Anticoccidial and Biochemical Effects of Artemisia Brevifolia Extract in Broiler Chickens

ABSTRACT

This study was conducted to evaluate the anticoccidial potential of Artemisia brevifolia extract (ABE) and its effect on biochemical parameters against experimental coccidiosis in broiler chickens. For this purpose, a total of 150 broiler chicks were procured and divided into 5 equal groups (30 birds per group). At one week of age, the first three groups (A, B and C) were orally fed with ABE at dose rates of 100, 200 and 300mg/kg respectively for seven consecutive days. Group D served as infected/medicated and was treated with Baycox^® (1ml/L of water). Group E served as the infected/non-medicated control group. At two weeks of age, all groups were infected with 50,000 sporulated oocysts of Eimeria tenella. The anticoccidial potential was evaluated based on lesion score, oocyst score, fecal score, feed conversion ratio, and mortality (%). Furthermore, the effect of ABE on hematological parameters and serum chemistry was also evaluated by following standard procedures. The anticoccidial effects were compared with the standard drug Baycox^® (p>0.05). Results of the study showed the anticoccidial activity of ABE in terms of lesion diminishing, oocyst and fecal scores, better feed conversion ratios, and mortality reduction (%). The ABE also improved hematological parameters and stabilized the serum enzymes (ALT, LDH, Creatinine) of infected chicks (p>0.05).

Keywords:
Artemisia brevifolia; Eimeria: Resistance: Control

INTRODUCTION

Avian coccidiosis is caused by protozoa of genus Eimeria, which belongs to family Eimeriidae and has multiple species with complex life cycle stages (Abbas et al., 2015, 2019; Khater et al., 2020). Eimeria species mostly affect bird intestines and cause bloody diarrhea, poor feed conversion ratios, and high mortality. Among all Eimeria species, E. tenella and E. necatrix are the most pathogenic, causing high mortality in chickens. Naturally, coccidian oocysts are present everywhere and infection starts with the bird’s ingestion of sporulated oocysts, which then produce millions of oocysts in the host’s body (Fall et al., 2016; Lin et al., 2020; Zhang et al., 2020). Coccidiosis causes heavy economic losses to the world’s poultry industry annually (Chapman, 2014; Abbas et al., 2017a). Synthetic anticoccidial drugs are generally used to control coccidiosis, including a variety of anticoccidial drugs. However, due to the irrational use of anticoccidial drugs, resistance has been developed and these drugs are losing their effectiveness (Abbas et al., 2008, 2011a; Bachaya et al., 2015; Abbas et al., 2019). It takes time to find some alternative agents for coccidiosis control. In the past decade, several alternative approaches including use of plants and their products have been reported for their anticoccidial potential (Abbas et al., 2011a, 2011b, 2011c, 2017b; Idris et al., 2017; Sun et al., 2019; Ayatollahi et al., 2019; Tammam et al., 2020). Because of frequent reports on parasites’ resistance against chemical drugs (Saddiqi et al., 20006; Abbas et al., 2008, 2011a), the experimental use of plant extracts especially rich in antioxidants as antiparasitic agents has gained special importance (Abbas et al., 2018, 2020; Ashraf et al., 2020; Asrar et al., 2020; Khater et al., 2020; Aboubakr et al., 2019; Fayyaz et al., 2019; Elghobashy et al., 2020; Gohar et al., 2020; Hassan et al., 2020; Salman et al., 2020; Yasmin et al., 2020; Zaman et al., 2020). Antioxidant (phenols, flavonoids, tannins, and saponins) rich plants (Khaskheli et al., 2020; Mahmoud et al., 2020; Qamar et al., 2020) are being used as an alternative strategy to treat coccidiosis (Abbas et al., 2017b; Batool et al., 2019; Yi et al., 2020).

Artemisia brevifolia, known as worm wood in English and as Afsanteen locally, bears diverse pharmacological and therapeutic properties against different parasitic and bacterial diseases, due to the action of various antioxidant compounds (Allen et al.,1997; Zeb et al., 2018). Based on the diverse therapeutic properties of Artemisia brevifolia, the present study was planned to assess the anticoccidial activity of its extract against Eimeria infection in broiler chickens.

MATERIALS AND METHODS

Plant Material

Artemisia brevifolia (afsanteen) leaves were procured from a local market. The plant material was dried out under a covered place and then ground into powder using an electric mill. The aqueous methanolic extract of Artemisia brevifolia was prepared using Soxhlet’s apparatus (Velp Italy) (Abbas et al., 2017b). The prepared Artemisia brevifolia extract was stored at 4°C in a refrigerator until further use.

Parasite

The Eimeria parasite was collected from outbreak cases in Faisalabad and guts of naturally infected chicken with Eimeria. Oocysts of E. tenella were collected from caeca of chickens obtained from coccidiosis outbreaks in Faisalabad, Pakistan. The sporulation of oocysts was conducted in a potassium dichromate solution (2.5%) at 25-29°C with 60-80% humidity level (Ryley et al., 1976). The sporulated oocysts were counted using the modified McMaster technique. The slides were examined under microscope at low (10x) and high magnification (40x).

Experimental Design

The experiment was approved by the Board of studies committee members, Department of Parasitology, University of Agriculture, Faisalabad, Pakistan. A total of 150 broiler chicks were purchased and divided into 5 equal groups (30 birds per group). At one week of age, groups (A, B and C) were orally fed with Artemisia brevifolia extract at 100, 200, and 300 mg/kg respectively for seven consecutive days. All doses of plant extract were dissolved in PBS and fed orally using a soft plastic tube attached to a 0.5 ml sterile syringe. Group D served as infected/medicated, being treated with Baycox^® added to the drinking water (1 ml/L of water). Group E served as the infected control group. At the same day, all groups were orally infected with 50,000 sporulated oocysts of Eimeria tenella.

Evaluation Of Anticoccidial Activity

The anticoccidial potential of Artemisia brevifolia extract (ABE) was evaluated on the basis of parameters such as mortality (%), which was calculated till the end of experiment, with death cause being confirmed by postmortem analysis. Feed Conversion rate (FCR) was evaluated on the 30^th day of the experiment (Abbas et al., 2019), while lesion scores were recorded on the 14^th (Johnson & Reid, 1970). An oocyst score ranging from 0 to 5 was also recorded on the 14^th day of trial, calculated through a microscopic examination of caecal scrapings (Hilbrich, 1978). A fecal score (1 to 5) was recorded between the 4^th and 6^th day after the infection (Youn et al.,1993). Score of 1 indicated normal feces and score of 5 indicated the presence of severe diarrhea or profuse blood. These parameters were monitored in order to estimate the intensity of disease.

Serum Chemistry

Serum samples were analyzed using different imported kits (Merck, Germany) for serum enzymes levels (ALT, LDH, Creatinine) to check the toxic effects of plant extract and cellular injuries, if any (Abbas et al., 2019).

Hematological Parameters

Blood and serum samples were collected at the 40^th day of experiment. Collected blood samples were analyzed for Packed cell volume (PCV) by the microhaematocrit method. Hemoglobin level (Hb) was checked using Sahli’s apparatus (Benjamin, 1978). Erythrocyte (RBCs) and leukocyte (WBCs) counting was done using an haemocytometer (Natt & Herrick, 1952).

Statistical Analysis

Statistical analysis was done through the ANOVA technique using the SAS software (SAS, 2004). The data was considered statistically significant with p-value <0.05.

RESULTS AND DISCUSSION

All the groups administered with Artemisia brevifolia extract (ABE) exhibited better feed conversion ratio (FCR) at graded doses (Figure 1). The best FCR was observed in the group treated with the highest dose of plant extract. There was no statistical analysis for FCR due to the group feeding of chicks. A lower mortality (%) was observed in ABE administered groups and the lowest mortality was observed in the groups treated with the highest dose of ABE (Figure 2). Lower lesion scores were observed in ABE treated groups (Table 1). Groups treated with ABE showed reduced lesion scores, with no significant difference to the Baycox^® treated group (p>0.05) and significantly different to the infected/non-medicated group (p<0.05).

Table 1
Lesion scores of chickens infected with Eimeria tenella and treated with different doses of Artemisia brevifolia extract.

Groups	0	+1	+2	+3	+4	Mean±SD
ABE@ 100 mg/kg	-	1	2	2	1	2.50±0.5b
ABE @ 200 mg/kg	-	1	2	3	-	2.33±0.5b
ABE @ 300 mg/kg	-	2	2	2	-	2.00±0.5c
Baycox®	-	3	2	1	-	1.66±0.5c
Infected/non-medicated	-	-	1	2	3	3.33 ±0.5a

• ABE = Artemisia brevifolia extract

Figure 1
Feed conversion ratios (FCR) of chickens infected with Eimeria tenella and treated with different doses of Artemisia brevifolia extract.

Figure 2
Mortality rates (%) of chickens infected with Eimeria tenella and treated with different doses of Artemisia brevifolia extract.

All ABE administered groups showed reduced oocyst scores in infected chicks. However, the oocyst scores of groups treated with a higher dose of ABE was non-significantly different (p>0.05) from Baycox^® (Table 2). The fecal score of ABE treated groups was significantly different to those treated with standard medicine (Baycox®) (Table 3). However, the best results were obtained in the group administered with the highest dose of ABE. Artemisia brevifolia extract showed improved serum chemistry in infected birds (Table 4). At a higher dose of ABE, the serum enzyme values were non-significantly different (p>0.05) from the Baycox® group.

Table 2
Oocyst scores of chickens infected with Eimeria tenella and treated with different doses of Artemisia brevifolia extract.

Groups	0	+1	+2	+3	+4	+5	Mean±SD
ABE@ 100 mg/kg			1	2	2	1	3.5 ± 0.5b
ABE @ 200 mg/kg		1	1	2	2		2.8 ± 0.75b
ABE @ 300 mg/kg	1	1	3	1			1.6 ± 0.5c
Baycox®	2	2	2				1.00 ± 0.5c
Infected/non-medicated				2	2	2	4.00 ± 0.50a

• ABE = Artemisia brevifolia extract

Table 3
Fecal scores of chickens infected with Eimeria tenella and treated with different doses of Artemisia brevifolia extract.

Groups	4th day	5th day	6th day
ABE @ 100 mg/kg	3.21±0.8b	3.30±0.7a	2.89±0.6b
ABE @ 200 mg/kg	3.15±0.5b	3.22±0.7a	1.90±0.5bc
ABE @ 300 mg/kg	3.00±0.5bc	3.08±0.75bc	1.81±0.6c
Baycox®	1.71±0.8c	1.66±0.5c	1.37±0.5c
Infected/non-medicated	3.80±0.44a	3.89±0.5a	3.00±0.88a

• ABE = Artemisia brevifolia extract.

Table 4
Serum enzyme values of chickens infected with Eimeria tenella and treated with different doses of Artemisia brevifolia extract.

Groups	ALT	LDH	Creatinine
ABE@ 100 mg/kg	14.90 ± 1.23b	575.76 ±16.74b	0.36 ±0.03a
ABE @ 200 mg/kg	12.90 ± 1.71b	556.91±18.30b	0.28 ±0.04b
ABE @ 300 mg/kg	12.80 ±1.14c	533.03 ±24.35c	0.26 ±0.03c
Baycox®	11.50 ±1.11c	518.50 ±20.13d	0.17 ±0.01c
Infected/non-medicated	25.61 ± 2.32a	889.94 ±22.16a	0.54 ±0.03a

• ABE = Artemisia brevifolia extract; ALT = Alanine transaminase; LDH = Lactate dehydrogenase.

ABE also improved the blood parameters of the infected groups (Table 5). Hematological values (including PCV, RBCs, WBCs, and Hb) were significantly different from the infected/non-medicated group (p<0.05), and were non-significantly different from Baycox®(p>0.05).

Table 5
Hematological values of chickens infected with Eimeria tenella and treated with different doses of Artemisia brevifolia extract.

Groups	PCV%	Hb g/dl	RBC106/µL	WBC 103/µL
ABE@ 100 mg/kg	15.33±1.63c	9.58±1.35c	2.88±0.48b	20.01±3.54b
ABE @ 200 mg/kg	17.20±1.47c	10.44±0.59c	3.13±0.61a	19.86±2.31b
ABE @ 300 mg/kg	18.19±2.31b	10.90±1.01a	3.20±0.48a	21.16±2.94b
Baycox®	24.23±1.75b	12.00±0.11a	3.05±0.65a	23.77±1.86a
Infected/non-medicated	19.15±1.47c	8.20±0.55d	2.00±0.24c	16.85±3.98c

• ABE = Artemisia brevifolia extract.

Previous and recent research reports have shown that plants rich in antioxidant compounds (phenols, flavonoids, tannins, and saponins) are being used as alternative agents to treat avian coccidiosis and have shown promising anticoccidial effects (Allen et al.,1997; Jang et al., 2007; Molan et al.,2009; Abbas et al., 2020). Based on the results obtained from the current research, it may be concluded that Artemisia brevifolia extract has anticoccidial activity in terms of different parameters, including lesion score, fecal score, mortality (%), and FCR. The ABE also improved the hematology and serum chemistry of infected chicks. Results were non-significantly different to standard medicine (Baycox^®) (p>0.05). The results of this study revealed that Artemisia brevifolia at 100, 200, 300 mg/kg showed dose-dependent anticoccidial activity against Eimeria tenella infection in broiler chickens. Similar types of dose dependent trends have also been reported in previous studies on the evaluation of anticoccidial potential of different herbal extracts (Dkhil et al., 2011; Yang et al.,2015; Wang et al., 2016; Abbas et al., 2019, 2020). In one study, ethanolic extract from Carica papaya showed remarkable effect on the weight gain in broiler chickens infected with Eimeria (Nghonjuyi et al.,2015). In another study, oral administration of Ageratum conyzoides extract showed a positive effect in reducing lesion score and oocyst count, while also improving hematological parameters in challenged treated birds (Nwezeand & Obiwulu, 2009).

Recently, Zhang et al. (2020) have reported a similar type of anticoccidial effects with the use of Camellia sinesis (green tea) extract in broiler chickens. Camellia sinensis extract reduced the Eimeria infection by reducing mortality (%), oocysts count, and lesion score in experimentally infected broiler chickens, with effects that were non-significantly different to those of the standard anticoccidial drug Toltrazuril^® (p>0.05).

In another recent study, the anticoccidial effects of Trachyspermum ammi (Ajwain) have been observed in broiler chickens: Trachyspermum ammi was supplemented in the feed of chickens, which reduced lesion and oocyst scores of infected groups (Abbas et al., 2019).

Artemisia brevifolia is enriched with various antioxi-dant compounds (phenols, flavonoids, conyzorium, methexnebilitin, and quercetin), essential oils and some other important derivatives, such as artemisinin, which are commonly used to cure lethal diseases such as malaria and cancer (Zeb et al., 2018).

The anticoccidial potential of Artemisia brevifolia based on different parameters may be due to the action of different antioxidant compounds present in the plant, which can decrease coccidiosis by interfering with the life cycle of Eimeria. During this study, the stabilization of different serum enzyme values in plant administered groups indicates that the extract has no toxic effect. ALT and LDH are refer to hepatotoxicity, while creatinine is refers to nephrotoxicity. Artemisia brevifolia extract also improved hematological parameters (PCV, RBCs, WBCs and Hb) in infected chicks. Plants rich in antioxidants have gained special experimental importance as antiparasitic agents. Based on the aforementioned studies, it may be concluded that plants-derived extracts like that of Artemisia brevifolia may be helpful in controlling avian coccidiosis.

CONCLUSION

The present study showed the anticoccidial effects of Artemisia brevifolia extract in broiler chickens and showed it to be dose-dependent. It suggests that Artemisia brevifolia derived antioxidant compounds may be helpful in controlling avian coccidiosis and treating chickens’ internal injuries. However, further studies are needed to characterize the active novel compounds of Artemisia brevifolia that are involved in enhancing the anticoccidial potential against avian coccidiosis.

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Publication Dates

• Publication in this collection
  10 Sept 2021
• Date of issue
  2021

History

• Received
  13 Jan 2021
• Accepted
  06 June 2021