PBS HEPES
H. contortus T. circumcincta Lectin
5.10 Future directions
These experiments have shown that it was possible to recover L3 H. contortus and T.
circumcincta off herbage and then to distinguish the species by lectin binding. Larvae were recovered more consistently from herbage, but the recovery required a long incubation period and was still low. To assess the use of the modified recovery method as a field method, much larger sample sizes of herbage will have to be examined to evaluate if the recovery percentage and consistency are still reliable.
The lectin binding was also incomplete on the surface of larvae of H. contortus and T.
circumcincta. The differentiating lectin for sheathed H. contortus was AAL, which bound to a high percentage of, but not all larvae besides the lectin binding being weak and only visible on the head region of the larvae at high magnification. For this method of speciation to be reliable, lectins which bind all the larvae of one species but none of the larvae of other species tested would be ideal, but may not be achievable. In addition, stronger fluorescence based on lectin binding is desirable, as this would allow better imaging and therefore faster and possibly automated counting of the larvae. At this stage, differentiation of H. contortus and T. circumcincta larvae using lectin binding offers little advantage over traditional methods based on morphological differentiation. For this method to be a field method, another way of visualising lectin binding rather than fluorescence is required, since a fluorescence microscope is specialised equipment. One approach could be to visualise lectin binding with coloured beads, which could probably be developed on eggs, as they show more binding to the surface. It has been demonstrated that lectin binding offers opportunities for a novel method of speciation and possibly for other sheep parasitic nematodes and life cycle stages.
6 Summary
Katharina Hillrichs (2010)
Investigations on the binding capacity of lectins to sheep parasitic nematodes to improve recovery and differentiation of infective larvae
To maximise growth, fecundity and animal health of livestock, it is important to understand, control and manage nematode parasite burdens. A central issue regarding infection of livestock with gastrointestinal parasites is the degree of contamination of pasture with the infectious larval stages of these nematodes, often referred to as larval challenge. Assessing the degree of larval challenge is essential in managing the degree of infection in grazing animals. Traditionally it has been difficult to assess the pasture contamination for two reasons: firstly because of the difficulty of identification and differentiation of nematode larvae and secondly, because of poor and inconsistent recovery of these larvae of pasture. Both problems were addressed by using a panel of 19 lectins to differentiate L3 of H. contortus and T. circumcincta and by modifying the Baermann method as a recovery method of larvae from grass. When counted numbers of larvae were incubated on grass, recovery was most consistent when the incubation period was extended to 48 hours, which resulted in recovery of larvae of 55 % (CV 7 %). Lectin binding to the surface of L3 of both species was weak, patchy and inconsistent. Sheathed larvae could be distinguished by AAL binding to the head of H. contortus only. Exsheathed larvae did not show any binding to the surface, but binding to the mouth region, amphids and phasmids was more consistent. Exsheathed L3 could be differentiated by binding of BGS I to the head of H. contortus only. In comparison to L3, other life cycle stages like eggs and adults showed intense lectin binding and could be distinguished easily.
When the two methods were combined and a mixed population of H. contortus and T. circumcincta were placed in grass, 53 % of larvae could be recovered with Baermann separation and the H. contortus distinguished by AAL binding.
7 Zusammenfassung
Katharina Hillrichs (2010)
Untersuchungen zur Lektinbindungskapazität von Nematoden des Schafes zur Speziesdifferenzierung und Gewinnung von infektiösen Larven aus Weidegras
Um Gewichtszunahmen, Fruchtbarkeit und Gesundheit einer Schafherde zu maximieren, ist es wichtig, den Parasitenbefall mit Nematoden des Magen-Darm-Traktes zu verstehen und zu kontrollieren. Ein bedeutender Punkt in Bezug auf das Infektionsrisiko der Herde ist die Kontamination der Weide mit dem infektiösen Larvenstadium (L3) der Nematoden. Um die Infektionsrate der weidenden Tiere zu kontrollieren, ist es wichtig, den Infektionsdruck zu bemessen. Traditionsgemäß ist es aus zwei Gründen schwierig, die Kontamination der Weide einzuschätzen: erstens wegen der Schwierigkeit, die verschiedenen Larven zu identifizieren und zu differenzieren und zweitens wegen der niedrigen und inkonsistenten Rückgewinnungsrate der Larven vom Weidegras. Beide Probleme wurden bearbeitet, indem eine Auswahl 19 fluoreszierender Lektinen auf ihre Eignung untersucht wurden, Larven von H. contortus and T. circumcincta leichter zu unterscheiden und die Baermann Methode zur Rückgewinnung der Larven vom Weidegras modifiziert wurde. Die konstanteste Rückgewinnungsrate wurde nach einer Auswanderzeit von 48 h erzielt. Die Rückgewinnungsrate lag bei 55 % (CV 7
%). Die Fluoreszenz der Lektine, die an die Oberfläche der L3 beider Spezies gebunden haben, war schwach, ungleichmäßig und unregelmäßig. Bescheidete Larven konnten durch Bindung von AAL nur an der Kopfregion von H. contortus unterschieden werden. Kein Lektin zeigte Bindungsfähigkeit zur Oberfläche von entscheideten Larven, aber die Bindung zu Amphiden und Phasmiden war konsistenter. Entscheidete Larven konnten durch Bindung von BGS I nur an der Kopfregion von H. contortus differenziert werden. Im Vergleich zum Larvenstadium, zeigten die Lektine höhere Bindungsfähigkeit zu anderen Entwicklungsstadien, wie
Eiern und Adulten. Beide Methoden wurden kombiniert und eine gemischte Population von bescheideten H. contortus und T. circumcincta Larven wurden auf Gras präinkubiert. Mit der modifizierten Baermann Methode konnten 53% der Larven zurück gewonnen und mit AAL unterschieden werden.
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