In Vivo Anthelmintic Activity of Whole Plant Powder of Striga Hermonthica (Deli.) Benth

DICKO A., Almamy KONATE, Basile TINDANO, Arnaud S. R. TAPSOBA, Moumouni SANOU, Adama KABORE, Amadou TRAORE, Balé BAYALA, Hamidou H. TAMBOURA 1, Laboratoire de Biologie et Santé Animale (LaBioSA), Institut de l’Environnement et de Recherches Agricoles (INERA) 04BP 8645, Ouagadougou 04 Burkina Faso. 3,8 Laboratoire de Physiologie Animale (LaPA), Unité de Formation et de Recherches en Sciences de la vie et de la Terre, Université OUAGA I Joseph KI-ZERBO, 03 BP 7021, Ouagadougou 03, Burkina Faso,


INTRODUCTION
Parasitosis due to gastrointestinal strongyles is a worldwide problem that can lead to significant productivity losses in small ruminants (Okombé, 2011;Francesco and al, 2014). These parasites can cause mortality of up to 50% of lambs and economic losses beyond 50% of production capacity (Bodji and al, 2017). There are a large number of Gastro-Intestinal Nematodes's species that thrive under diverse climatic conditions and consequently there is a wide geographical distribution of nematodes threatening the sustainability of livestock farms around the world. Haemonchus contortus is one of the most widespread and pathogenic species causing severe anemia in small ruminants (Burke, 2007). This hematophagous parasite located in the abomasum of small ruminants can measure between 10 and 16 mm for the male and 18 and 30 mm for the female that can lay up to 5,000 to 10,000 eggs per day (Sicard and Robert, 2010;Mongellaz, 2019;Ruiz-Huidobro, 2018). Gastrointestinal parasites's control has been based for several years on the use of synthetic anthelmintics which are unfortunately inaccessible and increasingly ineffective against these parasites (Kaboré andal, 2009). Also, these molecules have been found to be ecotoxic, thus presenting a high environmental risk (Saccareau, 2016). Several control alternatives, including biological control through the use of medicinal plants with anthelmintic properties, have been developed. Studies in Burkina Faso and Benin have shown the anthelmintic activity of certain woody species (Kaboré, 2009;Awohouedji, 2014 hermonthica is an annual herb that parasitizes the roots of several cereals (Kiendrebeogo and al, 2006;Hamisou and al, 2020). Many studies have mentioned the use of S. hermonthica in human and veterinary pharmacopoeia and the presence of some secondary metabolites such as polyphenols, tannins and flavonoids in this plant (Djerro, 2002, Baba and al, 2012, Garba and al, 2019. The objective of our study is to evaluate the anthelmintic activity of the whole plant powder of S. hermonthica through the measurement of the feacal eggs count in sheep artificially infested with L3 larvae of H. contortus.

MATERIAL AND METHODS Plant material
The whole plant of S. hermonthica was collected in the villages of Katchari and Mamasiol in the urban commune of Dori early in the morning between the end of September and mid-October. Whole plant samples were dried in a room at DRREA-Sahel for 2 weeks at room temperature and then transported to CREAF Kamboinsin in the commune of Ouagadougou. One sample was identified at the National Herbarium of Burkina Faso (HNBF) under number 8759. The samples were then ground using a Retsch type SM 100 grinder with a mesh size of 5 before being used as whole plant powder for the in vivo test.

Animal material Sheep
Twenty-four (24) Mossi sheep between 12 and 24 months of age and with an initial weight between 16.6 and 27.7 kg were used for the in vivo tests. All animals were paid at the livestock market of Kaya, capital of the Centre-Nord region. The animals were not dewormed during the three months preceding the experiment.

L3 larvae
The infesting L3 larvae of H. contortus were obtained by culturing fresh eggs in the sterilized's feacal of sheep mixed with wood's sawdust for 14 days at 31° C. After 14 days the culture was mounted in gauze and then placed on the Baermann device to get the L3 larvae.

Experimental device.
Thirty (30) days before the beginning of the experiment, all animals were artificially fed 3200 H. contortus larvae orally through a naso-esophageal tube in two phases of 1600 larvae each, spaced one week apart. One week before the start of the in vivo tests, the animals were weighed and the degree of infestation was determined and then the animals were divided homogeneously into 4 lots of 6 sheep each according to the weight and the feacal eggs count (FEC). The lots included, 2 test lots treated with two different doses of S. hermonthica whole plant powder, 1 untreated negative control lot and 1 positive control lot treated with levamisole. The experiment lasted 21 days during which all animals received concentrate (SN Citec cattle feed) in the form of pellets in the morning at 6:00 am. For the treated lots, the concentrate was mixed with the equivalent amount of the set treatment dose. In the afternoon, all animals were fed corn bran for the duration of the experiment. The animals were provided with water and the mineral lick ad libitum during the experimental period. The treatment in the form of S. hermonthica whole plant powder was administered to the treated lots on the first four days (D1, D2, D3, D4) and then repeated on the 7th day after the end of the first treatment (D11, D12, D13, D14). At the end of the first treatment the animals grazed on natural pasture for 6 days before the second treatment was repeated. After the last day of the second treatment, the animals were still grazing on natural pasture until the 21st day of the experiment.

MEASURED PARAMETERS
The main parameters collected for this study are: clinical data concerning the general condition of the animals, parasitological and hematological data.
-During the period of the experiment, the clinical data concerning the general condition of the animals after the administration of the whole plant powder of S. hermonthica were evaluated by direct observations and recorded on a form.
-The weight growth of the animals of different lots was measured from individual weighing at D0, D4, D7, D14 and D21. Weighing was done to jeun using a MARECHALLE-PESAGE cattle scale for small ruminants equipped with an electronic indicator.
-Parasitological data were obtained by individual sampling of feces directly from the rectum of the animals at D0, D4, D7, D14, D21. Individual coproscopies were performed and -The hematocrit rate variation of the animals in each lot was determined by drawing blood through the jugular vein of the animals using heparinized tubes at D0, D7, D14, and D21 of the experiment. Then each sample was used to fill a capillary tube with 4/5th hematocrit. The filled capillary tubes were centrifuged at 9000 rpm for 5 minutes using a HAEMATOKRIT 210 hematocrit centrifuge. In conjunction with the blood collection with heparinized tubes, a collection with dry tubes was performed on D0 and D21 of the experiment for each animal. The dry tubes were centrifuged using a SIGMA 3-15K centrifuge at 3000 rpm for 10 minutes and the serum was collected in 1.8 ml cryotubes for biochemical analysis.
-The degree of anemia of each animal was determined by evaluating the FAMACHA score by comparing the coloration of the ocular conjunctiva of each animal with a card showing different colorations of the ocular conjunctiva in relation to the degree of anemia of the animal.

STATISTICAL ANALYSIS
The collected data were entered into Excel 2016 which was used to calculate the means and standard deviations of the different measured parameters as well as the animals's Feacal Eggs Count reduction (FECR) rate. Then the data of the different days of follow-up were subjected to a onefactor analysis of variance (ANOVA I) followed by a multiple comparison of means at the threshold of 5% by the method of Tukey's using the interface Rstudio Version 1.4.1717 with the packages Rcmdr Version 2.7-1 of the software R Version 4.1.0. Prism 5.0.0.288 software was used to produce the graphics.

Clinical and parasitological data
No apparent clinical signs of toxicity (salivation, skin reaction, diarrhea) in the animals during the treatment time were observed. The evolution of FEC showed a significant decrease over time compared to the negative control P (<0.05)  StrigaB: dose 10g/kg body weight.

FECAL EGGS OF H. CONTORTUS REDUCTION RATE
A significant reduction of fecal excretion rate of H. contortus eggs compared with the negative control P (<0.05) was observed from the first day of follow-up and throughout the experiment period (Table II).

WEIGHT EVOLUTION
The animals weight evolution in the different treated lots was not significant P (>0.05) compared to the both controls lots.
A low average daily gain was observed during the treatment (Table  III).  Figure 3 shows us a slight increase in FAMACHA score in animals at D21 compared to D0. Statistical analysis of the comparison of means of the FAMACHA score shows a strong significance at D14 compared to the negative control (P<0.05). values, which is one of the most reliable biochemical markers of glomular function, were within the range of the different usual and reference values at D0 and D21 (Table  IV). Statistical analysis of these parameters at D0 and D21 and between treated batches and negative controls was not significant (P>0.05).  Table V shows that the treatments with the two doses of S. hermonthica powder did not lead to an imbalance of the energetic and mineral metabolism with a normal range obtained for all the values of the different parameters at D0 and D21. The statistical analysis of the different parameters at D0 and D21 and between the treated lots and the negative controls was significant (P<0.05) except for the triglyceride parameter which was only measured at D21 (P>0.05).  Kaneko and al., (2008), D: Deghnouche and al., (2011); E: Ndoutamia and Ganda (2005), P: Probability, a, and al, 2011). Also aqueous extracts of Annona senegalensis stems, roots and leaves administered to Djallonké sheep at dose of 22 mg/kg live weight resulted in a decrease in FEC similar to our results although the nature of the remedy administered was not the same in our two studies (Bodji andal, 2017). In contrast to our results, Mossi breed sheep treated with dried leaves of Calotropis procera (Will). R. Br had a decrease in FEC reduction rate at D3 and D7 and then the level of FEC rate increased from D14 to D28 of the Follow-up (Kanazoé and al, 2017). The difference with our present study would be due to the content of secondary metabolites in C. procera. The Average Daily Gain (ADG) of the treated animals in our present study was low over the follow-up time and statistical tests showed no significant difference compared to the two controls performed. In Congo, goats treated with Vitex thomasii root powder did not obtain a statistically significant difference in weight evolution after 126 days of treatment which corroborates our results (Okombé, 2011). Our results are in agreement with those obtained on sheep treated with aqueous extracts of Myrsine africana by doses of 50 and 125 g/kg of weight but which did not obtain a significant difference between the different batches (Githiori and al, 2002). Treatment with both doses of S. hermonthica resulted in an improvement of the hematocrit level in animals of the different lots with a statistically significant difference (P<0.05). Indeed, we note a mean hematocrit level varying from 24 at D0 to 30.5 at D21 for the treatment at a dose of 17g/kg of body weight and from 25.83 at D0 to 31 at D21. In conjunction with the hematocrit, there was an improvement in the FAMACHA score in the animals of the different lot. Sheep treated with Newbouldia laevis leaf powder at a dose of 1.6g/kg body weight achieved improved hematocrit levels similar to our study (Olounladé and al, 2017). Sheep treated with Newbouldia laevis leaf powder at dose of 1.6g/kg body weight achieved improved hematocrit levels similar to our study (Olounladé andal, 2017). In contrast to our study, Mendonça-Lima and al. (2016) did not obtain a significant difference in hematocrit levels in goats treated with different doses of Cratylia mollis leaf decoction in Brazil. The area and/or the different species incriminated in these goats could explain this difference. During treatment, no signs of toxicity (salivation, skin reaction, diarrhea) were observed in the animals. The transaminases, creatinine and the different energy and mineral parameters measured at D0 and D21 are within the range of the usual reference values, which shows non-injury of the liver and kidneys of the animals treated with the whole plant powder of S. hermonthica.

CONCLUSION
The results of the present study show a significant reduction in FEC of animals artificially infested with H. contortus after treatment with the tested doses of S. hermonthica powder. Treatment with S. hermonthica powder resulted in improved hematocrit levels in treated animals between D0 and D21, but weight changes were not elevated in animals from different lots. The liver and kidneys of the animals were not affected by the treatment of S. hermonthica powder at the tested doses. Thus, S. hermonthica can be used as an alternative to chemical treatment of GIN in powder form.