At low temperatures third stage trichostrongylid larvae survIve on pasture for protracted periods, presumably due to the Iow rate of utilization of stored energy (Seghetti, 1 948; Kates, 1 950; Rose, 1 963; Gibson and Everett, 1 967; Donald, 1 968; Boag and Thomas, 1 970; Andersen et aI. , 1 970; Levine and Andersen, 1 973 ; Gibson and Everett, 1 976; Southcott et aI. , 1 976; Donald et aI. , 1 978). Similarly, prolonged period of snow during winter tends to preserve the larvae rather than killing them (Gibson and Everett, 1 967). As the temperatures and resultant metabolic rates rise, energy is utilized with increased speed, leading to a progressively reduced longevity (Kates, 1 950; Levine, 1 963 ; Rose, 1 963 ; Andersen et aI. , 1 965; Anderson, 1 972 & 1 973 ; Callinan, 1 978; Banks et aI., 1 990). The larvae of Ostertagia spp. survive longer than T colubriformis (Kates, 1 950; Gibson and Everett, 1 972; Pandey, 1 972; Boag and Thomas, 1 977; WaIler and Thomas, 1 978a; Boag and Thomas, 1 985; Jasmer et aI. , 1 987). A trial carried out with T colubrtformis revealed that the optimum survival temperature of all free-living stages was 4°C with 95% of infective larvae surviving for 3 1 2 days (Andersen et aI, 1 966). The infective larvae survive better than other free-living stages and embryonated eggs survive longer than unembryonated eggs (Stewart and Douglas, 1 93 8 ; Andersen et aI, 1 966)
The infective larvae of 0. circumcincta survive better at lower relative humidity (RH
of 30 and 50%) compared to higher relative humidity (RH of 75 and 95%) at 1 6°C,
25°C and 3 5°C (Pandey et aI. , 1 993). In other trichostrongylids such as T
colubriformis (Andersen and Levine, 1 968) and O. ostertagi (Pandey, 1 976), it has been shown that desiccated infective larvae survive better than non-desiccated larvae. However, age of the infective larvae (storage at 4°C) of T colubriformis can decrease their ability to withstand desiccation (Andersen and Levine, 1 968).
Genetic differences between the different strains of the same species also influence the survival of infective larvae. The Weybridge isolate of 0. circumcincta (Pandey et al. , 1 993 ) is more resistant to low temperatures than the Washington (Jasmer et al., 1 987) and the California isolates(Furman, 1 944) .
Larvae can survive in soil and have been shown to reappear on herbage at various times (Bairden et al. , 1 979; Al S aqur et al. , 1 982). A study with T vitrinus (Rose and Small, 1 985) demonstrated that infective larvae buried in the soil of grass plots, to a depth of 1 0 cms, migrated to the herbage throughout the year. However, only a limited
amount of upward migration of T colubrtformis larvae was observed (Sturrock, 1 965).
The type of the soil can also influence the upward migration of the larvae. In a study
with 0. circllmcincta, Furman ( 1 944) showed that larvae migrated upward most
readily in sandy soil, less so in sandy loam and least in clay loam. They failed to migrate through dry soil.
1. 1 4.5. MIGRATION OF LARVAE
Infective third stage larvae use the surface tension of a moisture films on leaves to migrate vertically on herbage (Croll, 1 970). A higher relative humidity and higher temperature also favour the upward migration of larvae (Silangwa and Todd, 1 964). Moisture above 0. 1 5 ml/cm2 on the leaf surface will obstruct the migration of most
common species of gastrointestinal nematodes (Rogers, 1 940). The amount of
moisture on the leaf surface also have differential effect on migration of different nematode species; for example, H. contortus require less moisture on the leaf surface to migrate than 0. circllmcincta (Rogers, 1 940). A good pasture transmission for T.
colllbriformis migration occurred when total monthly precipitation was more than 2 5
mm and the mean monthly temperature at the soil surface beneath 7- 1 0 cm vegetation
was above 1 6°C (Levine and Andersen, 1 973). Similarly, Wal lace and Doncaster ( 1 964) demonstrated that migration of T. colubriformis was most rapid in water saturated soil at 20°e.
The type of herbage species has also been shown to influence larval migration. The vertical migration ofL3 of T. colubriformis and 0. circumcincta was found to be better on lucerne and white clover whereas few larvae migrated up the swards of 'iorkshire fog (Niezen et al. , 1 998). These differences were considered to be due to different
amounts of moisture on the leaf surfaces of different plant species (Niezen el a!., 1 998). Similar studies also observed that larvae could migrate further on some grass species than others (Crofion, 1 948; Knapp, 1 963 ; Silangwa and Todd, 1 964; Moss and Vlassoff, 1 993).
1 .15. FITNESS OF ANTHELMINTIC-RESISTANT NEMATODES
There are very few reported studies of fitness differences between resistant and susceptible genotypes of nematodes. Most studies are aimed at investigating the fitness status or pattern of reversion to susceptibility, generally involves benzimidazole resistant strains. A fitness study with benzimidazole-resistant H. contortus showed that the benzimidazole-resistant strains were more infective for sheep than the benzimidazole-susceptible strain. Faecal egg output, development and survival of eggs and free-living stages on pasture were also higher in the benzimidazole-resistant
ex..
strains (Kelly et aI., 1 978). Similarly, �higher establishment rate was recorded in sheep
infected with phenothiazine-resistant H. contortus as compared to phenothiazine
�t
susceptible strain (Drudge� 1 957b). Recently, in a fitness study of benzimidazole- resistant and -susceptible worms of 0. circumcincta, '" PCR technique was used to determine the genotypes (RR, SS, RS) and fitness of each was compared within the same strain (Elard et a!., 1 998). There was no significant difference in egg production, development rate from to eggs to infective larvae under laboratory conditions, establishment rate at 3 5 days post infection, survival rate of adult worms in lambs at 60 days post infection and survival of infective larvae at 8°C in laboratory conditions. In contrast to these findings, Maingi et a!. ( 1990) demonstrated that thiabendazole susceptible strain of H. contortus had a higher establishment rate, faecal egg count and more severe pathology than a thiabendazole-resistant strain of H. contorlus. However, further selection of thiabendazole-resistant strain for four generations produced similar establishment rate, faecal egg counts and pathology comparable to tho5Ji! produced by susceptible strain.
In a study with H. contortus, the control ofr-resistant strain on pasture was tried by
replacing the resistant-strain with a susceptible strain (Van Wyk and Van Schalkwyk, 1 990). Reversion to susceptibility occurred in only three out of five camps indicating that fitness of the resistant genotype was not at a selective disadvantage. Similarly, in a
fi eld study, Martin et a!. ( 1 988a) did not observe any significant reverSIOn to susceptibility of a benzimidazole-resistant strain of Ostertagia spp . over 4-years with
no anthelmintic selection indicating that the resistant phenotype was as fit as the susceptible phenotype. A similar study showed that on a farm where benzimidazole resistance was encountered for the first time in 1 980, a resistant population was still present in 1 988, despite the use of levamisole for 6 years (Borgsteede and Duyn, 1 989). Another experiment conducted to investigate the potential for benzimidazole resistant strains of H. contorlus and T colubrtformis to revert towards susceptibility monitored the changes in anthelmintic resistance status throughout 1 2 generations (Hall et al., 1 982). No reversion towards susceptibility was recorded for either nematode species. The studies with insecticide resistance (McKenzie et a!. , 1 982; McKenzie and Purvis, 1 984) also suggest that accompanying intense selection, other alleles are concurrently selected to produce a reorganised genome such that the resistant phenotype is as fit as the susceptible phenotype, even in the absence of the selecting agent.
However, there are some reports of nematode populations, previously resistant to an anthelmintic group, showing some reversion towards susceptibility. Based on an egg hatch assay, Simpkin and Coles ( 1 978) demonstrated a fall in resistance to thiabendazole in H. contonus and 1: coluhriformis, if worms were passaged for one or two generations without thiabendazole. However, treatment of lambs harbouring passaged strains of worms with thiabendazole resulted in resistance similar to the original unpassaged-resistant strain. A similar study aimed at investigating reversion to susceptibility in a thiabendazole-resistant strain of H. contortus found that first treatment with thiabendazole after five years of Ievamisole use removed 95% of the worms. However, after the third passage thiabendazole resistance reappeared and only
50% worms could be removed (Kelly and Hall, ] 979).
Obvious reversion to susceptibility in benzimidazole-resistant Ostertagia spp was observed after a single dose (Donald et ai. , 1 980) or repeated doses of levamisole (Wall er et al., 1 983). Similarly, another study demonstrated a change to benzimidazole susceptibility in a mixed population of H. contortus and T. coluhriformis after two years of levamisole use (WaIler et aI. , 1 989). However, benzimidazole resistance increased rapidly following the re-introduction of thiabendazole. The results of these
studies may not actually indicate reverSIOn to susceptibility because levamisole treatment might have resulted in replacement of highly resistant benzimidazole worms by one reflecting a lower frequency of resistant individuals. Therefore, it is not clear yet to what extent reversion to susceptibility occurs.
CHAPTER TWO
OPTIMISATION OF THE LARVAL DEVELOPMENT ASSAY AND METHODS F OR LARVAL RECOVERY FROM PASTURE AND SOIL