Other methods for processing herbage washings .1 Sieving followed by floatation

Separation of larvae by floatation utilises the specific gravity of larvae to separate them from debris. Larvae are suspended in a high concentration of iodine, sucrose, sodium chloride or sodium sulphate and they then float to the top of this solution while debris remains in or below the solution. Generally, these methods are effective for extracting larvae from faecal samples, but become less effective for assays of herbage samples as the plant material as well as the larvae tend to float on the dense solution. KRECEK et al. (1991) used a modification of a floatation method described by CAVENESS and JENSEN (1955) in which the herbage samples are Baermannised for 24 h at 25 °C prior to sugar float ation. This method resulted in a recovery of 24 - 27 % from grass and 44 % from soil samples.

2.4.2.2 Sieving followed by agar migration

Migration of worms from an agar gel into an overlying solution has been reported for recovering larval and adult stages. The major advantage of worm migration through agar is that the emerging worms are clean and well separated from sediment. The disadvantage is that only very active worms are recovered, which reduces recovery of lethargic species or older larvae.

JØRGENSEN (1975a) found that in the absence of grass or soil, bile activated infective larvae of D. viviparus showed 98 % recovery after 24 h at 38 °C from 2 % agar. This optimum recovery temperature of 38 °C (J ØRGENSEN 1975a) was higher than the reported value in a Baermann apparatus (see 2.4.1.2). Even when used to

recover larvae from grass, recovery of D. viviparus larvae was 60 - 80 % (JØRGENSEN 1975b). The higher recovery using agar migration as opposed to a Baermann funnel alone as reported in other papers may be due to higher efficiency in separating larvae from herbage debris rather than from the grass itself. Although extraction with agar is efficient for the young larvae of Dictyocaulus species, recoveries are low with older larvae, dropping from 60 - 70 % to 3 - 6 %, and are lower with H. contortus and Trichostrongylus spp. (EYSKER and KOOYMAN, 1993) and induced exsheathment in these species.

2.4.2.3 Agitation of grass using a washing machine followed by floatation

As most larvae appear to be lost at the filtration stage of separation from herbage, separation of washing solutions from herbage using centrifugation has been attempted. Results from these experiments appear to give varying results, ranging from a consistent recovery of 50 % to 18 - 41 % of larvae on herbage. EYSKER and KOOYMAN (1993) report recoveries of 68 - 77 % of L1 D. viviparus and 28 - 35 % of L3 H. contortus from herbage by washing the herbage with a 0.2 % teepol solution, followed by concentration of the washed herbage sediment by centrifugation and resuspension of the pellet in a sucrose solution with a density of 1.17. AUMONT et al. (1996) reported 55 % recovery of L3 H. contortus and Trichostrongylus spp. from herbage sediment using a sucrose solution, when the sieving step was replaced by washing herbage and extracting larvae in a washing machine. MATTHEE et al.

(2002) also reported the use of a washing machine to mechanically wash herbage to remove larvae followed by a sugar centrifugal floatation technique described by KRECEK et al. (1991). Unfortunately, recovery data were not reported for this study, but KRECEK and MAINGI (2004) used this method and reported systematically higher recoveries of 18 - 41 % of larvae from grass compared to 0 - 27 % with the Baermann method. The difference in recoveries between studies reported by AUMONT et al. (1996) and KRECEK and MAINGI (2004) may be due to the inclusion of a 25 µm sieve in the latter study, which was specifically avoided by AUMONT et al.

(1996).

2.5 Conclusions

It is well established that carbohydrate groups which could bind lectins are present on the surface of nematodes in some life cycle stages with variations between nematode species and life cycle stage. However, it is not clear whether the L3 stage of T. circumcincta and H. contortus express enough specific carbohydrate moieties on their surface to allow identification and differentiation by lectins, as the L3 stage in other species examined seems to bind lectins least. Experiments in this thesis will therefore examine the lectin binding by L3 in simple phosphate buffer. The lectin binding of adult worms will also be tested as this stage should bind lectins strongly due to heavy surface glycosylation and therefore act as a positive control for the lectin binding methods used for L3. Similarly, lectin binding to eggs will be tested as lectins have been shown to bind to eggs of sheep parasitic nematodes. Lectin binding to exsheathed L3 will also be tested, in case this stage binds lectins better than sheathed L3. In the case of weak binding to sheathed and exsheathed L3, attempts will be made to optimise to lectin binding conditions, to maximise possible binding of lectins to L3, exsheathed L3, adult and egg stages alike.

For the recovery of larvae from herbage, experiments will test whether floatation of larvae or filtering through Baermann funnel give the best recovery as a field method.

The most effective method reported for recovery of larvae from herbage seems to be the use of centrifugation to remove larvae from grass. However, this technique requires specialised equipment, so is not ideal for crude field testing. For all other methods tested filters or sieves are used to separate herbage from washing, but the greatest loss of larvae is at this stage. Therefore, this process rather than the separation of larvae from wash sediment needs to be optimised. Floatation methods will be tested using sucrose and iodine gradients. Baermann filtering using different detergents, different incubation times and temperatures and different filter types will be examined. Finally the best recovery method will be used to recover a mixed larval population which will then be examined using identifying lectins to show lectin binding and recovery methods can be combined as a field method.