Introduction
Coccidiosis is an enteric parasitic disease caused by a protozoan Eimeria species of the phylum Apicomplexa. It is the major parasitic disease of poultry causing substantial economic losses due to malabsorption, poor feed conversion rate, reduced weight gain, and high mortality [1,2]. Eimeria species widely recognized as the causative agents of coccidiosis in chickens include E. tenella, E. necatrix, E. maxima, and E. brunetti, which are highly pathogenic. Eimeria acervulina and Eimeria mitis are less pathogenic, while Eimeria praecox is regarded as the least pathogenic [3,4]. These species have been detected in Nigeria [5–7]. Other Eimeria species identified include E. lata, E. nagambie, and E. zaria, in Australia and Nigeria [8], Ghana, India, Tanzania, Uganda, Unites States of America, Venezuela, and Zambia [9,10].
Good animal husbandry measures such as maintaining good litter quality, controlling house temperature, ventilation, and humidity, and thorough cleaning between flocks were recommended to minimize losses due to coccidiosis. These measures reduce the sporulation of oocysts and decrease the population of the parasite’s infective stage [11]. Although the control of coccidiosis using various drugs (synthetics and ionophores) has long been a mainstream strategy in modern poultry production, this is limited by the development of drug resistance. This resistance has resulted in the adoption of several alternatives for chemotherapeutic purposes with consequent desirable outcomes. These alternatives include extracts of mushroom (Ganoderma lucidum) and plant materials such as Vernonia amygdalina and Vitellaria paradoxa [12,13]. The methanol extract of G. lucidum and V. paradoxa stem bark decreased the negative effects of E. tenella infection in broiler chickens [12]. Also, the methanol extract of V. amygdalina leaves exerted positive anticoccidial effects in broiler chickens [13,14].
Despite the chemotherapeutic benefits of these materials in coccidiosis, there is scanty data on their prophylactic anticoccidial effects. Hence, this study assessed the clinico-pathological changes and performance indices in E. tenella-infected broiler chickens following the prophylactic administration of methanol extracts of G. lucidum, V. amygdalina, and V. paradoxa.
Materials and Method
Ethical approval
Ethical approval was obtained for this study was obtained from the Ahmadu Bello University Committee on Animal Use and Care (ABUCAUC) with an approval number: ABUCAUC/2022/056.
Location of the study
The study was carried out at the Poultry Research Pens of the Veterinary Teaching Hospital, Ahmadu Bello University (ABU) Zaria, Nigeria.
Collection and identification of pharmaceutical materials
The G. lucidum and V. paradoxa stem bark used in the study were provided by Professor P. A. Abdu, of the Department of Veterinary Medicine, ABU Zaria, Nigeria. Leaves of V. amygdalina were harvested at the flowering stage from a private vegetable garden in Makurdi, Benue State, Nigeria. These pharmaceuticals collected were validated at the Department of Botany, ABU Zaria.
Processing, preparation, and phytochemical analyses of extracts
The materials were processed [15] and the methanol extract of each material was prepared using the method described by Youn et al. [16]. The methanol extracts of the G. lucidum, V. amygdalina leaf, and V. paradoxa stem bark were subjected to chemical tests for identification of phytochemical constituents using the method described by Trease and Evans [17].
Experimental chickens
One hundred, 1-day-old Abor acre broiler chickens were obtained from a commercial hatchery and reared in pens containing ten wire-floored battery cages each measuring 1,000 × 1,000 × 800 mm and raised to a height of 100 mm from the floor of the pen. The cages were cleaned, washed with detergent and water, and disinfected 7 days prior to the arrival of the birds. Borehole water and commercial broiler starter feed were given to the birds ad libitum without drugs or supplements. The chicks were brooded together for 2 weeks, screened, and confirmed negative for E. tenella oocysts.
Source of E. tenella oocysts used for the study
Eimeria tenella previously isolated and characterized by Jatau et al. [6] obtained from the Department of Veterinary Parasitology and Entomology Laboratory, ABU Zaria, Nigeria.
Prophylactic treatment of birds
Ganoderma lucidum and V. paradoxa extracts were administered at 250 mg/kg body weight [12] while V. amygdalina extract was administered at 2,000 mg/kg body weight [13] orally for seven consecutive days. Also, 100 gm of amprolium HCl (Amprolium 250 wsp), manufactured by KEPRO B. V.—Maagdenburgstraat 17—7421 ZA Deventer—Holland was purchased from a Veterinary Distributor in Kaduna, Kaduna State, Nigeria, was administered at 100 gm/100 l of drinking water for seven consecutive days.
Challenge of birds with E. tenella oocysts
Birds were challenged at day 21 of age by oral administration of 104 sporulated oocysts of E. tenella using an insulin syringe.
Experimental design
The 100 1-day-old Abor acre broiler chickens were allotted into 10 groups: A1, B1, C1, D1, A2, B2, C2, D2, E, and F (n=10/group) at 14 days of age (doa). Methanol extracts of G. lucidum; V. amygdalina; and V. paradoxa at a dosage of 250; 2,000; and 250 mg/kg body weight was administered orally to chickens in group A1 and A2; B1 and B2; C1 and C2; respectively for seven consecutive days. Group D1 and D2 were administered with amprolium at 100 gm/100 l of drinking water for seven consecutive days. At 21 doa, groups A2, B2, C2, D2, and E (positive control) were challenged orally with 104 sporulated oocysts of E. tenella. Birds in group F (negative control) were untreated and not challenged. The birds were monitored for clinical signs which were graded [12]. Also, morbidity and mortality rates and performance indices were determined [18,19]. At 28 doa [7 days post-challenge (dpc)], five birds from each group were euthanised for gross caecal lesion scoring [20] and histopathological examination.
Data analyses
Descriptive statistics were used to express clinical signs, morbidity and mortality rates, and gross and histopathological lesions. Data for some performance indices were expressed as the means and standard errors of means (±SEM), and statistically analyzed by one-way analysis of variance. SPSS13.0 Data Editor software (SPSS Inc., Chicago, IL) was used for the analyses. Values of p ≤ 0.05 were considered significant.
Results
Phytochemical constituents of extracts
The methanol extracts of G. lucidum contained flavonoids, saponin, anthraquinones, carbohydrates, cardiac glycosides, steroids, and tritepenes; V. amygdalina leaves contained carbohydrates, alkaloids, flavonoids, saponins, tannins, steroids and triterpenes, cardiac glycosides, and anthraquinones; V. paradoxa stem bark contained alkaloids, carbohydrates, saponins, tannins, flavonoids, anthraquinones, cardiac glycosides, steroids, and tritepenes.
Clinical signs
Clinical signs of coccidiosis were observed in broiler chickens of groups A2, B2, C2, D2, and E. The signs observed were dullness, somnolence, ruffled feathers, decreased feed consumption, and bloody and mucoid diarrhea. These signs were observed from 3 dpc, peaked at 5 dpc, and decreased by 7 dpc. Based on severity, the signs were mild in groups A2 (19.3%), B2 (16.7%), and C2 (15.7%), moderate in D2 (29.7%) but severe in group E (41.3%). No clinical signs were observed in groups A1, B1, C1, D1, and F (Table 1).
Morbidity and mortality rates
Caecal lesion score
The caecal lesion score was 0 in broiler chickens of groups A1, B1, C1, D1, and F as there were no visible gross lesions (Table 2). In broiler chickens of groups A2, B2, C2, and D2, the caecal lesions observed were a few scattered petechiations on the serosal wall with normal caecal content and the lesion score was 1. The lesions observed in the caeca of broiler chickens in group E were large amounts of blood present and thickened caecal walls, and the score was 3.
Feed consumed, live body weight gain, and feed conversion ratio (FCR)
The mean (±SEM) feed consumed and live body weight gain of broiler chickens showed no significant (p > 0.05) difference in all groups but was nonsignificantly (p > 0.05) higher in groups A1, B1, C1, D1, and F (Table 3). In the challenged groups of broiler chickens, there were nonsignificantly (p > 0.05) higher mean (±SEM) live body weight gain in groups A2 (586.93 ± 14.83 gm), B2 (577.13 ± 17.83 gm), C2 (581.40 ± 15.96 gm) and D2 (578.07 ± 18.93 gm) compared to group E (562.40 ± 17.01 gm) (Table 3).
Table 1.
Clinical signs observed in broiler chickens administered methanol extracts of G. lucidum, V. amygdalina leaves and V. paradoxa stem bark from 14 to 20 days of age, and challenged with E. tenella oocysts at 21 doa.
| Group | Prophylactic treatment (dose) | Percentage (%) | Grade |
|---|---|---|---|
| A1 | G. lucidum (250 mg/kg) | Nil | Nil |
| B1 | V. amygdalina (2,000 mg/kg) | Nil | Nil |
| C1 | V. paradoxa (250 mg/kg) | Nil | Nil |
| D1 | Amprolium (100 gm/100 l water) | Nil | Nil |
| A2 | G. lucidum (250 mg/kg) + challenge | 19.3 | Mild |
| B2 | V. amygdalina (2,000 mg/kg) + challenge | 16.7 | Mild |
| C2 | V. paradoxa (250 mg/kg) + challenge | 15.7 | Mild |
| D2 | Amprolium (100 gm/100 l water) + challenge | 29.7 | Moderate |
| E | Positive control | 41.3 | Severe |
| F | Negative control | Nil | Nil |
0=no clinical sign; 1–20=mild; 21–40=moderate; ≥41=severe.
Table 2.
Morbidity and mortality rates, and caecal lesion scores of broiler chickens administered methanol extracts of G. lucidum, V. amygdalina leaves and V. paradoxa stem bark from 14–20 doa, and challenged with E. tenella oocysts at 21 doa.
| Group | Prophylactic treatment (dose) | Morbidity rate (%) | Mortality rate (%) | Caecal lesion score |
|---|---|---|---|---|
| A1 | G. lucidum (250 mg/kg) | 0.0 | 0.0 | 0 |
| B1 | V. amygdalina (2,000 mg/kg) | 0.0 | 0.0 | 0 |
| C1 | V. paradoxa (250 mg/kg) | 0.0 | 0.0 | 0 |
| D1 | Amprolium (100 gm/100 l) | 0.0 | 0.0 | 0 |
| A2 | G. lucidum (250 mg/kg) + challenge | 100.0 | 0.0 | 1 (mild) |
| B2 | V. amygdalina (2,000 mg/kg) + challenge | 100.0 | 0.0 | 1 (mild) |
| C2 | V. paradoxa (250 mg/kg) + challenge | 100.0 | 0.0 | 1 (mild) |
| D2 | Amprolium (100 gm/100 l) + challenge | 100.0 | 0.0 | 1 (mild) |
| E | Positive control | 100.0 | 0.0 | 3 (severe) |
| F | Negative control | 0.0 | 0.0 | 0 |
0=no lesion; 1=mild; 2=moderate; ≥3=severe.
Table 3.
Mean (±SEM) body weight gain, feed consumed, and FCRs of broiler chickens administered methanol extracts of G. lucidum, V. amygdalina leaves and V. paradoxa stem bark from 14 to 20 days of age and challenged with E. tenella at 21 days of age.
| Group | Treatment | Initial weight (gm) | Final weight (gm) | Weight gain (gm) | Feed consumed (gm) | FCR |
|---|---|---|---|---|---|---|
| A1 | G. lucidum (250 mg/kg) | 1,004.73 ± 9.17a | 1,622.33 ± 21.22a | 617.0 ± 14.44a | 1,960.29 ± 80.84a | 3.20 ± 0.08a |
| B1 | V. amygdalina (2,000 mg/kg) | 1,005.73 ± 11.62a | 1,628.80 ± 24.29a | 623.07 ± 14.02a | 1,968.14 ± 81.11a | 3.18 ± 0.07a |
| C1 | V. paradoxa (250 mg/kg) | 1,005.27 ± 7.71a | 1,628.00 ± 23.01a | 622.73 ± 16.69a | 1,966.57 ± 81.29a | 3.19 ± 0.09a |
| D1 | Amprolium (100 gm/100 l) | 1,005.13 ± 15.47a | 1,627.47 ± 26.96a | 622.33 ± 15.16a | 1,964.71 ± 81.38a | 3.18 ± 0.07a |
| A2 | G. lucidum (250 mg/kg) + challenge | 1,005.13 ± 10.44a | 1,592.07 ± 23.71a | 586.93 ± 14.83a | 1,948.29 ± 75.34a | 3.35 ± 0.09a |
| B2 | V. amygdalina (2,000 mg/kg) + challenge | 1,005.87 ± 8.36a | 1,583.00 ± 24.87a | 577.13 ± 17.83a | 1,943.00 ± 73.64a | 3.41 ± 0.11a |
| C2 | V. paradoxa (250 mg/kg) + challenge | 1,005.40 ± 10.80a | 1,586.80 ± 25.33a | 581.40 ± 15.96a | 1,943.43 ± 74.29a | 3.38 ± 0.09a |
| D2 | Amprolium (100 gm/100 l) + challenge | 1,004.73 ± 8.97a | 1,582.80 ± 25.83a | 578.07 ± 18.93a | 1,941.14 ± 74.05a | 3.41 ± 0.11a |
| E | Positive control | 1,005.53 ± 10.19a | 1,567.93 ± 25.44a | 562.40 ± 17.01a | 1,893.42 ± 57.08a | 3.41 ± 0.11a |
| F | Negative control | 1,005.93 ± 8.54a | 1,634.93 ± 21.92a | 629.00 ± 15.15a | 1,962.43 ± 82.17a | 3.15 ± 0.08a |
Mean (± SEM) values with the same superscript alphabets along the same column do not differ significantly at p > 0.05.
There was no significant (p > 0.05) difference in the mean (±SEM) FCR of all groups of chickens, but in unchallenged groups, this was nonsignificantly (p > 0.05) lower (Table 3). In the challenged groups, FCR was nonsignificantly (p > 0.05) lower in A2 (3.35 ± 0.09) and C2 (3.38 ± 0.09) than in B2 (3.41 ± 0.11), D2 (3.41 ± 0.11) and positive control (3.41 ± 0.11) (Table 3).
Oocyst output and decrease ratio, and anticoccidial index
The oocyst output was significantly higher in group E than in A2, B2, C2, and D2 (Table 4). The oocyst decrease ratio was 79.1%, 82.9%, 83.5%, 68.4%, and 0.0% in broiler chickens in groups A2, B2, C2, D2, and E, respectively (Table 4). The anticoccidial index was 178.3, 176.8, 177.4, 176.9, and 119.4 in broiler chickens in groups A2, B2, C2, D2, and E, respectively (Table 4).
Table 4.
Oocyst outputs and decrease ratios, and anticoccidial indices of broiler chickens administered methanol extracts of G. lucidum, V. amygdalina leaves, and V. paradoxa stem bark from 14 to 20 days of age and challenged with E. tenella at 21 doa.
| Group | Prophylactic treatment (dose) | Oocyst output (× 105) | Oocyst decrease ratio (%) | Anticoccidial index (ACI) |
|---|---|---|---|---|
| A1 | G. lucidum (250 mg/kg) | 0 | NA | - |
| B1 | V. amygdalina (2,000 mg/kg) | 0 | NA | - |
| C1 | V. paradoxa (250 mg/kg) | 0 | NA | - |
| D1 | Amprolium (100 gm/100 l) | 0 | NA | - |
| A2 | G. lucidum (250 mg/kg) + challenge | 2.20 ± 1.13a | 79.1 | 178.3 |
| B2 | V. amygdalina (2,000 mg/kg) + challenge | 1.80 ± 0.34a | 82.9 | 176.8 |
| C2 | V. paradoxa (250 mg/kg) + challenge | 1.74 ± 0.56a | 83.5 | 177.4 |
| D2 | Amprolium (100 gm/100 l) + challenge | 3.33 ± 2.49a | 68.4 | 176.9 |
| E | Positive control | 10.55 ± 2.45b | 0.0 | 119.4 |
| F | Negative control | 0 | NA | - |
Mean (±SEM) values with different superscript alphabets along the same column differ significantly at p < 0.05. NA=Not applicable.
Figure 1.
Photomicrographs of sections of caeca of unchallenged broiler chickens pretreated with methanol extracts of G. lucidum (A1), V. amygdalina (B1), and V. paradoxa (C1), and Amprolium (D1); untreated and challenged with E. tenella oocysts (E); untreated and unchallenged (F). Note intact mucosal glands (m) and tissue architecture in A1, B1, C1, D1, and F; desquamated mucosal glands with developmental stages of E. tenella (arrow) and distorted tissue architecture in E. H&E × 200.
Outcomes of histopathological examination of the caeca
The caeca of unchallenged broiler chickens (groups A1, B1, C1, D1, and F) showed intact mucosal glands and tissue architecture (Fig. 1). In broiler chickens of group E, there was severe desquamation of the mucosal glands and distortion of tissue architecture (Fig. 1). There was slight desquamation of the mucosal glands in the caeca of broiler chickens in groups A2, B2, C2, and D2 (Fig. 2).
Discussion
In this study, the findings of phytochemical analysis of G. lucidum methanol extract are consistent with that reported by Usman et al. [15] in Lafia, Nassarawa State, and Kilyobas [12] in Zaria, Kaduna State Nigeria. Another study in India revealed the presence of tannin in the methanol extract of G. lucidum [21]. The phytochemical constituents of V. amygdalina leaf are similar to those reported in other studies [22–24]. However, Okoduwa et al. [25] reported the presence of polyphenols and triterpenes, and the absence of anthraquinones in the methanol leaf extract of V. amygdalina. Udochukwu et al. [26] in addition, reported the presence of oxalate, phytate, phenols, and cyanogenic glycosides in the aqueous and ethanolic leaf extracts of V. amygdalina. The phytochemical constituents of the methanol extract of V. paradoxa stem bark are similar to those reported by Kilyobas [12]. However, Ndukwe et al. [27] reported the absence of cardiac glycoside and flavonoid in V. paradoxa collected from Zaria Kaduna State, Nigeria. Variations in climatic conditions, geographical locations, soil type, extraction method, type and volume of solvent, part of plant used and season in which plant was collected might be responsible for the discrepancies between the phytochemical constituents in plants used in the present study compared to those of other studies [28,29].
Figure 2.
Photomicrographs of sections of caeca of challenged broiler chickens pretreated with methanol extracts of G. lucidum (A2), V. amygdalina (B2), and V. paradoxa (C2), Amprolium (D2); untreated and challenged with E. tenella oocysts (E); untreated and unchallenged (F). Note slightly desquamated mucosal glands (m) in A2, B2, C2, D2; intact mucosal glands (m) and tissue architecture in F; desquamated mucosal glands with developmental stages of E. tenella (arrows) and distorted tissue architecture in E. H&E × 200.
The clinical signs of coccidiosis in challenged groups (A2, B2, C2, D2, and E) of broiler chickens in this study were dullness, somnolence, ruffled feathers, decreased feed in-take, bloody diarrhea, and mucoid diarrhea. This confirmed that the characterised E. tenella isolate [6] used in this study was pathogenic to the broiler chickens. However, the morbidity, and mortality rates due to coccidiosis were 100.0% and 0.0%, respectively in all groups of broiler chickens. These findings are consistent with the reports of other studies [12,29–32].
In the present study, the clinical signs were mild in broiler chickens pretreated with methanol extract of G. lucidum, V. amygdalina, and V. paradoxa, and moderate with amprolium after challenge with E. tenella. A previous study documented mild clinical signs of coccidiosis in broiler chickens challenged with E. tenella at a dose of 36,250 sporulated oocysts per bird and 42 doa following administration of aqueous extract of G. lucidum at 49 doa for 7 days [33]. Vernonia amygdalina leaf administered in the feed of broiler chickens inhibited the invasion of caecal endothelial cells by sporozites; this supports its anticoccidial role as Eimeria parasites must invade and multiply in the endothelial cells of the caecum before the infection is established [14,34]. In another study, mild clinical signs of coccidiosis were reported in broiler chickens challenged with 20,000 sporulated E. tenella oocysts at 21 doa and administered methanol extracts of G. lucidum and V. paradoxa at 250 mg/kg body weight each from 24 to 28 doa [12]. In these studies [6,12], the extracts were administered post-challenge while prophylactic administration of the extracts was performed in the present study. The similar result of mild clinical signs suggests that these extracts had a prophylactic effect against the E. tenella challenge.
The clinical signs and caecal lesions due to coccidiosis were associated with the destruction of the caecal epithelium and underlying connective tissue of the mucosa resulting from the proliferative stages of E. tenella and this may lead to hemorrhage into the lumen, catarrhal inflammation, and diarrhea [35]. The mild clinical signs and caecal lesions in this study might be due to the protective effects of the extracts on the caecal mucosa and submucosa thus preventing the destructive effect of the E. tenella oocysts. The protective effect is speculated as these extracts were administered prior to the challenge. The immunomodulating effects of the constituents of the extracts in response to the challenge with the E. tenella oocysts might be another possible mechanism. These immunomodulating effects might be due to the saponin [36], alkaloid [37], tannin [38], anthraquinone [39], flavonoid [40], and cardiac glycoside [41] present in the extracts. However, extracts of V. paradoxa and V. amygdalina resulted in milder clinical signs and caecal lesions than G. lucidum and this might be associated with the absence of tannin and alkaloid in G. lucidum. Although nonsignificant, the higher feed intake live weight gain, and lower FCR in the pretreated challenged groups compared to positive control might be associated with the prophylactic anticoccidial effect of the extracts.
The oocyst output was absent in unchallenged groups and significantly lower in the pretreated challenged groups than in positive control. The absence of Eimeria oocysts in the unchallenged groups was expected as they were not challenged with E. tenella oocysts. This indicated that there was no accidental challenge during the course of the study. The significantly lower oocyst output with corresponding higher oocyst decrease ratio and anticoccidial index in the pretreated challenged groups compared to the positive control might be due to the protective effects of the extracts on caecal epithelia and submucosa. This protection thus negatively interfered with the proliferation of E. tenella and consequently decreased oocyst output. Thus, based on the ACI of G. lucidum, V. amygdalina, and V. paradoxa extracts in this study, they could be said to be effective against caecal coccidiosis following their prophylactic administration.
Conclusion
The prophylactic administration of G. lucidum, V. amygdalina leaf, and V. paradoxa stem bark methanol extracts decreased the clinical signs, caecal lesions, and histopathological changes, and improved performance in the E. tenella-challenged broiler chickens. Studies involving the identification of the bioactive substances in these extracts responsible for their prophylactic anticoccidial activities and their possible mechanisms are therefore recommended.
Conflict of interest
The authors declare no potential conflict of interest.
