Distinction of responder types with a single oral dose of 30000 L3

A priori, it was expected that an infection with 30000 L3 would induce less protective immunity than with 100000 L3 and we expected a reduced ability to distinguish between the responder types with this lower dose. Priming with 100000 L3 resulted in a significantly higher egg output at day 21 p.i. but the significant difference in egg excretion between both doses did not prevail after the peak. The differences between the infection doses in the current experiment resembled more what was described for the comparison of 50000 and 100000 L3 (Frankena, 1987), than for comparison of 20 000 and 100000 L3 (Nieuwland et al. 1995). However, a specific problem in all these experiments was the high variation between the animals, which cannot easily be overcome in an outbred population. The capacity to discriminate between low, intermediate and high responders after a single oral infection with 30000 L3 suggested that the antigenic threshold required to provoke the development of acquired immunity in intermediate responder animals was reached with this infection dose. Analysis of the egg output in the responder animals revealed that independent of the infection dose, low responders were still excreting eggs at day 49 p.i., while intermediate and high responders were not.

Worm establishment is predominantly influenced by host responder type

Expulsion of adult nematodes in ruminants is a common manifestation of acquired immunity as a consequence of repeated infection and was reported for sheep infected with Haemonchus contortus (Barger et al. 1985; Balic, Bowles & Meeusen, 2002), Teladorsagia circumcincta (Seaton et al. 1989), Trichostrongylus colubriformis (Barnes & Dobson, 1990) and calves after continuous larval exposure to O. ostertagi (Michel, 1963). However, these studies did not investigate if the decrease in adult worm burden resulted from the development of immunity against larval stages or if a higher larval challenge resulted in a higher worm burden and hence a faster build up of immunity against adult stages. Alternatively, immunity can alter establishment by an effect on the larval stages. This can be by rapid expulsion of the incoming L3 in the first days after the challenge or by a delayed effect on L4 and L5 stages (reviewed by Miller, 1984; and Rothwell, 1989). Depending on the host–parasite system resistance to adult and larval stages develops concurrently (Seaton et al. 1989) or independently (Michel, 1963; Emery et al. 1992), but expulsion of adult nematodes might also occur as a result of immune mechanisms triggered by larval stages (Rothwell, 1989). The results of these studies indicate that the term ‘reduced establishment’ can refer to both larval and adult stages and that the distinction is not always very clear. In the current experiment reduced establishment was defined as the ability to establish as (juvenile) worms beyond the early L4 stage. Previous studies demonstrated that reduced establishment and survival of worms depended on the density of the infection or the rate of incoming larvae (Anderson & Michel, 1977; Barger et al. 1985; Barnes & Dobson, 1990). Herein, we were not able to measure a differential effect on worm establishment induced by the low (30000 L3) and the high (100000 L3) priming dose, but we cannot exclude that this was caused by the variability in response among the animals. This was further confirmed by the observation that analysis of the host responder types did reveal differences. The adult worm burden in the intermediate but not in the low responders was significantly reduced compared to the challenge control animals. Moreover, irrespective of the infection group, a low worm burden concurred with a relatively higher % L4. These findings indicated that animals which were more resistant to a primary infection remained more resistant after re-infection as indicated by reduced establishment. Nevertheless, the small reduction in worm burden of the low responders clearly suggested an effect of priming. Thus low responders showed an enhanced resistance as a result of the primary infection, but this occurred at a lower rate or with less efficiency than in intermediate responders.

A common mechanism of immunity is that, upon re-infection worms are redistributed proximal to or distal from their normal niche (Miller, 1984). Worm expulsion in the course of a primary infection with C. oncophora was characterized by a distal shift in the worm population at day 42 p.i. (Kanobana et al. 2001). In the current experiment worms were recovered from the distal gut of low and intermediate responders as early as day 14 p.c., which denoted that expulsion was ongoing in these animals. In contrast, at this time-point the establishment in challenge control animals was still around 100% and all worms resided in their predilection site, the proximal gut. At day 28 p.c. the proportion of worms recovered from the distal gut of the primed animals was lower than at day 14 p.c. Interpretation of the results of the low responders is difficult as we had only 1 low responder at day 14 p.c. but, the altered distribution and the lower total worm burden in intermediate responders at day 28 p.c., suggested that a high proportion of worms was already expelled. Logically, the expelled worms were residing distally from their usual niche at day 14 p.c. The accelerated expulsion of the adult worms in primed animals was further supported by the variation in sex ratio in the proximal and the distal gut of these animals.

In summary, the results discussed above demonstrated that the main effects on worm establishment differed between low and intermediate responders. Whether the immune mechanisms associated with a reduced establishment are also regulated by the infection dose when doses of 30000 and 100000 L3 are compared, cannot be excluded but would require a higher number of animals within one infection group to yield conclusive evidence.

Worm length is predominantly influenced by priming infection dose

The GLM procedure with repeated measures indicated no effect of priming dose, nor even an effect of priming as such. Subsequent analysis was performed in which individual worm lengths within one group were included in the analysis. This may give insight into biological processes that, independently of the individual properties of the animals, have an influence on worm length.

Whereas with this model the processes influencing establishment seemed to be largely primary dose independent, analysis of individual worm lengths within infection group after challenge revealed a significant effect of the primary infection dose but not of the host responder type. At day 14 p.c. a gradual decrease in worm length was observed when comparing worms of the proximal gut of challenge control animals with animals primed with 30000 and 100000 L3, suggesting that the growth rate of the worms (and hence their length) was influenced in a dose-dependent way. Interestingly, at day 28 p.c. no differences in length were observed between both primed groups. This suggests that the effect of the priming dose relates more to the temporal kinetics of worm development than to the attainable adult worm length as such. This conclusion only applied for the proximal gut of the animals, as in the distal gut, worm length was always comparable between the challenge control animals and both primed groups.

Comparison of the proximal and the distal gut among primed and challenge control animals revealed interesting differences. Although in the early part of the infection only a few worms were recovered from the distal gut of the challenge control animals, we assume that the observation that worms in the proximal gut were longer than worms in the distal gut reflected the normal situation. By day 28 p.c. the situation was reversed in all infection groups, however, this was already observed in the animals primed with 100000 L3 at day 14 p.c. The seminal observation that the morphology of the worms was altered (longer but thinner) suggests either (i) that the worms were damaged by the detrimental environment of the proximal gut and on their way to be expelled or (ii) that within the physiological environment of the distal gut worms do not succeed to develop similarly to within their preferential niche.

Is there a link among effects on worm length and fecundity in C. oncophora-infected calves?

There have been extensive studies on worm length and fecundity of T. circumcincta in sheep (Stear et al. 1997a; Stear, Strain & Bishop, 1999). In these studies worm length and fecundity were very well correlated and particularly worm length was affected by acquired immunity in a density-dependent manner (Stear et al. 1997 b). Based on our current observation i.e. longer worms with low fecundity were found in the distal gut compared to shorter worms with high fecundity in the proximal gut, we propose that at least part of the mechanisms involved in the regulation of worm length and worm fecundity differ, and consequently also their effect.

The fecundity of the worms of the primed animals was surprisingly high as only 2 animals excreted eggs in their faeces. Beside factors such as an unaltered sexual behaviour, spermatogenesis and oögenesis, faecal egg output also requires a functional egg-laying machinery of the worms. This merely depends on contractions of uterine and somatic muscles that make the eggs go down the uterus (Bird & Bird, 1991). The lack of relationship between egg output and worm fecundity in the primed animals suggests that, in contrast to our observations in primary infected animals (Kanobana et al. 2001), during the development of resistance to re-infection to C. oncophora these mechanisms may be influenced independently.

Conclusions

The data presented here suggest that the memory responses to re-infection with C. oncophora consist of immunological processes that may be influenced by the primary infection dose and that different components of parasite fitness are affected differently by prior exposure. In the current experiment, reduction in establishment did not differ between animals primed with 30000 or 100000 L3, yet, abrogation of development resulting in a stunted growth and reduced fecundity did. These observations seem contradictory to a recent report that described density-dependent effects of prior exposure to Strongyloides ratti on worm survival and establishment (Paterson & Viney, 2002). The level of prior exposure quantitatively reduced the establishment after subsequent challenge, but the authors did not find an effect of protective immunity on the fecundity or length of S. ratti. Similar observations were done following infection with Ostertagia spp. in calves (Ploeger et al. 1995). But as the effect of the primary infection dose in the current experiment mainly referred to the kinetics of an effect on worm length and fecundity, the different conclusions among the experiments might result from the analysis of variable time-points after infection or from variable kinetics inherent to each specific host–parasite system. Similarly, we currently only compared infections with 30000 or 100000 L3, hence, we do not exclude a different outcome with more, or less divergent dose levels.

Finally, the effect on the worm burden seemed to be regulated by the host's responder types. Based on the differences in worm establishment and distribution it is clear that the ability of intermediate responder animals to mount a more effective and faster immune response as compared to low responders is sustained after a secondary infection. A better distinction and understanding of the mechanisms which are regulated by an effect of the dose and the mechanisms which depend on the responder types, and likely the genetic background of the animals, will contribute to improved preventive control strategies.