Inconsistency of in vitro exsheathment triggers for gastrointestinal nematode parasites of sheep, cattle and deer.

Exsheathment is crucial in the transition from free-living to parasitic phase for most strongyle nematode species. A greater understanding of this process could help in developing new parasitic control methods. This study aimed to identify commonalities in response to exsheathment triggers (heat acclimation, CO2 and pH) in a wide range of species (Haemonchus contortus, Trichostrongylus spp., Cooperia spp., Oesophagostomum spp., Chabertia ovina, and members of the subfamily Ostertagiinae) from sheep, cattle and farmed deer. The initial expectation of similarity in pH requirements amongst species residing within the same organ was not supported, with unexpected pH preferences for exsheathment of Trichostrongylus axei, Trichostrongylus vitrinus, Trichostrongylus colubriformis and Cooperia oncophora. We also found differences between species in their response to temperature acclimation, with higher exsheathment in response to heat shock observed for H. contortus, Ostertagia ostertagi, T. axei, T. vitrinus and Oesophagostomum sikae. Furthermore, some species showed poor exsheathment under all experimental conditions, such as Cooperia curticei and the large intestinal nematodes C. ovina and Oesophagostomum venulosum. Interestingly, there were some significant differences in response depending on the host from which the parasites were derived. The host species significantly impacted on the exsheathment response for H. contortus, Teladorsagia circumcincta, T. vitrinus and T. colubriformis. Overall, the data showed variability between nematode species in their response to these in vitro exsheathment triggers, highlighting the complexity of finding a common set of conditions for all species in order to develop a control method based on triggering the exsheathment process prematurely.

Host effects on the free-living stages of Haemonchus contortus.

A group of 5 lambs (Host 1-5) was infected with the same batch of Haemonchus contortus and after patency individual faecal samples were collected, separately incubated at 23 °C for 14 days and third stage larvae collected through Baermannisation. Life-history traits were compared between larvae from different hosts: the length of the larvae was measured by microscope image analysis, larval survival in water at 35 °C, larval susceptibility to ivermectin (EC50) in a migration assay, the proportion of larvae exsheathing in vitro and the proportion establishing to the adult stage in young lambs. For all traits there were significant differences between the host animals, with larvae from specific hosts following a consistent pattern of displaying the highest or lowest trait results. Compared with larvae from Host 1 the larvae from Host 5 were () shorter (741-692 µm, p < 0.05), had a longer median survival at 35 °C (3.6-6.4 days, p < 0.05), were less susceptible to ivermectin (EC50 of 1.2 v 4.5 µM, p < 0.05), exsheathed to a lesser degree (83.6-58 %, p < 0.05), but showed a higher establishment rate in the consecutive host (15.2-31.4 %, p < 0.05). Regarding the survival time, anthelmintic susceptibility (under most commercial farming practices) and establishment rate as indicators for fitness, the parasites populating Host 5 produced progeny of higher fitness. The findings indicate that the host animal of the parental parasite generation has a significant effect on the parasite progeny.

Abomasal nematode species differ in their in vitro response to exsheathment triggers.

A crucial step in the infection process of grazing ruminants by gastro-intestinal nematodes is the exsheathment of the infective third-stage larva following ingestion. Recently, heat shock was shown to play an important role in the carbon dioxide (CO2)-dependent exsheathment response in Haemonchus contortus. The current in vitro study set out to evaluate the role of heat shock in other abomasal species. In rumen fluid, all species tested exsheathed rapidly and efficiently in response to heat shock and CO2. This response was significantly higher compared to slow temperature changes, supporting the hypothesis that heat shock plays an important role in vivo. However, in artificial buffer, the effect of heat shock was species-dependent. For H. contortus and Ostertagia leptospicularis, the response in artificial buffer was similar to rumen fluid. In contrast, Ostertagia ostertagi and Teladorsagia circumcincta exsheathment was significantly lower and/or slower in artificial buffer, and there was no benefit of heat shock. For these two species, it appears that there are co-factors in the rumen fluid, in addition to heat shock and CO2, contributing to exsheathment. Overall, the data indicate that there are significant differences between abomasal species in their response to exsheathment triggers.

Carbon dioxide is an absolute requirement for exsheathment of some, but not all, abomasal nematode species.

The first step in the infection process of grazing ruminants by gastrointestinal nematodes is the exsheathment of the third-stage larva (L3). Exsheathment of various species can be achieved in vitro using carbon dioxide (CO2) under the appropriate temperature and pH conditions. However, it remains unclear whether elevated CO2 levels are an absolute requirement for exsheathment. Exsheathment of four abomasal species was investigated in both the presence and absence of CO2, in either rumen fluid (cow or sheep) or buffer (standard or enriched). Exsheathment of Ostertagia ostertagi, Teladorsagia circumcincta and Ostertagia leptospicularis was observed in CO2-depleted rumen fluid and enriched buffer (respectively 46%, 22% and 15% in rumen fluid and 28% 18% and 26% in enriched buffer after 24 h). The level of this response was dependent on the species as well as the medium, and exsheathment was significantly higher in the presence of CO2. For Haemonchus contortus, exsheathment could only be achieved under CO2-saturated conditions. In conclusion, even though these parasite species exsheath in the same environment, there were significant differences in the minimal requirements to trigger their exsheathment. Some abomasal species were capable of exsheathment in the absence of CO2, which is likely facilitated by cofactors present in the rumen fluid and/or enriched buffer.

Heat shock, but not temperature, is a biological trigger for the exsheathment of third-stage larvae of Haemonchus contortus.

Gastrointestinal parasites are an important health issue in grazing ruminants. Understanding the processes involved in the transition from the free living to the parasitic life stage of these nematodes is one avenue to identifying new targets amenable to future intervention. The transition to parasitism is initiated by exsheathment and is triggered by the sudden change in environment after ingestion of the infective larva by the host. Two major changes in environment are the increases in temperature and carbon dioxide (CO2) levels. For CO2 a role in exsheathment has been described previously, but the exact role of temperature was unclear. The current study is the first to investigate the importance of temperature in triggering exsheathment of Haemonchus contortus. Carbon dioxide induced exsheathment in H. contortus proved to be temperature dependent, as no exsheathment was observed at room temperatures. However, the temperature requirement to trigger exsheathment was quite specific. A rapid change in temperature (heat shock) very efficiently induced high levels of exsheathment. In contrast, when the larvae were exposed to a slow increase in temperature, the exsheathment response was smaller and delayed. Further investigation revealed that timing of the heat shock in relation to the CO2 administration was crucial, as well as the final temperature and magnitude of the heat shock. In conclusion, these data indicate that heat shock rather than temperature itself is a crucial aspect in triggering the biological exsheathment cascade, and thus infection process, of H. contortus.