Ethical approval /statement of animal ethics
In this study, all procedures involving the use of broiler chicken were carried out in accordance with the Ahmadu Bello University Committee on Animal Use and Care’s ethical standards and approval (No: ABUCAUC/2020/63) was obtained.
Housing and management of experimental chickens
The birds were housed at the experimental Poultry Research Pen of the Department of Veterinary Parasitology and Entomology, Ahmadu Bello University Zaria, in cages with wire-floored batteries raised to a height of 10 cm off the floor of the pen. Electric bulbs and large netted wire-meshed windows were used to maintain the recommended temperature. Feed and water were provided ad libitum throughout the study period. Feeders and drinkers (3 each) were provided per group with a 200-watt bulb in each of the 6 compartments to provide light and heat. Feeders and drinkers were washed on a daily basis to minimize risks of contamination. Faecal droppings were screened for coccidia oocysts using simple floatation technique as described by Dryden et al. (2005) before experimental infection.
Eimeria tenella parasite
The Eimeria tenella oocysts isolated and characterized by Jatau et al. (2016), maintained in 2.5 % potassium dichromate in the refrigerator (+ 4 o C) at the Department of Veterinary Parasitology and Entomology, Ahmadu Bello University, Zaria was used for this study.
Experimental birds and infection
The study was performed in a 35-day period with experimental birds grouped into six (6) having fifteen (15) birds each, each group was further divided into three (3) replicates, containing five (5) birds. A total of ninety (n = 90) commercial broiler chicks purchased at day old were randomly assigned into the six (6) groups A, B, C, D, E and F. Group A was not supplemented or infected but served as the negative (non-infected) control. Group B birds were not supplemented but infected to serve as the positive (Eimeria tenella -infected) control. Groups C and D were supplemented with prebiotics and probiotics at 1 ml/L of drinking water while Group E was supplemented synbiotic at 1 g/L of drinking water from day old till end of the study. Lastly, Group F was not supplemented but administered Amprolium prophylactic treatment; 1 g/L of drinking water prior to infection and 0.25 g/L of drinking water thereafter for 5 and 7 days respectively. All birds in the challenged groups B to F were gavage with 0.15 ml of water containing 2.0 × 104 Eimeria tenella sporulated oocysts at 21 days of age as described by Kumar et al. (2014). All birds were fed a basal diet and water ad libitum. Feeds used were purchased from a commercial poultry feed distributor with the following composition (Table 1):
Table 1: Composition of the broiler chicken diets used for the study.
Feed Composition
|
Starter
|
Finisher
|
Ingredients (%)
|
|
|
Crude protein
|
22.00
|
18.0
|
Crude fat
|
4.50
|
5.50
|
Crude fibre
|
5.00
|
5.00
|
Calcium
|
1.10
|
1.0
|
Phosphorus
|
0.50
|
0.43
|
Lysine
|
1.33
|
1.05
|
Metabolizable energy (kCal/kg)
|
3000.00
|
3200.00
|
Supplements/drug
Sugarcane molasses (prebiotic) used was acquired from a local market in Zaria, Nigeria and it comprised; dry matter, sugar, crude protein, crude ash, potassium, phosphorus, magnesium, and sodium. Antox® (probiotic) used comprises; Saccharomyces cerevisiae 4.125 X 10− 6 CFU/ml, then Citric acid, Lactic acid, B complex, Calcium chloride, Potassium iodide, Sodium selenite, Zinc chloride, Iron chloride, Magnesium chloride, Manganese chloride, Copper sulphate, and Cobalt chloride. En-florax® (synbiotic), had a mixture of strains of probiotic bacteria (Enterococcus faecium 1.5×1011CFU/Kg, Lactobacillus casei 1.5×1011CFU/Kg, L. plantarum 1.5×1011CFU/Kg, Pedicoccus acidilactici 1.5×1011CFU/Kg) and prebiotic substances: maltodextrin, fructo-oligosaccharides, oligofructose, and inulin then Crude protein, Crude fiber, Crude fat, Crude ash, Vitamin B12, Lysine, Methionine, Kwas fohoway, Vitamin B1 and Vitamin B2. The standard drug; Amprolium, is sold as ‘Amprolium 250 wsp’, a saturated powder of Amprolium hydrochloride (HCl), water-soluble for use in drinking water of poultry; used according to the producer’s instruction. 100–150 g per 100 litres of drinking water for 5–7 days followed by 25 g per 100 litres for 1 or 2 weeks.
Observation of clinical signs, morbidity and mortality
Following infection onset of clinical signs, morbidity and mortality were monitored, recorded and calculated for each group. The morbidity and mortality rates were calculated as described by Bai et al. (2017):
![](https://myfiles.space/user_files/83064_0857a92044b57365/83064_custom_files/img1627652894.png)
![](https://myfiles.space/user_files/83064_0857a92044b57365/83064_custom_files/img1627652911.png)
Determination of feed intake, weight gains and feed conversion ratio
Live body weight and weight gain were assessed on a weekly basis while the feed intake, feed conversion ratio and performance index for each group were determined as described by Jahan et al. (2006) using the formulae below:
![](https://myfiles.space/user_files/83064_0857a92044b57365/83064_custom_files/img1627652943.png)
![](https://myfiles.space/user_files/83064_0857a92044b57365/83064_custom_files/img1627652964.png)
![](https://myfiles.space/user_files/83064_0857a92044b57365/83064_custom_files/img1627652981.png)
Faecal oocyst shedding
Faecal samples collected from each pen from 4–13 days post infection was kept in separate airtight plastic bags for determination of oocysts per gram (OPG) of faeces as described by Dalloul et al. (2003). The samples for each day were homogenized and stored at 4°C until processed and oocysts were counted microscopically using a McMaster counting chamber (Hodgson, 1970); all oocysts under the grid of each chamber in the McMaster were counted using a ×10 magnification. Results were then expressed as oocysts per gram of faeces (Dryden et al. 2005).
Caecal lesion scoring
At day 7 post infection, 3 chickens randomly selected from each group were humanely euthanized by cervical decapitation and the paired caeca were exteriorized for lesion scoring as described by Johnson and Reid (1970), caecal tissues were then harvested for microscopic examination.
Histopathology
Caecal tissues harvested were fixed in 10 % formolsaline for 24 h. Fixed tissues were histologically processed according to the method of Bancroft et al. (1990). Tissues were dehydrated through ascending grades of alcohol (70 %, 90 % and 100 %) for 2 h each. The tissues were then cleared in xylene for 2 h (1 h each, 2 changes). Tissues were impregnated/infiltrated in molten paraffin wax 2 h each (1 h each, 2 changes) and embedded in paraffin wax. The tissue blocks were sectioned at 5-micron thickness using Rotary microtome machine (Leica RT 25 made in England). Sectioned tissues were mounted on slides, dried and stained using Hematoxylin and Eosin (H and E) stain. Photomicrograph of stained slides of the tissues were taken using Amscope Digital Camera for microscope version 3.0, made in Japan.
Data analyses
Data obtained were subjected to two-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test, lesion scores were analyzed using Kruskal-Wallis non-parametric test; using GraphPad Prism version 8.0.2 (GraphPad software, San Diego, CA, USA) for windows. Values of P ≤ 0.05 were considered significant. The analyzed data were then expressed as Mean ± SEM and presented in charts.