Assessment of probiotics supplementation via feed or water on the growth performance, intestinal morphology and microflora of chickens after experimental infection with Eimeria acervulina, Eimeria maxima and Eimeria tenella

Abstract

In this study, the effect of probiotic supplementation via drinking water or feed on the performance of broiler chickens experimentally infected with sporulated oocysts of Eimeria acervulina (5 × 10⁴), Eimeria maxima and Eimeria tenella (2 × 10⁴ each one) at 14 days of age was evaluated. Two hundred and forty 1-day-old Ross 308 male chicks were separated into eight equal groups with three replicates. Two of the groups, one infected with mixed Eimeria oocysts and the other not, were given a basal diet and served as controls. The remaining groups were also challenged with mixed Eimeria species and received the basal diet and either water supplemented with probiotic (three groups) or probiotic via feed (two groups); the probiotic used consisted of Enterococcus faecium #589, Bifidobacterium animalis #503 and Lactobacillus salivarius #505 at a ratio of 6:3:1. Probiotic supplementation was applied either via drinking water in different inclusion rates (groups W1, W2 and W3) or via feed using uncoated (group FN) or coated strains (group FC). The last group was given the basal diet supplemented with the anticoccidial lasalocid at 75 mg/kg. Each experimental group was given the corresponding diet or drinking water from day 1 to day 42 of age. Throughout the experimental period of 42 days, body weight and feed intake were recorded weekly and feed conversion ratios were calculated. Seven days after infection, the infected control group presented the lowest weight gain values, while probiotics supplied via feed supported growth to a comparable level with that of the lasalocid group. Probiotic groups presented lesion score values and oocyst numbers that were lower than in control infected birds but higher than in the lasalocid group. In the duodenum, jejunum and ileum, the highest villous height values were presented by probiotic groups. In conclusion, a mixture of probiotic substances gave considerable improvement in both growth performance and intestinal health in comparison with infected control birds and fairly similar improvement to an approved anticoccidial during a mixed Eimeria infection.

Introduction

Avian coccidiosis represents a major detrimental issue of the poultry industry with important problems such as mortality, malabsorption, inefficient feed utilization and affected growth rate in broilers.

Chickens are host to seven species of Eimeria and each species is responsible for a different form of coccidiosis. The three economically most significant species are Eimeria tenella, Eimeria acervulina and Eimeria maxima. The cost of clinical and sub-clinical disease, together with the cost of drugs and vaccines, was estimated as £40 million for the year 2000 in the UK, equivalent to 4.5% of the revenue from sales of live birds (Williams, 2002). The annual cost worldwide was probably in excess of £2 billion in 2000, and was over £3 billion in 2012, according to the annually increasing number of broilers raised worldwide (Bozkurt et al., 2013).

Coccidiosis control relies heavily on chemoprophylaxis, which is a tremendous cost to the industry. The continuous emergence of drug-resistant strains of Eimeria coupled with the increasing regulations and bans on the use of anticoccidial drugs in commercial poultry production, the increasing costs of developing new drugs and the public's distrust of drug-treated meat demonstrate the urgent need for novel approaches and alternative control strategies (Giannenas et al., 2003; Applegate et al., 2010; Peek & Landman, 2011; Bozkurt et al., 2012). Consumers also demand food products of animal origin without drug residues or pharmaceutical metabolites (Dalloul & Lillehoj, 2006).

Probiotics are viable, non-pathogenic bacteria that contribute to the health and balance of the intestinal tract. Probiotics have been used in broiler chickens (Mohan et al., 1996; Patterson et al., 1997; Dalloul et al., 2002, 2003; Mountzouris et al., 2007) in order to assess their beneficial effect on health and performance, mediated through bacterial antagonism, immunomodulation or histological alteration of the intestinal epithelium. The addition of a mix of probiotics containing Lactobacillus, Bifidobacterium, Enterococcus and Pediococcus strains into the diet has been found to improve growth performance and feed conversion in broilers (Mountzouris et al., 2007). The mode of action of probiotics in poultry includes: maintaining normal intestinal microflora by competitive exclusion and antagonism; altering metabolism by increasing digestive enzyme activity and decreasing bacterial enzyme activity and ammonia production; improving feed intake and digestion; and neutralizing enterotoxins and stimulating the immune system (Jin et al., 1998a).

Numerous substances have been tested as potential alternatives to coccidiostatic feed additives, which might provide protection against or modulate the effects of coccidial infections. The information in the current literature on the usage of probiotics as alternatives to coccidiostatic drugs is rather rare in the chicken (Dalloul et al., 2003; Lee et al., 2007) or in the rabbit (Simonova et al., 2009) and there appears to be no information on whether probiotics provide significant protection against lesions due to mixed Eimeria spp. infection in chickens.

As a consequence, there is a challenging idea to evaluate the potential of probiotics in restricting the depression of performance parameters attributed to coccidiosis. The aim of our study was to investigate the potential protective use of five probiotic preparations in the diet or water of broiler chickens infected with E. tenella, a highly pathogenic Eimeria species that causes caecal coccidiosis. The probiotic strains were assessed in vitro for anticoccidial activity prior to the infection trial (Henikl et al., 2010) and in vivo against E. tenella (Giannenas et al., 2012).

Materials and Methods

This trial was carried out in accordance with the principles and regulations of the local Public Veterinary Service and the Authorities of the Veterinary Faculty of the University of Thessaly. The health of the birds was monitored by a veterinary surgeon. Birds were vaccinated against Newcastle disease, infectious bronchitis and Gumboro disease at 12 days of age (National Research Council, 1996).

Experimental design and dietary treatments

A total of 240 1-day-old Ross 308 male chicks were randomly allocated into eight equal groups with three subgroups of 10 birds each. All subgroups were housed in separate floor pens, each equipped with an infrared lamp. The temperature was gradually decreased from 36°C on day 1 to 24°C on day 21 and then kept constant. The lighting regime provided 24 h of continuous light per day until day 2 of experiment, and 23 h thereafter.

To meet the nutrient requirements of the broilers during the experimental period, a complete basal diet was formulated. The diet was in mash form, and was analysed according to the Weende system. Based on this basal diet, additional diets were prepared by incorporating either probiotic preparations at a chosen level or lasalocid at 75 mg/kg feed (group LA) respectively to the corresponding groups. Two of the groups that were given a diet without coccidiostatic or other antimicrobial feed additives served as control; one infected with Eimeria spp. (group UI) and the other not (group UU). The remaining groups that were all infected with Eimeria spp. were administered the multispecies probiotic mix PoultryStar^® (BIOMIN GmbH, Tulln, Austria) consisting of Enterococcus faecium #589, Bifidobacterium animalis #503 and Lactobacillus salivarius #505 at a ratio of 6:3:1. Probiotic supplementation was applied either via drinking water in different inclusion rates (groups W1, W2 and W3) or via feed using uncoated (group FN) or coated strains (group FC). Each experimental group was given the corresponding diet or drinking water from day 1 to day 42 of age (Table 1). Feed and drinking water was offered ad libitum to the birds. Table 2 presents details about labelling and dietary or water supplementation of the experimental groups.

Table 1. Description of experimental groups and tested probiotic products.

Group	Description	Inclusion rate	Infected
UU	Unsupplemented, uninfected control		−
UI	Unsupplemented, infected control		√
LA	Lasalocid	75 mg/kg	√
W1	Probiotic mix^a via drinking water	2.5 × 10^7 CFU/l	√
W2	Probiotic mix via drinking water	5.0 × 10^7 CFU/l	√
W3	Probiotic mix via drinking water	2.5 × 10^8 CFU/l	√
FN	Probiotic mix via feed	5.0 × 10^8 CFU/kg	√
FC	Probiotic mix, coated, via feed	5.0 × 10^8 CFU/kg	√

A multispecies probiotic mixture was applied in different inclusion rates and by different routes. CFU, colony-forming units. ^aProbiotic mix consisting of Enterococcus faecium #589, Bifidobacterium animalis #503 and Lactobacillus salivarius #505 at a ratio of 6:3:1.

Table 2. Composition of the basal diet in mash form.

Ingredient	Starter (1 to 14 days of age)	Grower-finisher (15 to 42 days of age)
Maize grains (g/kg)	591.1	624.9
Soybean meal −46.5 (g/kg)	326.2	277.3
Fish meal (g/kg)	25.0	25.0
Soy oil (g/kg)	23.0	39.0
Limestone (g/kg)	18.7	17.9
Monocalcium phosphate (g/kg)	8.0	8.0
l-Lysine HCl (g/kg)	2.0	2.0
d,l-Methionine (g/kg)	2.0	2.0
Vitamin premix^a (g/kg)	1.0	1.0
Trace mineral premix^b (g/kg)	1.0	1.0
Salt (g/kg)	2.0	1.9
Proximate analysis^c (g/kg)
Dry matter (g/kg)	882.2	884.1
Crude protein (N × 6.25, g/kg)	220.2	200.2
Crude fat (ether extract, g/kg)	53.3	69.5
Crude fibre (g/kg)	33.0	31.1
Ash (g/kg)	50.9	48.1
Starch (g/kg)	366.1	370.3
Calculated analysis (g/kg)
Calcium	9.3	9.0
Phosphorus (total)	6.9	6.6
Sodium	1.8	1.7
Chloride	2.4	2.3
Lysine total	14.0	12.7
Methionine + cystine	9.8	9.3
Energy (kcal/kg)	3000	3100

^a Supplied per kilogram of diet: retinol, 12,000 IU; cholecalciferol, 5000 IU; tocopherol, 30 IU; menadione, 3 mg; thiamin, 1 mg; riboflavin, 8 mg; pyridoxine, 3 mg; cyanocobalamin, 0.02 mg; niacin, 20 mg; pantothenic acid, 20 mg; folic acid, 2 mg; biotin, 0.2 mg; ascorbic acid, 10 mg; choline chloride, 480 mg.

^b Supplied per kilogram of diet: zinc, 125 mg; manganese, 100 mg; iron, 62 mg; copper, 7,5 mg; cobalt, 0.2 mg; iodine, 2 mg; selenium, 0.2 mg.

^c According to AOAC International (1995).

Eimeria infection

Infection of chickens with Eimeria spp. was carried out at 14 days of age. To induce sporulation, oocysts were preserved in 2% potassium dichromate solution, and kept refrigerated at 3 to 5°C until use. Infection of each bird was carried out by administering a 2-ml suspension of 5 × 10⁴ sporulated oocysts of E. acervulina, 2 × 10⁴ sporulated oocysts of E. maxima and 2 × 10⁴ sporulated oocysts of E. tenella directly into the crop via an oral gavage by a plastic tube. The number of coccidian oocysts per millilitre was controlled before being used for oral inoculation of the birds through standard enumeration techniques.

Performance parameters

All chicks were individually weighed at the time of their placing into the pens and on days 7, 14, 21, 28, 35 and 42 of age. Four hours prior to bird weighing, diets were removed and feed consumption within each subgroup was determined. Feed conversion ratio values were calculated weekly as the ratio of feed intake to weight gain. Mortality was recorded daily in each subgroup.

Seven days after infection, the lesion score was estimated in all groups by evaluating caecal intestinal lesions of six chicks per group. Lesion scoring was performed at both ileal and caecal level and scores were assigned from 0 to 4, where 0 corresponds to the normal status with no gross lesions, 1 corresponds to small scattered petechiae, 2 corresponds to numerous petechiae, 3 corresponds to extensive haemorrhage, and 4 corresponds to extensive haemorrhage giving the caecal intestine a dark colour according to Johnson and Reid (1970). Dead birds were given a score of 4.

Bloody diarrhoea was determined daily from days 17 to 21 of age. The extent of bloody diarrhoea was determined according to Youn and Noh (2001) by assigning it one of five levels, where zero is the normal status, and <25%, 26 to 50%, 51 to 75%, or >75% bloody faeces in total faeces are the remaining levels.

Oocyst counts were determined in excreta samples taken daily from each subgroup from days 20 to 26 of age and at 7, 14, 35 and 42 days of age. Collection of excreta for oocyst analysis was done three times daily. Samples from each subgroup were placed in separate airtight plastic bags, homogenized thoroughly by a domestic mixer, and kept refrigerated until assessed for total oocyst counts. Homogenized samples were 10-fold diluted with tap water to be further diluted with saturated NaCl solution at a ratio of 1:10. Oocyst counts were determined using McMaster chambers and are presented as the number of oocysts per gram of excreta.

Histology and morphometric analysis of the intestine

During necropsy of the selected birds on day 21 of age, the gastrointestinal tract was removed and ileal segments (from Meckel's diverticulum to the ileo-caeco-colic junction) 1 cm in length were taken from the central part and fixed in 10% buffered formalin for morphometric assays under light microscopy. These sections were then dehydrated before being embedded in paraffin wax. Formalin-fixed intestinal tissues were processed, sectioned at 3 µm and stained by the haematoxylin and eosin methods. Histological sections were examined with a Nikon phase-contrast microscope coupled with a Microcomp integrated digital imaging analysis system (Nikon Eclipse 80i; Nikon Co., Tokyo, Japan). Images were viewed (at 4× magnification) to measure morphometric parameters of intestinal architecture. From the stained sections, the villous height and crypt depths were determined manually. For that purpose, three favourably orientated sections cut perpendicularly from villous enterocytes to the muscularis mucosa were selected from each bird and measurements were carried as follows. The villous height was estimated by measuring the vertical distance from the villous tip to the villous-crypt junction level for 10 villi per section. The crypt depth (the vertical distance from the villous-crypt junction to the lower limit of the crypt) was estimated for 10 corresponding crypts per section (Giannenas et al., 2010).

Enumeration of intestinal microbiota in the ileum and caecum

Intestinal samples were collected and fresh digesta samples from the ileum and caecum were taken for bacterial analyses within 1 h from collection on day 42. Digesta samples were serially diluted in 0.85% sterile saline solution for enumeration of total aerobes, total anaerobes, lactic acid bacteria, coliforms and Clostridium perfringens by conventional microbiological techniques, using selective agar media according to Engberg et al. (2004). All microbiological analyses were performed in duplicate and the average values were used for statistical analysis. Results were expressed as base-10 logarithm colony-forming units per gram of ileal or caecal digesta.

Statistical analysis

Experimental data were subjected to analysis of variance in the general linear model using the statistical package of SPSS version 17.0 for Windows (SPSS, Inc., Chicago, IL, USA). For data on growth performance, individual birds were considered to be nested within pens and data were analysed by a nested analysis of variance in repeated measurements. Effects of sex and experimental room were found not to be significant and were not included in the model. Feed intake and feed conversion ratio values were analysed by analysis of variance in repeated measurements, the pen being the experimental unit. Mortality was analysed by chi-squared test. As bacterial and oocyst numbers were not normally distributed, they were log-transformed to create a normal distribution prior to analysis. Levene's test was performed to check homogeneity of variances and Tukey's test was carried out to assess any significant differences at a probability level of 0.05 among the experimental treatments.

Results

Performance parameters

Growth performance

Feed intake was similar among the experimental groups during the whole trial period (Table 3). During the first experimental period (0 to 14 days), there were no significant differences in either the feed conversion ratio values of the birds fed on any of the diets or the body weight among the experimental groups. On day 21, 7 days after the infection, the control infected group presented the lowest body weight and body weight gain values, while probiotic substances administered via feed supported growth to a level comparable with that of the lasalocid group. On day 42, the control infected group again presented the lowest body weight and body weight gain values, while probiotics through feed gave similar body weight values to those of the lasalocid group. Probiotics given by drinking water improved body weight and body weight gain only slightly, with significant improvement compared with group UI only on day 35. No dose–response effects were apparent due to different inclusion rates. Mortality was highest in the control infected group and lowest in the lasalocid group, while probiotic groups showed intermediate values.

Table 3. Effects of probiotics via feed or drinking water on growth performance of infected chickens with mixed Eimeria species.^a

Growth performance	Group UU	Group UI	Group LA	Group W1	Group W2	Group W3	Group FC	Group FN	SEM	P value
BW 1^b (g)	43.3	43.4	43.7	43.2	42.9	42.9	42.8	42.5	0.11	0.366
BW 7^b (g)	144.4	148.6	143.7	144.5	146.8	142.4	146.9	145.2	0.12	0.344
BWG (g)	101.1	105.2	99.9	101.3	103.9	99.6	104.1	102.7	0.12	0.345
FCR^c	0.916	0.912	0.915	0.911	0.909	0.906	0.908	0.911	0.005	0.644
FI (g)	92.6	96.0	91.4	92.2	94.5	90.2	94.5	93.5	0.12	0.575
BW 14^b (g)	326.8	336.1	348.8	317.5	316.5	319.8	344.8	343.5	9.5	0.454
BWG (g)	283.4	292.7	305.0	274.3	273.7	277.0	302.0	301.0	9.6	0.433
FCR^c	1.057	1.061	1.057	1.060	1.058	1.056	1.053	1.063	0.007	0.408
FI (g)	300.0	310.4	322.3	290.6	289.4	292.6	317.7	320.0	15.6	0.596
BW 21^b (g)	813.6^A	662.7^C	738.5^B	681^BC	703.6^B	685.2^BC	694.7^B	701.3^B	12.1	0.000
BWG (g)	770.2^A	619.3^C	694.8^B	637.8^BC	660.7^B	642.3^BC	651.9^B	658.8^B	12.2	0.000
FCR^c	1.218	1.313	1.239	1.275	1.261	1.273	1.259	1.255	0.01	0.102
FI (g)	938.0	812.8	860.8	813.4	832.9	817.6	820.8	827.0	23.4	0.185
BW28^b (g)	1388.1^A	1105.2^C	1240.7^B	1150.4^BC	1151.2^BC	1158.6^BC	1208.6^B	1231.1^B	18.5	0.000
BWG (g)	1344.8^A	1061.6^C	1197.0^B	1107.2^BC	1108.3^BC	1115.7^BC	1165.8^B	1188.6^B	18.6	0.000
FCR^c	1.461^C	1.595^A	1.561^B	1.573^B	1.573^B	1.577^B	1.561^B	1.551^B	0.005	0.000
FI (g)	2028.3	1762.5	1936.3	1809.3	1811.4	1827.5	1886.4	1908.8	32.6	0.253
BW 35^b (g)	1886.4^A	1646.6^C	1839^A	1727.2^B	1710^B	1719.4^B	1812^A	1833.8^A	20.2	0.000
BWG (g)	1843.1^A	1603.1^C	1795.2^A	1684.0^B	1667.1^B	1676.6^B	1769.2^A	1791.3^A	20.3	0.000
FCR^c	1.644^B	1.912^A	1.648^B	1.672^B	1.677^B	1.678^B	1.658^B	1.656^B	0.006	0.024
FI (g)	3030.9	3065.5	2957.5	2816.4	2795.4	2813.8	2933.2	2967.2	36.9	0.466
BW 42^b (g)	2420.0^A	2097.2^C	2290.5^B	2158.3^BC	2123.6^BC	2153.6^BC	2224.5^B	2239.4^B	36.5	0.000
BWG (g)	2376.7^A	2053.8^C	2246.7^B	2115.1^BC	2080.8^BC	2110.8^BC	2181.7^B	2196.9^B	36.7	0.000
FCR^c	1.739^C	2.012^A	1.755^C	1.886^B	1.862^B	1.845^B	1.837^B	1.826^B	0.005	0.000
FI (g)	4134.3	4132.3	3941.9	3989.9	3876.2	3893.0	4007.2	4012.7	111.3	0.567

^a Groups of chickens fed the corresponding diet. Results presented as means of groups (n = 3 = subgroups). BW, body weight; BWG, body weight gain; FCR, feed conversion ratio; FI, feed intake; SEM, standard error of the mean. Values in the same row with an uppercase superscript letter in common do not differ significantly (P > 0.05).

^b bIndicates the day of age when performance parameters were measured.

^c FCR is calculated cumulatively.

Diarrhoea and lesion scores

Bloody diarrhoea was observed on day 18 of age among all infected groups, except for the lasalocid group. The extent of bloody diarrhoea was highest in the infected control group and similar to other infected birds. The lasalocid group gave lower bloody faeces compared with all infected groups. The non-infected control group had no blood in faeces at all. Lesion scores in the caecum and ileum were highest in the infected control group and zero in the non-infected control group. Probiotic supplemented groups presented lesion score values that were lower than those of infected control birds but higher than those of the lasalocid group (Table 4).

Table 4. Intestinal lesion score, bloody faeces and mortality of chickens after Eimeria mixed challenge at 21 days of age.^a

	Group UU	Group UI	Group LA	Group W1	Group W2	Group W3	Group FC	Group FN	SEM	P value
Caecal lesion score	0.00^D	3.00^A	1.56^C	2,89^AB	2,72^AB	2,78^AB	2,67^BC	2,56^B	0.21	0.012
Ileal lesion score	0,00^D	3,28^A	1,61^C	2,94^AB	2,89^AB	2,78^AB	2,56^B	2,44^B	0.22	0.011
Bloody faeces	0^C	3 + ^A	1 + ^B	3^A	3^A	3^A	2 + ^AB	2 + ^AB	0.19	0.012
Total mortality^b	0^B	4^A	1^B	3^AB	3^AB	3^AB	2^AB	2^AB		0.017

^a Groups of chickens fed the corresponding diet. Results presented as means of groups (n = 3 = subgroups). SEM, standard error of the mean. Values in the same row with an uppercase superscript letter in common do not differ significantly (P > 0.05).

^b Values in the column with an uppercase superscript letter in common do not differ significantly (P > 0.05) by non-parametric Kruskal–Wallis analysis.

Eimeria oocyst output

Oocyst shedding was highest in the infected control group and zero in the non-infected control group. Probiotic groups presented oocyst numbers that were lower than those of infected control birds but higher than those of the lasalocid group (Table 5).

Table 5. Oocyst output after Eimeria challenge transformed in a log₁₀ base.^a

	Group UU	Group UI	Group LA	Group W1	Group W2	Group W3	Group FC	Group FN	SEM	P value
Day 7 of age	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Day 14 of age	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Day 5 p.i. = day 19 of age	0.00^C	3.18^A	0.00^C	3.03^B	2.94^B	2.95^B	2.94^B	3.00^B	0.12	0.000
Day 6 p.i.	0.00^D	5.54^A	3.34^C	4.11^B	4.15^B	4.11^B	3.96^B,C	4.04^B	0.23	0.000
Day 7 p.i.	0.00^D	5.40^A	3.64^C	5.17^A	5.16^A	5.11^A	5.03^A	5.05^A	0.12	0.000
Day 8 p.i.	0.00^D	5.19^A	3.37^C	4.65^B	4.91^A,B	4.92^A,B	4.83^B	4.87^B	0.14	0.000
Day 9 p.i.	0.00^D	4.99^A	3.07^C	4.85^A,B	4.84^A,B	4.84^A,B	4.75^A,B	4.80^A,B	0.16	0.000
Day 10 p.i.	0.00^D	4.59^A	3.03^C	4.24^B	4.18^B	4.20^B	4.11^B	4.11^B,C	0.18	0.000
Day 11 p.i.	0.00^D	4.26^A	2.87^C	3.73^B	3.73^B	3.82^B	3.41^B,C	3.46^B,C	0.12	0.000
Day 12 p.i.	0.00^D	4.03^A	2.82^C	3.10^B	3.03^B	3.16^B	2.98^C	2.95^C	0.14	0.000
Day 28 of age	0.00^C	3.66^A	2.77^C	2.97^B	2.91^B	2.92^B	2.73^B,C	2.80^B,C	0.11	0.000
Day 35 of age	0.00^D	3.39^A	1.92^C	2.67^B	2.65^B	2.77^B	2.45^B,C	2.67^B	0.13	0.000
Day 42 of age	0.00^C	3.29^A	0.00^C	2.47^A,B	2.28^B	2.28^B	2.15^B	2.26^B	0.11	0.000
Average cocci number	0.00^D	4.32^A	2.44^C	3.73^B	3.71^B	3.73^B	3.58^B	3.64^B	0.000	0.11

^a Groups of chickens fed the corresponding diet. Results presented as means of groups (n = 3 = subgroups). p.i., post infection; SEM, standard error of the mean. Values in the same raw with an uppercase superscript letter in common do not differ significantly (P > 0.05). Values in the same row with an uppercase superscript letter in common do not differ significantly (P > 0.05).

Morphometric analysis of the gut

The Eimeria infection had a strong impact on the small intestine of the infected birds, especially at the ileal site where E. maxima mainly is located. In the duodenum and jejunum, the highest villous height values were presented by groups W1 and FN. In the ileum, highest villous height values were presented by groups FN and FC (both were fed applications of probiotics). The ratio of villous height to crypt depth in the duodenum and jejunum was highest in the FN group. The ratio of villous height to crypt depth in the ileum was highest in the FC group (Table 6).

Table 6. Effects of probiotics via feed or drinking water on intestinal morphology of broiler chicken at 21 days of age.^a

	Group UU	Group UI	Group LA	Group W1	Group W2	Group W3	Group FC	Group FN	SEM	P value
Duodenum
Villous height (µm)	1770.2^A	994.6^C	1401.5^B	1562.9^B	1472.3^B	1361.1^B	1318.7^B	1471.1^B	24.2	0.044
Crypt depth (µm)	197.7^A	101.8^C	145.3^B	194.3^A	143.8^B	132.6^B	130.6^B	134.7^B	6.64	0.048
Villous height to crypt depth ratio	8.95	9.77	9.64	8.04	10.24	9.81	10.09	10.92	0.044	0.332
Jejunum
Villous height (µm)	871.3^B	940.8^A	799.0^B	968.6^A	737.4^B	856.4^B	936.8^A	1003.9^A	22.1	0.022
Crypt depth (µm)	132.5	145.1	124.7	139.9	104.8	110.9	137.8	110.7	7.12	0.451
Villous height to crypt depth ratio	6.57^B	6.48^B	6.41^B	6.92^B	7.03^B	7.72^B	6.79^B	9.07^A	0.21	0.022
Ileum
Villous height (µm)	699.9^C	780.1^B	668.6^C	734.7^B	832.6^A	748.8^B	904.8^A	839.9^A	18.4	0.041
Crypt depth (µm)	104.5	117.3	110.5	100.8	102.2	109.5	107.1	109.9	3.94	0.566
Villous height to crypt depth ratio	6.69^CB	6.65^CB	6.05^C	7.29^B	8.15^A	6.84^B	8.44^A	7.64^B	0.18	0.046

^a Groups of chickens fed the corresponding diet. Results presented as means of groups (n = 3 = subgroups). SEM, standard error of the mean. Values in the same row with an uppercase superscript letter in common do not differ significantly (P > 0.05). Values in the same row with a different uppercase superscript letter differ significantly (P > 0.05).

Enumeration of intestinal microbiota composition

The composition of the ileal microbiota of chickens at the end of the experiment is shown in Table 7. In the ileum, total aerobes and total anaerobes were not different (P > 0.05) among the experimental groups. The lactic acid bacteria counts were higher (P < 0.05) and coliforms were lower (P < 0.05) in the probiotic supplemented groups compared with the control or lasalocid supplemented groups. C. perfringens counts were higher in the infected control group, intermediate in the control non-infected, lasalocid and probiotic via feed supplemented groups and lowest in the probiotic via water supplemented groups. In the caecum, an analogous profile of intestinal microbiota was noticed (Table 7). Total aerobes were higher (P < 0.05) in the control and lasalocid groups than in groups with probiotics via drinking water. Total anaerobes and lactic acid bacteria counts were lower in the control and lasalocid groups and higher in the probiotic supplemented groups. C. perfringens counts were higher in control groups, intermediate in lasalocid and W2 groups and were lowest in W1, W3 and probiotic via feed supplemented groups.

Table 7. Effect of dietary probiotic supplementation on ileum and caecum bacteria populations of broiler chicken at 42 days of age.^a

	Group UU	Group UI	Group LA	Group W1	Group W2	Group W3	Group FC	Group FN	SEM	P value
Ileum
Total aerobes	7.45	7.32	7.62	6.98	7.36	6.99	7.40	7.34	0.112	0.155
Total anaerobes	7.01	6.94	7.47	7.19	7.03	6.92	7.36	7.17	0.123	0.266
Lactic acid bacteria	5.87^B	5.81^B	6.11^B	6.78^A	6.84^A	6.69^A	6.49^A	6.88^A	0.065	0.031
Coliforms	4.87^A	4.24^A	4.22^A	3.18^B	3.31^B	3.17^B	3.36^B	3.08^B	0.011	0.031
Clostridium perfringens	3.54^AB	3.83^A	3.11^AB	2.33^B	2.39^B	2.44^B	3.06^AB	3.07^AB	0.037	0.003
CAECUM
Total aerobes	8.54^A	8.55^A	8.29^AB	7.52^B	7.83^AB	7.67^AB	8.41^AB	8.23^AB	0.052	0.047
Total anaerobes	8.01^B	8.08^B	8.52^AB	8.94^A	8.62^A	8.82^A	8.85^A	8.41^AB	0.014	0.025
Lactic acid bacteria	7.61^B	7.89^B	8.11^AB	8.63^A	8.67^A	8.54^A	8.77^A	8.17^A	0.018	0.015
Coliforms	6.11^A	6.53^B	5.85^AB	5.44^B	5.60^B	5.53^B	5.60^B	5.44^B	0.363	0.016
Clostridium perfringens	3.44^A	3.44^A	3.16^AB	3.03^B	3.11^AB	3.09^B	3.00^B	3.02^B	0.078	0.044

^a Groups of chickens fed the corresponding diet. Results presented as means of groups (n = 3 = subgroups). SEM, standard error of the mean. Values in the same row with a different uppercase superscript letter differ significantly (P > 0.05).

Discussion

Contamination of poultry houses with coccidian oocysts is difficult to control and attempts to eradicate infections with Eimeria spp. fail (Graat et al., 1994). It is noteworthy that some studies have indicated that E. tenella occysts, for example, can survive in the soil for up to 9 months (Marion & Wehr, 1949). Adverse effects of coccidiosis due to E. tenella include bloody diarrhoea, intestinal lesions, depressed growth rate and, sometimes, high mortality. In this study, birds were experimentally infected with a mix of three Eimeria spp. and compared with non-infected and infected birds fed the conventionally recognized anticoccidial drug lasalocid or a mix of probiotics via feed or drinking water. The major target of this study was to find a product with anticoccidial properties that could be used as a natural alternative without a withdrawal period or any adverse effects that may be associated with anticoccidial drugs.

In our study, the mucosal architecture in terms of villous height and crypt depth was influenced by the severe Eimeria infection and also by the different dietary supplements. The structure of the intestinal mucosa can reveal some information on gut health. Stressors that are present in the digesta can lead relatively quickly to changes in the intestinal mucosa, due to the close proximity of the mucosal surface and the intestinal content. Changes in intestinal morphology, such as shorter villi and deeper crypts, have been associated with the presence of toxins (Yason et al., 1987) or higher tissue turnover (Miles et al., 2006). The crypt can be regarded as the villous factory and a large crypt indicates rapid tissue turnover and a high demand for new tissue. Demand for energy and protein for gut maintenance is high compared with other organs. Cook and Bird (1973) reported shorter villi and deeper crypts when the counts of pathogenic bacteria increased in the gastrointestinal tract, which resulted in fewer absorptive cells and more secretory cells. In the present study, an increase in ileal villous height and crypt depth ratio at the ileum in probiotic-fed chickens was found compared with control birds.

An interesting finding of our study was that groups supplemented with probiotics had body weights higher than or similar to that of the lasalocid supplemented group. These data confirm that these substances protect chicken performance and may act as potential anticoccidial substances. Another very significant finding of the present study was that supplementation of broilers with probiotic substances gave lower oocyst values compared with the control infected group. However, the lasalocid group presented significantly lower oocyst shedding compared with infected birds. The above-mentioned results were in agreement with our previous work (Giannenas et al., 2012). An interesting finding is that group FN was exactly the same as with the previous work and gave positive performance results as in our previous paper concerning E. tenella infection (Giannenas et al., 2012).

The results of the study indicate that probiotic substances administered by feed exerted a coccidiostatic effect against Eimeria spp., especially in terms of performance comparable with that exhibited by lasalocid. Feed conversion ratio values were best in the non-infected control group and worst in the infected control group. All infected birds supplemented with probiotics gave similar feed conversion ratio values that were higher than the feed conversion ratio of the lasalocid group on day 42.

Similar effects were observed in coated strains as in the uncoated strains, indicating a proper release of the coated strains in the gut. Microencapsulation of probiotics can be used in order to enhance their viability during processing, to enhance their targeted delivery in the gastrointestinal tract and to withstand acidic conditions in the stomach in order to pass through the stomach (Anal & Stevens, 2005; Anal & Singh, 2007).

Pertinent studies with non-infected chickens clearly showed that the administration of probiotics via the drinking water had beneficial effects on broiler performance (Timmerman et al., 2006). In the field trials, probiotic treatment significantly improved feed conversion. In similar studies, evidence was provided that multispecies probiotics were more effective than monospecies probiotics (Timmerman et al., 2004) and also that species-specific probiotics elicit different health effects than do probiotics derived from another host species (Timmerman et al., 2005). In our trial, administration of probiotics through drinking water was no more effective than probiotics via feed. In the past, Timmerman et al. (2006) investigated the effect of chicken-specific probiotics that were administered in drinking water with similar results. In the literature, however, performance results are not consistent. Differences in the administration of probiotics might only be one factor affecting efficacy. Administration of probiotics in the drinking water (Watkins & Kratzer, 1984; Timmerman et al., 2006) generally resulted in a lower increase of average daily weight gain when compared with studies with probiotic administration via the feed (Yeo & Kim, 1997; Jin et al., 1998a, b, 2000; Abdulrahim et al., 1999; Zulkifli et al., 2000; Kalavathy et al., 2003). A possible explanation was related to an adverse taste response of the water due to the organic acids present in the chicken-specific probiotic preparations. Neutralization of the acids with calcium carbonate raised water intake (Timmerman et al., 2006).

Another determinant of probiotic efficacy may be the timing of administration. During early life, colonization patterns are unstable and chicks are then susceptible to environmental pathogens. Initial colonization is of great importance to the host because the bacteria can modulate expression of genes in epithelial cells (Hooper et al., 2001), thus creating a favourable habitat for themselves. In our previous study, it was shown that the potential of probiotics to improve growth performance and mortality in broilers was more pronounced by multispecies than monospecies (Giannenas et al., 2012).

In conclusion, during a mixed infection of E. tenella, E. acervulina and E. maxima, probiotics gave substantial improvement in both growth performance and intestinal health in comparison with infected control birds and similar improvement to lasalocid, which is an approved anticoccidial substance. Further research is needed to elucidate the mechanism of action both on chicken performance and on the parasite itself. Therefore, the possibility of rearing broilers without coccidiostatics is thought to be of promise for more extensive and large-scale studies.