In vitro effect of aqueous extract of fresh cassava leaves (Manihot esculenta, Crantz) on the larvae of Haemonchus contortus as an intestinal parasite of sheep.


 Cassava is frequently fed to animals. In the case of sheep, the producer relates consumption to a reduction in the parasite load. The literature has proven the effect of phenolic compounds as an anthelmintic in vivo, but no evidence for cyanogenic compounds, also present in all parts of the cassava plant, was found. A controlled in vitro bioassay was used to evaluate the aqueous extract of fresh cassava leaves. The efficiency parameter was the immobility of Haemonchus contortus larvae at the L3 stage, also used to evaluate commercial anthelmintics. Cell culture plates with 100 active L3-stage larvae per well were used, being each replicate constituted by three wells. Aqueous extract of fresh cassava leaves (FCL), Ivomec® 0.01% (PCI) as the positive control, and distilled water as negative control (NCW), were placed in the culture plate wells. Considering the immobility of the larvae as a positive anthelmintic effect, the results showed that in NCW treatments all larvae were mobile, while in PCI all 300 larvae were immobile. FCL produced a gradient of larval inactivation correlation (R2 0.996). The best-fit equation was y = -33.39ln(x) + 40.517, a logarithmic equation, which allowed the calculation of the Lethal Concentration (CL) of 3.44 µg CN- ml, or 80.0 mg of fresh cassava leaves per milliliter of water, with a performance equivalent to ivermectin. This concentration of free cyanide is compatible with the safe consumption of fresh leaves by live weight of sheep. The exact amount of cassava roots, leaves, or shoots, consumed to provide an effective dose for controlling H. contort should be established in vivo. Although phenolic compounds must also be present in the extract, the immobility was attributed to cyanogenic compounds since the correlation was proportional to the increase in the cyanide concentration. It can be concluded that the consumption of fresh cassava leaves has the potential as an anthelmintic agent to be evaluated in vivo by feeding sheep and goats. Local use could also add value to the production of fresh cassava leaves, with an average potential production of 2.5 tonnes ha-1, available throughout the year, with greater production at the beginning of cultivation and in the summer months. These leaves could be used after the roots harvesting or even after pruning for this purpose. Currently, this amount of good quality protein material remains without use in the field.


Introduction
Parasites are a limiting factor in raising sheep and goats. As stated by Kamaraj & Rahuman (2010), research on plants with anthelmintic potential has been carried out for years as alternative methods of controlling populations of parasites, mainly gastrointestinal, due to the resistance to traditional commercial compounds.
Among the main parasites present in sheep farming, Haeconchus contortus, a hematophagous parasite present in the abomasum of sheep, has been developing parasitic resistance to commercial compounds.
This parasite is a powerful hematophagous, feeding up to 0.05 ml of blood per day, causing lesions on the inner wall of the abomasum, in addition to anemia, diarrhea, emaciation, edema, supine position, and severe weakness, becoming a large problem in the production of these animals, as it impacts their performance and can lead to their death (Brik, et al. 2019).
Cassava (Manihot esculenta) has also been considered by several authors as an anthelmintic (Sokerya, 2009;Al-Rofaai et al (2010); Minatchy et al 2020). Some studies are carried out directly on the parasitized animals by including the plant in their feed (Sokerya, 2009;Minatchy et al, 2020) and others are carried out in vitro, observing its effects on eggs, larvae, and adults Kamaraj & Rahuman, 2010). These studies have shown promise concerning the anthelmintic effect of cassava, and researchers usually attribute this result to the presence of tannins in the plant. However, researchers have not furthered their research into the effect of cyanogenic glycosides such as linamarin and lotaustrarin, which are relevant to cassava (Ravindran, 1995).
When consumed by animals, cyanogenic glycosides are processed by enzymes present in the plant itself and by cellulosic microorganisms in the rumen, and the cyanide is then released into the body, which may or may not trigger intoxication (Gensa, 2019). Literature proves tolerance to 2.0-6.0 mg HCN kg -1 of fresh leaves per live weight for ruminants, which is explained by the presence of sulfur-based compounds in their feeds, which in contact with HCN forms the thiocyanate, which is released in the urine, promoting detoxi cation (Onwuka et al, 1992). Sokerya, (2009) obtained promising results regarding the use of fresh leaves and cassava silage with a reduction of eggs present in the animals' feces. In their experiment, the author highlighted the presence of HCN, within the limit for animal consumption, but did not attribute the anthelmintic effect to cyanide, as the target was phenolic compounds, and only highlighted the reduction from 585 mg to 170 mg of HCN by the ensiling process.
Due to the lack of information on the effect of water-extracted cyanogenic compounds, an in vitro protocol was tested to verify the effect of the aqueous extract of fresh cassava leaves (Manihot esculenta) on the mobility of H. contortus larvae at the L3 stage, as well as the amount of cyanide needed to reach the Lethal Dose under these circumstances.

Material And Methods.
Obtaining cassava leaf extract: cassava leaves were collected from the cassava cultivar Paraguainha, a traditional and commercial cultivar, widely used in the Central West region of Brazil. The leaves were washed, dried with paper towels, and weighed. Then, the leaves were immediately macerated with distilled water, obtaining an extract with a concentration of 80 mg of leaves for each ml. absence of gastrointestinal parasites was con rmed by coprological tests. The adult helminths collected from the donor lamb A were introduced into the abomasum of the donor lambs B, according to the surgical technique described by Paiva et al (1999). To con rm the presence of H. contortus eggs, several coprological tests were performed on fecal samples collected from the animals 25 days after the procedure. During the entire experimental period, the animals remained individually housed in pens, free from environmental contamination. To avoid external contamination, the pens were cleaned and sanitized daily with detergents and sodium hypochlorite. To obtain the third-stage larvae (L3) for the tests, the feces were collected in bags made of cotton fabric specially developed for their collection.
Following the guidelines of Tissier et al (1975), these collecting bags were xed for 12 hours directly in the animal's anal region. The material obtained was used to perform the coprological exams, following the methodology described by Roberts and O'Sullivan (1950), where feces and vermiculite were mixed in equal volume and placed in cylindrical glass containers with a capacity of 500 ml, but only half the container being used. With the aid of a glass rod, a hole was made in the center of the material to allow oxygenation. The container remained partially covered, and a piece of thick string was placed over its diameter to ensure that the container remained ajar. Once a day, distilled water was sprinkled over the material to keep the culture moist. The containers were labeled with the date and then incubated in a B.O.D chamber at the temperature of 28 °C for 7 days. After the incubation period, the L3-stage larvae were recovered using the Baermann Technique (Witola et al, 2016).
Sheathing and larval motility test of third-stage larvae: L3-stage larvae were sheathed in 0.15% sodium hypochlorite solution at 37 ºC for 5 min (Almeida, 2018). Then, the larvae were washed ve times in sucrose solution (12%) through centrifugation process to remove the remains of the sheaths and possible dead larvae (Paiva et al, 2001). The number of larvae by milligram was estimated by diluting L3-stage larvae in distilled water and doing the counting under an optical microscope. The larval motility test described by Hubert and Kerboeuf (1984) and Preston et al (2015), with adaptations, was used to conduct the in vitro bioassays. The unsheathed L3-stage larvae were added to 12-well culture plates (100 L3-stage larvae per well), followed by serial concentrations of 5.2 to 80.0 mg of cassava leaves ml -1 , being each well adjusted with distilled water for a total volume of 2 ml per well. A distilled water solution of Merial Ivomec® at the concentration of 0.01% was used as a positive control (FCL), and distilled water as a negative control (NCW). Subsequently, the plates were homogenized for 3 minutes and placed in a bacterial incubator at 28 °C for 24 hours. After this period, the mobile (viable) and immobile (inviable) larvae were counted with the aid of a stereomicroscope. All bioassays were performed in triplicate. The motility index was calculated for each group of different concentrations and positive and negative control, applying the Equation I: Total n o of L3 in the treatment Where: Unviable: the immobile L3-stage larvae Viable: the mobile L3-stage larvae Statistical analysis: the lethal concentration (LC) of the aqueous extract of fresh leaves of cassava (M. esculenta) enough to paralyze all larvae was estimated from the results, where the concentrations were transformed into Log10. Data from the larval motility index were compared using ANOVA and Tukey test, where the signi cance level adopted was 0.05%. All analyzes were performed using the software BioEstat®.

Results And Discussion
Sheep farmers believe, from experience and observation, that there is a reduction in the egg load of intestinal parasites, including Haemonchus contortus, in animals fed with cassava. The researchers decided to investigate the hypothesis that this reduction occurs due to the presence of free cyanide in the fresh cassava leaf extract that was prepared at the same time as the obtention of H. contortus for the mobility test of L3-stage larvae. The results con rmed the toxic effect on the sheathed larvae of H. contortus, an effect proportional to the concentration of extract of fresh cassava leaves. From the data presented in Table 1, a curve was tted relating the mobility of L3-stage larvae with the concentration of cassava leaf extract. The curve that best t the results was the logarithmic equation y = -33.39ln(x) + 40.517, R 2 =0.996 (Figure 1), with a positive and signi cant correlation of the larvae immobility with the increasing free cyanide concentration in the cassava leaf extract. The equation highlights that the greatest effect was caused between 0.0 and 0.5 µg of CN ml -1 when about 50% of L3-stage larvae lost their mobility. The curve becomes less accentuated with the increase in free cyanide content from this concentration, but it is not asymptotic before affecting the total of larvae, equaling the control of the commercial product based on ivermectin, which had an immediate impact on the larvae mobility.
The equation allowed us to calculate the dose that may cause total paralysis of one hundred L3-stage larvae (LD 100 ), which approximately occurred at 3.5 µg of CNml -1 , corresponding to 80 mg of cassava leaves ml -1 or 80 g l -1 . This result agrees with the results from Sokerya, (2009), who states that the consumption of cassava leaves can reduce the number of intestinal parasites in sheep, but it also may contradict the author's conclusion that a dose of 170 mg of HCN per kg can be innocuous as an anthelmintic. Onwuka et al, (1992) and Sokerya, (2009) reported the need for 246 to 248 mg 100 g of cyanide to achieve similar effects.
It is noteworthy that cassava leaves may vary in cyanide content depending on the cultivar or variety, age, and time of year (Ravindran, 1995). The results obtained in this experiment partially agree with those of Suteky and Ji (2019), where 47.87% of the larvae were immobilized with 12.5 mg ml of cassava leaf extract; this difference can be attributed to the fact that the authors were based on larval development and not on larval motility.  reported e ciency of 57.33% on L3-stage larvae by an extract of 12.5 mg of cassava leaf ml -1 , and that it would be necessary 22 mg to achieve total larvae immobility. Although even less than the 80 mg we found to immobilize all L3-stage larvae, it is necessary to consider that the experiment did not eliminate the effect of solvents used in the cassava leaf extract preparation. The author evaluated solvents, in addition to tannins, alkaloids, avonoids, steroids, and phenols. The solvents used were hexane, chloroform, ethyl acetate, and methanol (80%), precisely the ones that presented the best effect against the larvae, so it is impossible to separate the cyanide effect from the effect of the solvents used.
The same use of solvents was observed in several other studies carried out to evaluate the anthelmintic effect of cassava extracts. Marie-Magdeleine et al (2010), before processing the extract, removed the cyanogen glycosides and then dehydrated the extract. The resulting powder was then diluted with three different extraction solvents, dichloromethane, methane, and water, especially because the author focussed on the effects of tannins on the parasites.
In our experiment, we used only water, as the purpose was to evaluate the cassava leaves naturally, with minimal chemical interventions, so that we could have the closest scenario to the ingestion process of the plant leaves in the animal's rumen, ensuring the presence of all its compounds, even those not soluble in water.
In several studies carried out to evaluate the anthelmintic effect of cassava leaf extract, the authors use different solvents for the extraction, aiming to study the effect of phenolic compounds. Al-Rofaai et al (2012) evaluated tannins and phenols, substances known for their antiparasitic effects, not only against H. contortus, but also against other nematode species. Alkaloids, avonoids, steroids, and phenols, were also evaluated for the same purpose (Sokerya 2009;Suteky and Ji, 2019;Constant, 2020). In order to assess the effect of tannins on parasites, Marie-Magdeleine et al (2010) removed the cyanogen glycosides, and the resulting powder from the process was diluted with dichloromethane, methane, and water.
Another experiment carried out in Malaysia by Sokerya (2009) explores the potential of the cyanide present in cassava leaves, although focusing on goats and not sheep. The author evaluated the anthelmintic effects of cassava fed to animals contaminated with H. contortus but used cassava leaves in different ways such as fresh, ensiled, and dehydrated. The count of eggs present in the feces of these animals was conducted after a speci c period after the ingestion and showed positive results in all groups in which the animals were fed with cassava, but mainly with the consumption of fresh leaves. The authors justify that they did not carry out the detoxi cation of cassava by drying, as carried out by , precisely because the objective was to relate the forms of feeding the animals with cassava and their respective responses to the parasite load. However, the authors emphasize that the risk of poisoning the animal with cyanide should not be neglected, which is why they measured the amount of HCN present in the plants used, which presented 585 mg of CNper kg of fresh leaves, and 170 mg of CNper kg of silage made with the same leaves. The author also highlighted that the values observed are within the tolerance limits for ruminants, which is 2 to 6 mg kg of the animal's live weight Onwuka et al, (1992), reducing the risk of intoxication.
Although our bioassay con rms that the cyanide present in cassava leaves, and not only its phenolic compounds, has a direct effect against H. contortus larvae, these results must be evaluated and adjusted by feeding fresh cassava leaves to sheep.
The comparison of results with the literature showed the lack of information on the anthelmintic potential of cassava leaves through aqueous extract or that that highlights the cyanogenic compounds of the plant, although the literature pointing out the risks of cyanide consumption for human and animal health is very common.
The review also highlighted that the focus of anthelmintic use of cassava leaves for sheep and goats feed has always been hypothesized by the effect of the phenolic compounds, such as tannins, as reported in the literature for their antiparasitic effects, not just against H. contortus, but to other nematode species as well. Also, it would be important to compare the effect of cyanogenic and phenolic compounds, with and without the use of solvents in the same in vitro bioassay.
On the other hand, the research started from the principle that, if the anthelmintic effect is proven, the use of leaves is easier and more viable, since it is not necessary to use the cassava roots, which have commercial value, or the branches, which are used as planting material. Leaves can be obtained uninterruptedly throughout the year or the summer in some regions of southern South America, through pruning, as the literature con rms that up to 3 prunings in 12 months of cultivation do not affect root yields.
If cassava leaves contain compounds from the phenolic group, such as tannins and saponins, in addition to cyanogenic glycosides (Tao et al, 2019), they also have a high nutritional value (Pereira et al, 2018). The use of cassava leaves to feed sheep or goats is possible (Vilpoux et al. 2013) and may also be a powerful tool, diversifying its antiparasitic power and preventing the occurrence of resistance to the present anthelmintic principles. This type of local use could also add value to the production of cassava leaves, as assessed by Sagrilo et al. (2001), who weighted the leaves harvested in commercial cassava plantations for starch extraction, with 12 and 24 months of cultivation of the 5 main cultivars planted. The authors found out an average production of fresh leaves of 2.4 tonnes ha -1 , but ranging from 1.8 to 7.5 tonnes ha -1 , available throughout the year, with higher production at the beginning of the cultivation cycle and in the summer months in Brazil (September to May). Currently, this volume of good-quality protein material remains without use in the eld.

Conclusions
Considering that it is impossible to rule out the antiparasitic effect of phenolic compounds, and even though the investigation of the cyanogen compounds from cassava leaves was never approached indepth, the bioassay was planned with a different concept and perspective in the control of endoparasites, as an aqueous extract and targeting H. contortus larvae mobility as an index of parasitosis and as a target for the evaluation of anthelmintic substances.
It was possible to conclude that the aqueous extract of fresh cassava leaves was able to immediately inhibit the movement of one hundred L3-stage larvae (LD 100 ) of H. contortus with a concentration of 3.5 µg of CNml -1 , which corresponds to 80 mg of leaves per ml -1 or 80 g per liter.
The results obtained are promising but should be complemented with direct feeding tests with fresh, dried, and ensiled leaves, accompanied by dosing the free cyanide, which is generated by the rumen digestive process and circulates with the blood to reach the intestinal parasites, but also to study the effect on hematophagous ectoparasites such as ticks. Tests should be also carried out with a longer duration to verify the possibility of resistance to cassava compounds occurring over time. Only after these tests, rural producers will recommend the use of cassava leaves as natural anthelmintic medicine. v.24, p.595-602, 1975, https