Anticoccidial Activity of Coleus aromaticus Leaves Extract in Naturally Infected Goats

| The treatment and prevention of coccidiosis in goats is costly to backyard farmers. It is for this reason that the use of alternative medicinal plants to treat coccidiosis are explored. Thus, this study aimed to determine the anticoccidial activity of the ethanol extract of C. aromaticus leaves. A total of 20 naturally coccidia-infected goats at the University of Southern Mindanao Goat Project served as experimental animals for the study. In relation to this, three concentrations of Coleus aromaticus extract were tested for anticoccidial activity: 600, 800, and 1000 mg extract/ kg bodyweight. The efficacies of these concentrations were determined and evaluated by comparing with a commercial coccidiostat, toltrazuril. It was found that 21 days post-treatment, the efficacies of the three extract concentrations were significant but was lower than toltrazuril. A significant difference (p = 0.0394) existed in the efficacies of the extracts among the treatments on day 21. The efficacy rate of administering 600 mg/kg extract (92.29%) was comparable to that of toltrazuril (96.65%) at 21 days post-treatment. This shows that the extract has the potential to be as effective as toltrazuril in reducing the oocyst per gram counts of naturally-infected goats.


INTRODUCTION
C occidiosis remains to confront the goat industry affecting growth, health, and reproduction. It is a protozoan infection caused by several species of the genus Eimeria which develop in the small and large intestine. In particular, coccidiosis in goats leads to dehydration, diarrhea, weight loss, emaciation, weakness and anorexia that limit growth and result in decreased production when death occurs (Soulsby, 1982;Chartier and Paraud, 2012). It becomes an infection of economic importance due to intensive breeding conditions together with high animal density and high productivity (Foreyt, 1990;Chartier and Paraud, 2012). It often leads to morbidity and mortality, particularly in young animals up to 6 months of age (Ruiz et al., 2006;Majeed et al., 2015). In order to minimize the production losses and reduced productivity, good husbandry practices, hygiene maintenance, and medicines are important in reducing the contamination by infective oocysts .
Commercial drugs are commonly used in controlling coccidiosis because of their availability in the market. Drugs that have been used to control and treat coccidiosis in goats include amprolium (Young et al., 2011), decoquinate (Gibbons et al., 2016), diclazuril (Ruiz et al., 2012), halofuginone lactate (Giadinis et al., 2008), and toltrazuril . However, the continued use of commercial coccidiostat has resulted in the development of resistance of the parasite. Because of this and the cost of commercial drugs, alternative remedies to control coccidiosis are practiced and investigated.
Coleus aromaticus is a medicinal plant that has various reported bioactivities. It is a perennial and aromatic herb that is found in Asia, Africa, and South America where it is extensively used for a wide range of diseases (Lukhoba et al., 2006). Leaf extracts from C. aromaticus have been reported to exhibit antibacterial (Koba et al., 2011), antimicrobial (Deena et al., 2002), anthelmintic (Hussain et al., 2012), antidiabetic and antiproliferative activities (Govindaraju and Indra-Arulselvi, 2018). In addition, it also exhibited mosquitocidal and repellent activity (Baranitharan et al., 2017). Hence, the present work investigates the anticoccidial property of C. aromaticus leaf extracts in naturally-infected goats.

plAnt mAteriAl And extrAction
Fresh C. aromaticus leaves, shown in Figure 1, were collected in Kabacan, Cotabato, Philippines and were authenticated at the University of Southern Mindanao Department of Biological Sciences. The selected leaves were healthy and free of insect bites, discoloration, and diseases. Extraction of the plant material was done based on the method described by Eguale et al. (2011) with modifications and a different extracting solvent. The leaves were washed with water and were dried at room temperature. The dried leaves were then pulverized using a blender, which were then soaked in ethanol for 48 hours. For every 50 g of pulverized leaves, 300 mL of ethanol was added to ensure proper soaking. The solvent was evaporated in vacuo and the extract was stored at 4°C until use (Baranitharan et al., 2017).

AnimAls And experimentAl desiGn
The procedures used in the study were approved by the University of Southern Mindanao College of Veterinary Medicine Institutional Animal Care and Use Committee. The methods for research and animal handling are all in compliance with Republic Act 8485, The Animal Welfare Act.
A preliminary fecal examination using the modified McMaster method (Hutchinson, 2009) was performed to determine the number of oocysts per gram (opg) of feces and to identify which goats were infected with Eimeria that will be used for the study. Identification of the Eimeria species was based on morphometric characteristics of the oocysts and in some cases, based on the presence or absence of a micropyle and oocyst shape (Levine and Ivens, 1967;Saravia et al., 2021).
A total of twenty (20) naturally coccidia-infected goats with ages between three to four months, regardless of sex and breed, were obtained from the University of Southern Mindanao Goat Project from November to December 2020 to serve as experimental animals. The experimental animals weighed within 5.9-15.5 kg and the number of oocysts ranging from 1,400 to 20,150 opg. The animals were from the same herd in a semi-confined rearing system at the University of Southern Mindanao Goat Project facility.
The goats were divided into 5 groups (A-E) of four (4) goats each. The treatments, extracts and controls, were administered orally. Goats in groups A-C were given a single dose of 600, 800, and 1000 mg extract/kg bodyweight. Those in group D were treated with a single dose of 5% toltrazuril (0.4 mL/kg bodyweight) while those in group E were untreated and served as the negative control. The goats were fasted 12 hours prior to treatment to slow down gastrointestinal transit and allow better absorption of the drug administered orally (Lespine et al., 2007). The management of goats at the University of Southern Mindanao Goat Project was adapted. The animals were maintained in an elevated indoor area with wood-slatted floor and were given time to roam around the facility. Grass, hay, water, and mineral salt were provided ad libitum and the concentrate in the afternoon. All diets were free of coccidiostats and were prepared to ensure the nutritional requirements of the goats.

pArAsitoloGicAl exAminAtion
Fecal samples (3-5 g) were collected 7, 14, 21 and 28 days post-treatment (dpt). Fecal samples were scored using a correction factor based on a scale of 1-3 with 1, normal pellets; 1.5, soft formed but the pellets did not separate; 2, soft but there is no formation of pellets, and; 3, diarrheic (Skerman and Hillard, 1967;Le Jambre et al., 2007). Fecal examination was done using modified McMaster technique (Hutchinson, 2009) to determine the oocysts per gram (opg) of feces.

stAtisticAl AnAlysis
Statistical significance among treatments (p ≤ 0.05) was compared using one-way ANOVA and Tukey HSD test.

Advances in Animal and Veterinary Sciences
September 2021 | Volume 9 | Issue 9 | Page 1364

RESULTS AND DISCUSSION
Among the naturally coccidia-infected goats, there were fifteen (15) identified species of Eimeria, of which Eimeria arloingi was the most predominant. The mean oocysts per gram (opg) counts of goats in the different treatment groups are shown in Table 1. In general, there was an observed reduction of opg in naturally-infected goats. A similar reduction of opg was observed 21 days post-treatment.
The efficacy of the extracts in each treatment is shown in Table 2. Within 7 dpt, group A had the highest efficacy (62.96%) among the extracts. This was still lower than the efficacy of the commercial coccidiostat, toltrazuril. However, the efficacy of the extract given to group A increased at 21 dpt reaching 92.29%, which was comparable to that of toltrazuril (96.65%). This continued to increase 28 dpt.
However, the increase in efficacy was not observed for all doses of the extracts. Goats in groups B and C experienced an increase in efficacy only after 21 dpt. At 28 dpt, the efficacy of the extracts was inversely proportional to the dose administered. Statistical analysis shows that it was only 21 days post-treatment that there was a significant difference (p=0.0394) in the efficacy of the C. aromaticus extract. It should be noted that during this time, it was only the dose of 600 mg/kg extract resulted in an efficacy above 90% similar to toltrazuril. Since the goats were naturallyinfected, it is hypothesized that the variation in the efficacy of the extracts could be due to the different stages of Eimeria in the goat. Moreover, this may also potentially be accounted for by the phytochemical constituents present in the extract.
Bioactive phytochemical constituents are also present in the root extract of C. aromaticus. Hussain et al. (2012) reported that the methanol extract of the roots showed the presence of flavonoids, terpenoids, alkaloids, and tannins. It exhibited anthelmintic activity against the Indian adult earthworm (Pheretima posthuma), which may have been caused by the phytochemical constituents present, particularly tannins and phenolic compounds. However, compounds are yet to be isolated and identified from the root extracts.

Advances in Animal and Veterinary Sciences
September 2021 | Volume 9 | Issue 9 | Page 1365 Thymol, carvacrol, and saponins are known to exhibit anticoccidial activity (Felici et al., 2020). The thymol, carvacrol, saponins, and tannins present in the C. aromaticus leaf extract may have produced similar anticoccidial effects. Saponins tend to prevent the growth of protozoans by interacting with cell membrane cholesterol content resulting in the death of the parasite. The phenolic content which includes carvacrol (Sidiropoulou et al., 2020) may have affected the oocyst by interacting on the oocysts' cytoplasmic membrane and the permeability of hydrogen ions and potassium ions (Giannenas et al., 2003). Additionally, tannins have exhibited a significant effect on the reduction of the sporulation of the Eimeria species (Masood et al., 2013). Terpenoids, such as thymol, and tannins can bind to free proteins in the gastrointestinal tract or glycoproteins of the parasite leading to death. Further, tannins or their metabolites have a direct effect on the viability of the parasite during pre-parasitic stages (Hussain et al., 2012). This may support the variation in the efficacies of C. aromaticus leaf extract.
Coccidiosis causes reduced production and decrease in body weight in goats. Moreover, the tropical conditions promote the incidence of these parasitic diseases because the moist and warm environmental conditions stimulate sporulation of coccidia. It is through the timely use of medicines that the disease can be prevented. The chemical components of anticoccidial products target the parasite at varying stages of its life cycle. Amprolium, monensin, and lasalocide acts during the early stages of the life cycle while sulfonamides have an activity at the last stages. By contrast, toltrazuril acts on the whole cycle of the coccidia (Taylor, 2009), thus having both curative and preventive actions.
These drugs potentially face the problem of the parasite developing drug resistance. The long and repeated usage of the same drugs with no breaks or alternatives results to this resistance thus emphasizing the need for alternative anticoccidial drugs.

CONCLUSIONS AND RECOMMENDATIONS
The result of the study shows that the leaf extract of C. aromaticus has potential in vivo anticoccidial activity, with a similar activity to that of the commercial coccidiostat toltrazuril. The extract was able to reduce the fecal opg within 21 days post-treatment. Moreover, the extract can be developed into a medicine that can be used to treat coccidiosis.

NOVELTY STATEMENT
It was previously shown that root extracts of C. aromaticus exhibited anthelmintic activity. Leaves present a more viable source of secondary metabolites and have been reported to show antioxidant activity. However, its anthelmintic activity against coccidia, specifically against Eimeria, has not been reported. This study demonstrates that the extract from the leaves of C. aromaticus is able to elicit anthelmintic activity in naturally-infected goats. This means that the extracts contain sufficient amounts of secondary metabolites to produce such effect even after interaction with the diet and host metabolism.