of samples the use of the 90 micron sieve was omitted and only the 150 micron and the 38 micron sieve were used.

Results

The results are shown in Table 1. The detection limit for the examination of 10g of faeces from bovines or equines was 1 egg/g faeces or ½ egg/g respectively. If only samples of 4g of faeces were examined (cattle only), the detection limit was 10 eggs/g faeces. The percentage of eggs recovered varied from 0% to 68% depending on the total number of eggs present in the sample.

The inclusion of the additional 90 micron sieve did not decrease the sensitivity. Care has to be taken though to prevent overflow of the sieve when the samples are sieved through the series of sieves.