Date of Award

2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Science

First Advisor

Katherine Petersson

Abstract

With the growth of resistance to commercial dewormers in small ruminant gastrointestinal nematodes (GIN), alternative methods are needed for GIN control. It has been shown that plants containing certain secondary compounds (SC) suppress GIN infection, but there is a lack of knowledge concerning the mechanism behind this anthelmintic activity, as well as a lack of viable alternatives to commercial dewormers in the northeastern United States. Secondary compounds, such as proanthocyanidin (PAC), have complex chemical structures that vary between plant species, while SC concentrations can vary between different varieties of the same species. The relationship between specific SC and anti-parasitic efficacy is unknown. This study explores the use of PAC-rich cranberry vine (CV) and birdsfoot trefoil (BFT) as an alternative to commercial dewormers in the control of GIN in small ruminants.

In vitro techniques were used to test BFT against H. contortus eggs, first stage (L1) and third stage larvae (L3). The anti-parasitic activity of purified proanthocyanidin extract (Bruce-PAC) and crude aqueous extracts (BFT-AqE) from 51 strains of BFT were tested using the following in vitro assays: 1) egg hatching and viability of L1 Haemonchus contortus larvae and 2) exsheathment and viability of L3 H. contortus larvae. The BFT-AqE was prepared by soaking BFT powder in water at room temperature for 24 h, and then the plant matter was removed, leaving an aqueous extract. 1) For in vitro egg hatch inhibition and mortality of L1 larvae, H. contortus eggs were exposed to varying concentrations of BFT extracts for 24 h. 2) For in vitro exsheathment, 2,000 H. contortus L3 larvae were exposed to BFT extracts prior to exsheathment. Proanthocyanidin content ranged between 1.4 and 63.8 mg PAC/g powder across 51 BFT strains. Inhibition of egg hatch and L1 mortality were observed: at 3 mg/mL BFT-AqE, percent inhibition of egg hatch and L1 mortality ranged between 0 and 100% across 51 strains tested. Inhibition of exsheathment and L3 mortality was observed across thirteen tested strains: at 25 mg/mL, percent inhibition of exsheathment bridged between 0 and 75% inhibition. Incubation in Bruce-PAC exhibited 8% egg hatch inhibition, 98% L1 mortality, 4% percent L3 mortality, and 100% exsheathment inhibition at the highest concentration of 6 mg/mL Bruce-PAC. Results indicate that strains of BFT-AqE inhibited egg hatch and inhibited larval mortality, but the degree of inhibition varied. Results also indicate that Bruce-PAC did not inhibit egg hatch or mortality of L3 larvae, but did inhibit mortality of L1 larvae and inhibit exsheathment. When comparing PAC content of varietal BFT powders to anthelmintic efficacy of aqueous extracts, there were no significant correlations. Since PAC content was not related to activity, PAC structure was investigated for its potential influence on bioactivity. Further testing of PAC extracted from different varieties will provide additional information about the anthelmintic efficacy of strains of BFT for small ruminant GIN control. Inhibition of egg hatch, L1 and L3 mortality, and inhibition of exsheathment were observed with incubation in BFT-AqE; however, the concentration at which this inhibition was most effective varied among BFT strains. Correlations between the anthelmintic efficacy and PAC structure of each individual variety were determined based on these results.

This study tested CV extracts against H. contortus eggs, first stage larvae, third stage larvae, and exsheathment. This investigation is the first to examine the anthelmintic potential of cranberry vine against the GIN Haemonchus contortus, while advancing the study of plants containing PAC. The purpose of this study was to explore the anti-parasitic potential of cranberry vine proanthocyanidin extract (CVPAC) and cranberry vine aqueous extract (CV-AqE) on in vitro egg hatching, larval and adult worm mortality, and exsheathment of H. contortus, to observe cranberry vine treated H. contortus via scanning electron microscopy, and to investigate the efficacy of cranberry vine powder (CV) drench against an experimental infection of H. contortus in lambs. The in vitro anthelmintic effect on egg hatching and mortality of L1, L3, and adult worms was determined after 24-hour exposure to varying concentrations of CV-PAC and CV-AqE. The in vivo anthelmintic effect on an experimental infection in lambs was determined by administering 21.1 g CV or not to lambs (n = 9 per group) for three consecutive days, and collecting fecal egg count (FEC) data for four weeks post treatment. Inhibition of egg hatch was observed in concentrations ≥ 5 mg/mL CV-PAC and significant L1 mortality following hatching was observed at concentrations ≥ 0.3 mg/mL. Egg hatch inhibition occurred at ≥ 5 mg/mL CV-AqE (0.6 mg PAC/mL), and significant L1 mortality occurred in L1 exposed to ≥ 1.2 mg/mL (0.15 mg PAC/mL). Third stage larvae exhibited mortality when exposed to ≥ 2.5 mg/mL CV-PAC; no significant mortality was observed in CVAqE. Inhibition of L3 exsheathment was observed at concentrations ≥ 5 mg/mL CVPAC but was not observed at any concentration of CV-AqE. Adult worm mortality was observed at concentrations ≥ 0.6 mg/mL CV-PAC and ≥ 12.5 mg/mL CV-AqE (1.5 mg PAC/mL). Scanning electron microscopy revealed an accumulation of aggregate on the cuticle in the buccal area of adult worms incubated in both CV-AqE and CV-PAC. Both extracts (CV-PAC and CV-AqE) demonstrated anti-parasitic effects on all tested stages of H. contortus, but the extracts differed in their level of efficacy. In the lamb trial, no significant difference emerged between the weekly average FEC of the groups, but a significant treatment over time effect was observed.

Cranberry vine extracts were also tested in vitro against H. contortus adults and morphological changes in the adults incubated in extracts were observed. The purpose of this study was to investigate the effect that feeding cranberry vine (CV) to lambs had on 1) an established infection of H. contortus in lambs, 2) the establishment of a trickle infection of H. contortus in lambs, and 3) the morphology of H. contortus adult worms that were exposed to CV in vivo as determined by scanning electron (SEM), transmission electron (TEM), and light microscopy. Lambs were experimentally infected with L3 larvae in two separate trials; 1) CV trial: 21 lambs, stratified into three groups (n = 7) based on gender and fecal egg count (FEC) once the infection (10,000 H. contortus) matured; and 2) CVP trial: 14 lambs, stratified into two groups (n = 7) based on gender and twin separation, received trickle infection of 500 L3 H. contortus 3 times/week for 3 weeks. For the CV trial, lambs were fed CV0 (0 g CV, 250 g chopped alfalfa hay (AH)), CV100 (100 g CV, 150 g AH), or CV200 (200 g CV, 50 g AH). Fecal egg counts (FEC) were measured weekly for the duration of the trial. At the conclusion of the study, total worm burden was determined and five adult female H. contortus worms were collected from the abomasum of each lamb and preserved until viewed under SEM, TEM or light microscopy. There was no difference in FEC between CV0 and CV200. Evaluation of worms using SEM showed cracking in the cuticle of the adult worms from the CV200 group, compared to minimal or no cracking observed in CV0. Some accumulation of an aggregate was observed on worms collected from the CV200 group. Evaluation of vesicles internal to the microvilli lining of the lumen of the intestines worms using TEM showed a lower number of vesicles present in the worms collected from the CV200 group. For the CVP trial, lambs were fed CVP0 diet: 0 g CVP, 500 g grain; or CVP200 diet: 500 g CVP, 100 g grain for 8 weeks. Fecal egg counts (FEC) were measured weekly for the duration of the trial. At the conclusion of the study, total worm and larval burden were determined. There was no difference in FEC or total worm burden between CVP0 and CVP200 groups. Results indicate that feeding 200 g of CV to lambs experimentally infected with H. contortus for five weeks resulted in cuticular damage and possible malnutrition of the adult worms and may have potential as an antiparasitic against H. contortus. Further investigation feeding a higher intake of CV is warranted.

Available for download on Saturday, April 21, 2018

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