Acute Toxicity and Anthelmintic Effects of Gomphrena serrata L. (Amaranthaceae) Extracts on Adult Worms and Eggs of Haemonchus contortus

Gomphrena serrata L. (Amaranthaceae) is a medicinal plant widely used traditionally in rural areas to treat many diseases, including gastrointestinal helminthiasis of small ruminants. This work aims to estimate the plant extracts phytochemical potential and study the activities of the aqueous decoction (GS_AD), aqueous macerate (GS_AM), and hydroalcoholic macerate (GS_HEM) of Gomphrena serrata extracts on two developmental stages (eggs and adult worms) of Haemonchus contortus. Phytochemical screening was carried out. The acute toxicity test was performed according to the Organisation for Economic Co-operation and Development (OECD) test guideline 423. The Haemonchus contortus eggs hatching inhibition test was performed left to incubate for 48 hours in the presence of the different plant extracts at increasing concentrations of 0.125;0.25;0.5;0.625;1;2;3 mg/mL. For the motility inhibition test on adult worms, the worms were contacted with the extracts at concentrations of 0.75;1;1.25;1.5;2;3;4 mg/mL and left to incubate for 24 hours. Albendazole and levamisole were used as standard. Phytochemical screening revealed the presence of tannins, saponins, reducing compounds, coumarins and derivatives, anthocyanins, steroids, triterpenes, and flavonoids. An acute toxicity study showed that the oral LD50 value of G. serrata was estimated to be 5000 mg/kg. All extracts showed an effect on both stages of parasite development. The hatching inhibition rate was 100% from 3 mg/mL for all three extracts. The rate of inhibition of motility was 100% from 4 mg/mL for each extract. HEM is more efficient on eggs and Haemonchus contortus adult worms than other extracts with IC50 of 0.457 mg/mL and LC50 of 1.329 mg/mL, respectively. These results suggest that Gomphrena serrata, a plant used by farmers, has anthelmintic properties.

In vitro activities of acetonic extracts from leaves of three forage legumes(Calliandra calotyrsus,Gliricidia sepium and Leucaena diversifolia) on Haemonchus contortus

Objective:To assess ovicidal activity of three acetonic extracts from the leaves of three forage legume,Calliandra calotyrsus(C.calotyrsus),Gliricidia sepium(G.sepium) and Leucaena diversifolia(L.diversifolia) in vitro on Haemonchus contortus(H.contortus).Methods:Eggs were exposed for 24 hours to five different concentrations(0.075,0.15,0.3,0.6 and 1.25 mg/mL) of acetonic extracts at room temperature(24℃).Distilled water and 0.4%Tween were used in the bioassay as negative controls.Results:The later did not affect embryonation and egg hatching of H.contortus.Conversively,significant effects were obtained with the acetonic extracts of leaves of all three plants and the maximum activity was observed with the highest concentration (1.25 mg/mL).The acetonic extract of G.sepium was found to be more active(2.9%and 0.0% for embryonation and egg hatching,respectively) than the other substances 16.5%and 33.5%, respectively for C.calothyrsus,33.7%and 33.3%,respectively for L diversifolia.Conclusions: These results suggest that the three forage legumes do possess ovicidal properties and further studies on larvae should be carried out.

Single nucleotide polymorphisms in candidate genes are significantly associated with resistance to Haemonchus contortus infection in goats

Background: Haemonchosis is a major economic problem in goat production in humid, tropical and subtropical regions. The disease is caused by an abomasal nematode, Haemonchus contortus, which is highly pathogenic in small ruminants. The aim of this study was to identifying single-nucleotide polymorphisms(SNP) that were associated with fecal egg counts(FEC) and could be used as markers to identify resistance to H. contortus in goats.Results: Ten novel variants in the CI TA, ATP2 A3, HSPA8, STAT5 B, ESYT1, and SERPING1 genes were associated with FEC in goats with a nominal significance level of P < 0.05. Two missense mutation in the exon region of the caprine CI TA gene resulted in replacement of arginine with cysteine at position 9473550(R9473550 C) and aspartic acid with glutamic acid at position 9473870(D9473870 E). Chinese goat breeds had significantly higher FEC than Bangladeshi goat breeds within their respective genotypes. Polymorphism information content(PIC), effective al ele number(Ne), and heterozygosity(He)were greatest for the STAT5 B197A > G SNP locus in al goat breeds. Pairwise coefficients of linkage disequilibrium(D′, r2)revealed complete LD(r2= 1) between significant SNP polymorphisms in CI TA and SERPING1 and strong LD(r2= 0.93 and0.98) between polymorphisms in HSPA8 and ATP2 A3, respectively. Correlation coefficient(r) between FEC and body weight(BW) was significantly positive(r = 0.56***, P < 0.001) but that between FEC and packed cel volume(PCV) was negatively significant(r =-0.47**, P < 0.01) in the total population of goats. On the other hand, correlation coefficient(r)between BW and PCV was not significant in total population of goats. Association analysis revealed that haplotypes within ATP2 A3, HSPA8, and SERPING1 were significantly associated with FEC. Quantitative real-time PCR revealed that the relative expression of m RNA was higher(P < 0.001) for resistant, compared to susceptible, groups of goats for al candidate genes except CI TA.Conclusions: This study identified SNP markers that can potential y be used in marker-assisted selection programs to develop goat breeds that are resistant to H. contortus.

Anthelminthic Activity of <i>Moringa oleifera</i>Leaf Extracts Evaluated <i>in Vitro</i>on Four Developmental Stages of <i>Haemonchus contortus</i>from Goats

Haemonchus contortus is a blood-sucking abomasal helminth of small ruminants responsible for major economic losses to small farmers worldwide. Widespread resistance to synthetic anthelminthics has stimulated a need for alternative strategies of parasite control, among which is the use of medicinal plants with natural anthelminthic properties. This study assessed in vitro the efficacy of infused and macerated aqueous extract as well ethanolic extract of Moringa oleifera against fresh eggs, embryonated eggs, L1 and L2 larvae of H. contortus. For this purpose, five different concentrations (0.625, 1.25, 2.5, 3.75 and 5 mg/ml) were prepared from dry extracts via serial dilutions with distilled water. Fresh eggs obtained from artificially infected goat feces were exposed to these different concentrations for 48 hours, while embryonated eggs and larvae were exposed for 6 and 24 hours respectively. Distilled water and 1.5% DMSO were used as negative control. The results were expressed in terms of mean inhibition percentage of egg embryonation, mean inhibition percentage of egg hatch and mean percentage of larval mortality. An overview of results revealed that ethanolic leaf extract of M. oleifera was most efficient on eggs by inhibiting 60.3% ± 8.2% and 92.8% ± 6.2% eggs embryonation at 3.75 and 5 mg/ml respectively with a significant difference (P 0.05), which contributed to obtaining the lowest LC50 value of 0.985 mg/ml. This extract also inhibited 99% ± 2% egg hatching of H. contortus at 5 mg/ml with an LC50 value of 1.7 mg/ml. Concerning activity on larvae, the ethanolic extract was also most potent against them by inducing 98.8% ± 2.5% and 100% ± 0% mortality of L1 and L2 larvae at 5 mg/ml respectively. Infused aqueous extract was more efficient on eggs than on larvae with an IC50 value less than 2 mg/ml and an LC50 value more than 3.5 mg/ml. Macerated aqueous extract showed good activity against the four developmental stages with LC50 values ranging from 2.08 mg/ml for L2 larvae to 2.92 mg/ml for L1 larvae and 2.37 to 2.52 mg/ml for embryonated and fresh eggs respectively. The current study showed that all three extracts of M. oleifera tested possessed potential ovicidal and larvicidal activities against H. contortus. However, further in vivo studies are necessary to validate the anthelminthic property of this plant.

Partial characterization of superoxide dismutase activity in the Barber pole worm-Haemonchus contortus infecting Capra hircus and abomasal tissue extracts

Objective:To determine the activity of superoxide dismutase(SOD) in the male and female haematophagous caprine worms,Haemonchus contortus infecting Capra hircus,and their E/S products and also to analyse the effect of Haemonchus infection on the level of host SOD.Methods:The SOD activity was analysed by using the pyrogallol autoxidation assay and non-denaturing polyacrylamide gel electrophoresis followed by specific enzyme staining by riboflavin-nitroblue tetrazolium method.Results:The adult females were found to have higher enzyme activity than the male worms.Appreciable amount of SOD activity was also detected in the worm culture medium and female worms secreted more SOD in comparison to the male parasites.The SOD activity was negatively correlated to the worm burden.Statistically significant decrease in SOD activity(P<0.05) was observed in the heavily infected host tissue in comparison to the control non-infected host tissue.SOD profile of the crude extracts of both the sexes revealed polymorphism and a fast migrating activity band being characteristic of E/S products.The SOD activities were found highly sensitive to potassium cyanide indicating the Cu/Zn form of SOD.Conclusions:Haemonchus contortus is a key model parasite for drug and vaccine discovery.The presences of SOD activity in appreciable amount in the parasite as well as its E/S products indicate that it has a well-developed active antioxidant system to protect itself from the host immune attack.SOD could be the target for vaccine development which is the need of the hour as mass drug administration for parasite control has resulted in anthelmintic resistance across the globe and threatens the viability of sheep and goat industry in many regions of the world.The infection with Haemonchus causes a drastic reduction in SOD activity of the host tissue thus effecting its protective potential.One characteristic SOD band was found in the females which was not present in any other preparations and thus could be exploited for further studies on diagnostic/control measures.

Anthelmintic activity of Saba senegalensis(A.DC.) Pichon (Apocynaceae) extract against adult worms and eggs of Haemonchus contortus

Objective: To evaluate the anthelmintic property of Saba senegalensis(A.DC) Pichon(Apocynaceae)(S. senegalensis) on Haemonchus contortus that is traditionally used in Burkina Faso for its gastrointestinal parasites treatment.Methods: The lyophilized aqueous decoction of leaves of S. senegalensis at concentrations of 0.10, 1.00, 3.00, 10.00 and 15.00 mg/m L was used on eggs and adult worms of Haemonchus contortus collected from gastrointestinal tract of small ruminant.Results: The LC_(50) on adult worms was 6.79 mg/m L and 3.25 mg/m L for the leaves of S. senegalensis and the levamisole(reference drug), respectively. Inhibition of hatching assay showed a concentration-dependent manner with an inhibition of 93.63% at the concentration of 15.00 mg/m L of S. senegalensis.Conclusions: These results indicate that the aqueous extract of S. senegalensis possesses an anthelmintic property and may justify its use in traditional medicine for the treatment of gastrointestinal parasites.

Multiplex PCR Detection of Alleles Responsible for Benzimidazole- Susceptibility or -Resistance in Natural Populations of Haemonchus contortus

A multiplex PCR was developed to detect benzimidazole-resistance (BZ-R) or -susceptibility (BZ-S) in Haemonchus contortus by amplification with 4 primers of a sequence of the GRU-1 gene of β-tubulin of H. contortus making use of sequence information available in Genbank. The method was based on two allele-non-specific primers and two allele- specific primers. F1 (264 bp) and F3 (799 bp) should be produced in BZ-R, F2 (585 bp) and F3 in BZ-S. With this method, we demonstrated that H. contortus BZ-R strain from Australia showed F1 and F3, and the worm BZ-S strain from Shanghai did F2 and F3. Sequence analysis of the isotype 1 gene of β-tubulin of BZ-R from Australia and BZ-S from Shanghai showed the code in residue 200 of the gene was respectively TAC and TTC. The LD50 of albendazole of the Australian BZ- R strain was 0.54 μg mL-1, the Shanghai BZ-S strain was only 0.0023 μg mL-1 by EHA (egg hatch assay). The multiplex PCR could determinate the genotype of single adult worm or several third stage larvae and was performed on at least 50 ng of genomic DNA. BZ-R H. contortus were not detected in Shihezi and Yining of the Xinjiang, Wuhe of the Anhui Province, Nanjing and Xuzhou of the Jiangsu Province. The LD50 of the H. contortus from these locations to albendazole as determined by EHA varied between 0.0023-0.0032 μg mL-1. The result indicated that the multiplex PCR could be used to differentiate BZ-R and BZ-S of H. contortus and that the BZ-R situation of H. contortus was not serious in China.

<i>In Vitro</i>Ovicidal and Larvicidal Activities of Stem Bark of <i>Terminalia glaucescens</i>(Combretaceae) against <i>Haemonchus contortus</i>

Objectives: To evaluate the in vitro activity of aqueous and methanolic extracts of stem bark of Terminalia glaucescens on the eggs and larval stages of Haemonchus contortus of sheep. Methods: The eggs were exposed for 24 hours in five different concentrations (625, 1250, 2500, 3750 and 5000 μg/ml) of methanol, hot water (decoction) and cold water extracts at room temperature (25°C). Distilled water, 1.25% DMSO and Albendazole were used as negatives and positive control respectively in the bioassay. Results: A significant effect was obtained with all of the three extracts and differences were observed depending on the parasitic stage. Hot water extract (HWE), Methanol (MET) and Cold water extract (CWE) inhibited embryonic development by 98.1%, 96% and 86.5% respectively at 5000 μg/ml, meanwhile Albendazole had a 77.9% inhibition. For the mean inhibition rate of egg hatching, there was a general increase in the inhibition rate with increase in concentration of the extract from 625 to 5000 μg/ml. Methanol extract had the highest inhibition rate in all concentration from 625 to 5000 μg/ml. Concerning larval mortality, HWE had the highest effect in L1 larval mortality in all concentration above 1250 μg/ml. Distilled water had very little larval mortality on L1 and L2 larvae by 3.3 and 3.3% respectively. Conclusion: This in vitro study brought out the ovicidal and larvicidal properties of stem bark of Terminalia glaucescens.

The Anti-parasitic Effect of Extract of Ceiba pentandra (L.) Gaertn Is Related to Its Anti-inflammatory, Analgesic and Anthelmintic Activities on Haemonchus contortus

Background:Ceiba pentandra is a medicinal plant used as alternative therapy to control parasitic nematodes in livestock.Objective:This study aims to investigate anti-parasitic effect of aqueous stem back extract of Ceiba pentandra(L.)Gaertn through the evaluation of its anthelmintic,anti-inflammatory and analgesic activities.Methods:In vitro,the efficacy of aqueous extract(75 to 2400μg·mL−1)diluted in phosphate buffered saline(PBS)was tested against three stages of the life cycle of Haemonchus contortus through larval migration inhibition assay(LMIA),egg hatch assay(EHA),and adult worms motility inhibition assay(AMIA).In vivo,anti-inflammatory and antinociceptive properties of the aqueous extract(150 and 300 mg·kg−1)was evaluated on rodent using phlogistic and algic chemicals.Results:Significant inhibition activity(P<0.05)was obtained on EHA with the greatest inhibition of 46.63%obtained at 2400μg·mL−1.The plant treatment dramatically(P<0.05)inhibited L3 larval migration as compared to PBS.The highest inhibition rate was 53.33%at 1200μg·mL−1.Adding of polyvinylpolyrrolidone(PVPP)to the extract significantly(P<0.01)reduced at 38.6%the activity of the plant extract on larval migration compared to extract without PVPP.The Ceiba pentandra extract reduced(P<0.05)worm motility after 24 h post exposure as compared to control.In vitro,aqueous extract significantly(P<0.05)inhibited the paw inflammation induced by carragenine,with a significant(P<0.05)reduction of the number of abdominal contortions induced by acetic acid to 41.11%at 300 mg·kg−1 and the paw licking time induced by formaline in both phases to 57.22%and 63.59%at 300 mg·kg−1 likened to control.Conclusions:In vitro results suggest that,this plant possess anti-parasitic properties.Antinociceptive and anti-inflammatory effects of C.pentandra can contribute to its anti-parasitic property.

Anthelmintic activity of Securidaca longepedunculata(Family:Polygalaceae)root extract in mice,in vitro and in vivo

Objective:To elucidate the pharmacological bases of oral administration of Securidaca longepedunculata(S.longepedunculata) root extract as an anthelmintic in folkloric medicine. Methods:Albino mice were infected with infective third(L3) larval stage of Heligmosomoides polygyrus(H.polygyrus) by esophageal intubation.Following establishment of the adult worms in the intestine,the mice were treated with 0-2 000 mg/kg body weight(bw) of methanolic root extract of S.longepedunculata and 100 mg/kg bw of pyrantel embonate,the reference drug in vivo. Bioactivity and larvicidal effects of the extract were tested by exposing brine shrimps(Artemia salina) to 0.00-1.00 mg/mL and the L3 stage of Heligmosomoides contortus(H.contortus) and H.polygyrus to 0.00-2.50 mg/mL of the extract in vitro.Results:The percentage yield of the extract was 7.13%w/w dry matter.The brine shrimps toxicity bioassay resulted in an LC_(50) of 74.18 μ g/mL.The extract had a significant,dose-dependent larvicidal effect on the L3 stage of H.contortus and H.polygyrus with the terminal effect of 75%and 70%at the highest exposure concentrations,respectively.The extract however,did not affect the number of worm eggs per gram(epg) of fecal materials(P<0.05) and total worm burden(twb) of adult H.polygyrus in infected mice.Treatment with pyrantel embonate significant reduced both the fecal egg count and twb to 0 compared to the untreated control(P<0.05).Conclusions:These results indicate that S.longepedunculata root extract contains potent bioactive compounds and has larvicidal effect on L3 stage of H.contortus and H.polygyrus,substantiating its use as anthelmintic in alternative medicine.