Effects of vaccination against coccidiosis, with and without a specific herbal essential oil blend, on performance, oocyst excretion and serum IBD titers of broilers reared on litter

Abstract

The aim of the present experiment was to investigate the effects of oral administration of a live attenuated vaccine (VAC) and an essential oil blend (EOB), either alone or in combination, as a novel anticoccidial strategy for broiler chickens with a mixed Eimeria spp. infection. A total of 624 broiler chicks were randomly assigned to one of six treatments. Two of the groups, only one of which was challenged with coccidiosis, were given a basal diet and served as controls. The other two groups, also infected, were given a basal diet supplemented with monensin sodium (MON, 100 mg/kg) or the EOB (75 mg/kg). Of the remaining two groups, which were infected with coccidiosis, one was vaccinated against coccidiosis (VAC) and the other was both vaccinated and fed a diet with an EOB (VAC+EOB).

Birds treated with VAC and VAC+EOB had comparable live performance to MON-fed birds challenged with coccidiosis. Conversely, EOB diet supplementation had negative effects on growth, feed intake and feed conversion ratio throughout the growth period. None of the coccidial control strategies kept pace with the uninfected procedure in terms of performance during the course of the infection. There was no significant difference in mortality among treatments. All of the anticoccidial procedures kept serum infectious bursal disease titers at high levels after coccidial infection and reduced fecal oocyst excretion, with the exception of the MON-based procedure.

The results indicate that vaccination against coccidiosis, with or without EOB, demonstrated the same efficacy in promoting recovery from coccidial infection and in reducing oocyst shedding as MON.

Key Words:

• Coccidiosis
• Essential oil blend
• Vaccine
• Performance
• Oocyst excretion.

Introduction

Coccidiosis is one of the important diseases in poultry worldwide and causes annual global economic losses estimated at over 1.5 billion US dollars (Williams, 1999; Lillehoj and Lillehoj, 2000; De Gussem et al., 2008). When birds are infected by parasites, their ability to absorb nutrients is damaged, resulting in retarded growth performance, impaired feed conversion efficiency, poor flock uniformity, depressed immunity, and sometimes increased mortality (Cook, 1998; McDougold, 1998; 2003; Girgis, 2007).

Since the 1950s, various types of anticoccidials produced from different drugs and chemicals have been routinely and successfully used to prevent livestock suffering and limit economic loss. Current control of coccidiosis is achieved by including traditional coccidial drugs in feed. Years of commercial use have shown that anticoccidial drugs are safe if used at approved concentrations (Chapman, 1992; 1998). However, the emergence of drug-resistant strains of Eimeria, and the inefficacy that developed as coccidia became resistant to treatment, prompted the now common practice of alternating the use of anticoccidial drugs from different chemical families to avoid drug resistance. Because it is generally accepted that prolonged use of the same compound eventually results in resistance, it is important to limit usage as much as possible (McDougold et al., 1987; Thomke and Elwinger, 1998). Furthermore, consumer concerns about in-feed prophylactic drug inclusion, and the anticipated withdrawal of coccidiostats at the end of 2012, mean that alternative methods of preventing and controlling coccidiosis need to be urgently considered (Greathead and Kamel, 2006; Noirot, 2010).

One alternative way of controlling coccidiosis is vaccination. In recent years, coccidial vaccines have been developed and used commercially (Chapman, 2000; Chapman et al., 2002; Williams, 2002; Dalloul and Lillehoj, 2005). A limited number of studies have shown that the use of vaccines can control losses and replace drug-resistant field strains of Eimeria with drug-sensitive vaccine strains (Waldenstedt, 2003; Garcia and Bolis, 2005). Some field trials have shown that commercial vaccination programs could be as effective and cost-beneficial to antibiotic anticoccidials when broiler performance parameters are assessed (Williams et al., 1999; Williams and Gobbi, 2002). A recent experimental study also demonstrated that cocci-vaccinated broilers fed diets without anticoccidial feed additives showed excellent live performance and their lowest oocyst shedding after being infected with mixed Eimeria spp. (Oviedo-Rondón et al., 2005; 2006a).

On the other hand, scientific research during the last decade has proven the efficiency of plant-derived products as natural anticoccidial agents (Allen et al., 1997; Youn and Noh, 2001; Duffy et al., 2005). The extracts and essential oils of some selected herbs have shown promising results in the control of coccidial infections. The parameters obtained for the extent of bloody diarrhea, survival rate, lesion score, oocyst number, intestinal microbial ecology and even the production efficiency of broiler chickens have indicated that specific blends of these phytobiotic compounds could exert anticoccidial action against Eimeria infections (Saini et al., 2003; Giannenas et al., 2003; Christaki et al., 2004; Oviedo-Rondón et al., 2005; 2006a,^b). Moreover, unlike with chemical-based coccidiostat drugs, there has been no evidence of development of bacterial resistance to in-feed essential oil supplements, as the inhibitory effects of such supplements on coccidian parasites and pathogenic bacteria are due to the supplements’ phenolic action (Williams, 1997; Williams and Losa, 2002; Ultee et al., 2002; Brenes and Roura, 2010). Therefore, the purpose of this project was to evaluate the dietary supplementation of a specific blend of essential oils, either alone or in combination with a commercial cocci-vaccine, as an alternative to ionophore coccidiostat for broilers during a grow-out period of 42 days.

Materials and methods

Animals and housing

Six-hundred and twenty-four one-day-old sexed Ross-308 broiler chickens, purchased from a commercial hatchery, were randomly divided into 6 groups of 104 birds each. Each group was further randomly separated into 4 subgroups (replicates) of 26 birds each (13 males + 13 females). Each subgroup was housed in a separate floor pen (1.5 m × 2.7 m) equipped with one hanging bell drinker that was cleaned daily, two tube-type feeders, and an electric heater. Chickens were raised in pens (13 birds per m² floor space) floored with litter (pine wood shavings) to a depth of 5–6 cm throughout the experiment. The room temperature was gradually decreased from 33°C on Day 1 to 22°C on Day 21 and then kept constant until the end of the study (42 days of age). The house was naturally ventilated, with adjustable windows. Efforts were made to duplicate commercial conditions as much as possible. The birds were vaccinated against infectious bursal disease with Nobilis D 78 (Intervet®) at 14 days of age. Four of a total of 24 pens were allocated to uninfected birds and serviced separately each with individual equipment to prevent infection. The trial was terminated when the birds were 42 days of age.

Experimental diets

Complete basal starter, grower and finisher diets were given to broiler chickens during the experimental periods (from Days 1 to 14, 15 to 28 and 28 to Day 42, respectively) and in keeping with the specific nutritional requirements of broiler chickens at any age. Table 1 shows the ingredients and composition of the basal starter, grower and finisher diets, which contained no antibiotics, coccidiostat, growth enhancers or other forms of medication. The diets were isoenergetic and isonitrogenous. Chemical composition of basal diets was determined according to AOAC (1991). Diets were formulated to guarantee or exceed recommended nutritional requirements (NRC, 1994) and were presented in mashed form. Based on the basal diet, two additional diets were prepared with supplements of either a specific essential oil blend (75 mg/kg) or anticoccidial monensin sodium MON (100 mg/kg), at the expense of saw dust. The specific essential oil blend (EOB) was prepared and provided by Herba Ltd. Co. (İzmir, Turkey). The main active components of this EOB include carvacrol (24.5%), 1,8-cineole (20.1%), camphor (12.1%), thymol (6.0%), α-fenchone (4.5%), α-terpinyl acetate (3.6%) and sabinene (3.2%), and three totally different essential oils that are derived from selected herbs which grow in Turkey, i.e. oregano oil (Origanum sp.), laurel leaf oil (Laurusnobilis L.) and lavender oil (Lavandula stoechas). The active compounds of the essential oil mixture are presented in Table 2. Equal proportions of each of the essential oils were used in the mixture. The essential oil preparation used 925 g of zeolite as a feed-grade inert carrier for 75 g essential oil mixture. An in-feed ionophore anticoccidial preparation (MON) (Elancoban®, Elanco Animal Health Eli Lilly & Co., Ltd., Basingstoke, UK) contained 100 mg monensin sodium per kg premix. Feed and drinking water were offered to birds ad libitum.

Table 1 Ingredients and analyzed composition of the experimental starter, grower and finisher diets.

	Starter	Diets Grower	Finisher
Ingredients
Corn, g/kg	369.78	396.52	431.50
Wheat, g/kg	200.00	200.00	200.00
Soybean meal, g/kg	355.69	319.42	284.62
Soy oil, g/kg	34.50	45.76	49.27
DCP, g/kg	17.50	16.70	15.84
Limestone, g/kg	11.34	9.10	8.72
Sodium chloride	2.40	2.75	2.64
L-Lysine HCL, g/kg	1.00	0.00	0.00
DL-methionine, g/kg	2.05	2.75	2.21
L-threonine, g/kg	0.64	0.50	0.20
Vitamin premix°, g/kg	2.50	2.50	2.50
Mineral premix#, g/kg	1.00	2.50	1.00
Sodium bicarbonate, g/kg	0.60	0.50	0.50
Sawdust, g/kg	1.00	1.00	1.00
Analyzed composition
Dry matter, %	88.43	88.48	89.02
Crude protein, %	22.43	20.92	19.42
Ether extract, %	5.84	7.02	7.45
Crude fibre, %	3.24	3.29	3.19
Crude ash, %	6.27	5.81	5.52
Ca, %	1.05	0.94	0.89
Total phosphorus, %	0.70	0.67	0.64
Calculated composition
ME, kcal/kg	3013	3161	3203
Available phosphorus, %	0.46	0.44	0.42
Lysine, %	1.26	1.07	0.97
Methionine, %	0.55	0.52	0.49
Methionine+ cysteine, %	0.96	0.92	0.84
Threonine, %	0.89	0.82	0.73
Linoleic acid, %	2.81	3.45	3.69

DCP, digestible crude protein.

° Provides per kg of diet: vitamin A, 12,000 U; vitamin D³,1500 U; vitamin E, 75 mg; vitamin K³, 5 mg; vitamin B¹, 3 mg; vitamin B², 6 mg; vitamin B⁶, 5 mg; vitamin B12, 0.03 mg; nicotine amid, 40 mg; calcium-D-pantothenate, 10 mg; folic acid, 0.75 mg; D-biotin, 0.075 mg; choline chloride, 375 mg.

# Provides per kg of diet: Mn, 80 mg; Fe, 40 mg; Zn, 60 mg; Cu, 5 mg; I, 0.5 mg; Co, 0.2 mg; Se, 0.15 mg; ME, metabolizable energy.

Table 2 Bioactive components of the essential oil blend.

Compound	Compound
Carvacrol, %	24.5	α-Pinene, %	2.2
1,8-Cineole, %	20.1	p-Cymene, %	2.0
Camphor, %	12.1	Terpinen-4-ol, %	1.9
Thymol, %	5.9	α-Terpineol, %	1.6
α-Fenchone, %	4.5	α-Pinene, %	1.5
α-Terpinyl acetate, %	3.6	Camphene, %	1.4
Sabinene, %	3.2	α-Terpinene, %	1.1
Bornyl acetate, %	2.3	Unidentified, %	12.1

Experimental design

Six treatments were compared: four not cocci-vaccinated and two cocci-vaccinated. The four groups which were not cocci-vaccinated treatments included: i) uninfected chickens fed basal diets without anticoccidial feed additive (uninfected CNT-untreated control group); ii) infected chickens fed basal diets without anticoccidial feed additives (infected CNT-untreated control group); iii) infected chickens fed basal diets supplemented with MON, at 100 g/ton; iv) infected chickens fed basal diets supplemented with a specific essential oil blend at 75 g/ton.

Chickens in the other two treatments were vaccinated via drinking water at one day of age with a commercial vaccine for coccidiosis (Livacox® Q, Biopharm, Prague, Czech Republic) and then infected with coccidiosis at day 19. They were: i) vaccinated and fed basal diets without anticoccidial feed additives; and ii) vaccinated and fed basal diets including EOB at 75 g/ton. The oocysts used in the infecting inoculum were partly unrelated to those in the vaccine. The vaccine consisted of 30,000 to 50,000 sporulated oocysts of E. acervulina, E. tenella and E. maxima, and 10,000 sporulated oocysts of E. necatrix. Chickens were vaccinated at Day 1 of age, eight hours after being placed in experimental houses. Prior to vaccination, all groups were kept without water for two hours, including those who were not vaccinated. One bell-type drinker was used in each pen during vaccination. The vaccination dose was thoroughly mixed with 100 mL unchlorinated water per drinker. This was drunk within 60 min, after which the chickens were again given access to bell drinkers.

Performance parameters

All chicks were individually weighed at Days 1, 19, 30 and 42 to determine body weight. Body weight gain (BWG) was calculated for related periods. Feed intake (FI) within each subgroup was determined at Days 19, 30 and 42. Feed conversion ratio (FCR) was calculated as the ratio of feed intake to body weight gain (g feed/g gain) on a replicate basis. Mortality was recorded daily for each subgroup and calculated as the percentage of deaths to the initial number of chickens.

Eimeria infection and fecal oocysts measurement

Broilers in five of the six groups were infected at 19 days of age with a standard oral inoculum of sporulated oocyst from field isolates of E. acervulina, E. maxima, E. tenella, E. mitis, E. brunetti and E. praecox, at 19×10⁴ mixed oocysts, respectively, whereas the remaining group was not challenged. Thus, 19×10⁴ mixed oocysts were given to each of the five infected groups, while no oocysts were administered to the uninfected sixth group. Reference stocks of the six Eimeria species of sporulated oocysts in potassium dichromate were provided by the Department of Parasitology at the Veterinary Medicine Faculty of Ankara University, Turkey.

Coccidial infection of each bird was carried out by administering a 2 mL suspension of 19×10⁴ sporulatedoocysts of a mixture of E. acervulina, E. maxima, E. tenella, E. mitis, E. brunetti and E. praecox. The inoculum was washed several times with tap water to remove potassium dichromate, then directly given to the crop via an oral gavage, using a plastic syringe fitted with a plastic cannula.

At Days 15 and 19, oocyst counts in excreta samples taken from each subgroup of both the infected and uninfected groups were taken. Samples from infected birds were also evaluated daily from Days 19 to 34. Excreta samples were controlled again uninfected pens at Days 30 and 36, respectively. Sampling was carried out by collecting approximately 400–500 g samples of excreta from each replicate pen. Random collection of the samples taken from each pen was essential in order to obtain a precise evaluation of oocyst excretion. Therefore, every area of the pen floor was included and representative amounts of excreta were obtained. The samples collected daily from each subgroup were placed in separate, airtight plastic bags, homogenized thoroughly with a domestic mixer, and kept refrigerated until assessed for total oocyst counts (Christaki et al., 2004). Estimated homogenized samples were diluted 10-fold with tap water and further diluted with saturated NaCl solution, at a ratio of 1:10. Oocyst counts were determined using McMaster chambers and presented as the number of oocysts per bird (Hodgson, 1970). On Day 26 (Day 7 post challenge) lesion scores were given (scale from 1 to 4 where 4 is most severe; Johnson and Reid, 1970) for duodenum, jejenum, ileum, and ceca.

Blood samples for antibody titers

Blood samples were taken by puncturing the wing veins of 3 birds per pen (a total of 12 birds per group) at 17, 30 and 42 days of age, respectively. One milliliter samples in plastic tubes were immediately transferred to the laboratory. Serum was then isolated and stored at −20°C until used. Individual serum samples were analyzed for antibody responses against IBDV using the ELISA technique and commercial kits (Biochek®), and plates were read at 405 nm on an ELISA reader.

Measurement of carcass yield and some intestinal organs

At the end of the experiment (Day 42), 4 birds whose body weights were similar to the group average were selected from each replicate pen (16 birds per treatment group). A total of 96 sampled birds were then slaughtered by severing the bronchial vein to measure carcass yield and selected internal organs. Selected internal organs (liver, spleen and cecum) were individually weighed. Weight was expressed as a percentage of live body weight. Hot carcass yield was calculated as a percentage of preslaughter live body weight. Total length of small intestines (duodenum, ileum and jejunum) and large intestines (colon) was described as intestinal length.

Statistical analysis

Pen means were used as experimental units. A completely randomized statistical design was used. Data were subjected to oneway ANOVA analysis using the GLM procedure of the SAS system (SAS, 1991). Significant differences between treatment means were identified using Duncan’s multiple range test, with 5% probability. Mortality percentages were transformed by the arcsine method before analysis. Since the oocyst yields were not normally distributed, Kruskal-Wallis non-parametric analysis was used.

Results

Prior to infection, at 19 days of age, body weight in the VAC, EOB and VAC+EOB groups was lower than in the infected-untreated CNT and MON groups (Table 3). This clearly indicated that vaccination against coccidial infection on Day 1 induced growth depression, even though the chickens had not yet been infected. A similar pattern of retarded growth was also evident in birds given EOB or vaccinated and treated with EOB. Contrary to the tendency observed during the pre-infection period (Days 1 to 19), birds in the VAC+EOB treatment group managed to achieve remarkable weight gain recovery during the whole post-infection period (Days 19 to 42). There was no difference in body weight among the treatment groups subjected to coccidial infection at Days 30 and 42, with the exception of the EOM group. All infected birds were lighter than their uninfected counterparts during that period. Survival was excellent, in spite of one coccidial infection, and mortality was not significantly affected (P>0.05) by infection or any of the treatments evaluated (Table 3).

Table 3 Body weight and mortality of birds challenged with experimental coccidiosis at 19 days old of age.

Age of chickens, days	Uninfected	Infected					SEM	P
	CNT	CNT	MON	VAC	EOB	VAC + EOB
Body weight, g
Day 0	40.23	40.46	40.35	40.51	40.37	40.29	0.29	0.6649
Day 18	736^a	736^a	726a	665c	690b	660^c	7.86	0.0001
Day 30	1441^a	1352^b	1377^b	1340^b	1262^c	1341^b	20.67	0.0001
Day 42	2365^a	2251^b	2304^ab	2285^ab	2123^c	2276^ab	33.33	0.0001
Mortality, %
Day 0-18	0.50	0.95	1.55	0.00	0.00	0.50	0.542	0.3474
Day 19-30	0.00	0.00	0.00	0.00	1.00	0.00	0.400	0.4457
Day 31-42	1.00	0.00	0.00	0.00	0.00	1.00	0.576	0.5640
Day 19-42	1.00	0.00	0.00	0.00	1.00	1.00	0.708	0.7006
Day 0-42	1.50	0.95	1.55	0.00	1.00	1.50	0.826	0.7606

CNT, control; MON, monensin sodium; VAC, vaccinated against coccidiosis; EOB, essential oil blend;

a,b,c means in the same row with a different superscript letter are significantly different (P<0.05).

Coccidial challenge did not induce any remarkable reduction in feed consumption. As shown in Table 4, feed intake of all infected birds and the uninfected control group was comparable (P>0.05) during the entire post-infection period, except for the EOB group. The essential oil blend reduced feed intake throughout the experiment, compared to all other treatments (P<0.05). Vaccination at one day of age resulted in a slight reduction in feed intake during the pre-infection period, but not thereafter.

Table 4 Body weight gain, feed intake and feed conversion ratio of birds administrated with monensin sodium, live attenuated vaccine or essential oil blend.

Age of chickens, days	Uninfected	Infected					SEM	P
	CNT	CNT	MON	VAC	EOB	VAC + EOB
Day 0-18
BWG, g	697^a	697^a	686^a	625^b	650^b	620^b	10.44	0.0001
FI, g	1068	1072	1074	1032	1069	1052	13.28	0.2587
FCR	1.533^b	1.538^b	1.565^b	1.653^a	1.646^a	1.696^a	0.025	0.0005
Day 19-30
BWG, g	703^a	614^ab	651^ab	675^a	573^b	681^a	22.93	0.0498
FI, g	1329	1317	1378	1380	1254	1351	41.63	0.3163
FCR	1.904^b	2.158^a	2.123^ab	2.056^b	2.193^a	1.987^b	0.033	0.0416
Day 31-42
BWG, g	924	899	927	945	860	935	27.38	0.3140
FI, g	1956	1961	1969	1938	1847	1933	41.72	0.4018
FCR	2.123^ab	2.183^a	2.114^ab	2.052^b	2.149^ab	2.066^b	0.032	0.0381
Day 19-42
BWG, g	1628^a	1514^ab	1578^a	1620^a	1433^b	1616^a	46.30	0.0488
FI, g	3285	3278	3338	3318	3101	3284	70.98	0.2629
FCR	2.018^c	2.167^a	2.117^ab	2.051^bc	2.166^a	2.032^bc	0.029	0.0038
Day 0-42
BWG, g	2325^a	2211^ab	2264^a	2245^a	2083^b	2237^a	50.91	0.0483
FI, g	4353	4347	4412	4350	4170	4337	75.32	0.3542
FCR	1.872^c	1.967^ab	1.949^ab	1.939^b	2.003^a	1.939^b	0.018	0.0066

CNT, control; MON, monensin sodium; VAC, vaccinated against coccidiosis; EOB, essential oil blend; BWG, body weight gain; FI, feed intake; FCR, feed conversion ratio;

a,b,c means in the same row with a different superscript letter show significant difference (P<0.05).

Mixed coccidial infection worsened the feed conversion ratio, which was 13.2% soon after the infection (Days 19 to 30) and 7.4% during the entire post-infection period (Days 19 to 42), when unmediated treatment was administered. While birds showed no clinical signs of coccidial infection, feed conversion ratio was already impaired, along with live weight gain, in all infected, treated birds, compared with uninfected birds (Table 4). However, vaccination, either alone or in combination with EOB or MON, improved the adverse effects of coccidial infection on the efficiency of feed conversion during the entire post-infection period (Table 4). Conversely, individual EOB supplementation completely compromised the feed conversion ratio in both the pre-infection and post-infection periods compared to all other treatments.

Fecal oocyst count, measured daily between Days 23 and 34, is presented in Table 5. Overall mean values indicate that individual vaccination and the essential oil blend, and also their combined use, significantly reduced oocyst shedding compared to the untreated control program, after experimental coccidial infection. However, the pattern of reduced oocyst count observed with the treatments mentioned above was not seen with the MON program. The range of daily measurements of oocyst count in each treatment group generally displayed the same ranking of mean values, with some exceptions. Fecal oocyst counts peaked at Days 5, 6 and 7 post-infection, sharply declined at Days 8 and 9, and increased again in the following two days. They then demonstrated a steady decline throughout the remaining six days. No oocysts were detected in the uninfected control chickens during any of the test periods (data not shown).

Table 5 Effects of dietary anticoccidial treatments on excretion of oocysts in broiler chickens infected with mixed Eimeria spp. at 19 days old of age.

Age of chickens, days	Oocyst excretion, 10^3/g of faeces					SEM	P
	CNT	MON	VAC	EOB	VAC + EOB
23	22.60^a	17.30^a	17.90^a	12.45^ab	9.85^b	3.16	0.0471
24	23.25^a	15.32^a	5.37^b	6.66^b	5.77^b	2.83	0.0015
25	18.15^a	17.62^a	9.42^b	15.40^a	8.10^b	1.74	0.0019
26	9.82	9.72	6.80	11.07	7.32	1.54	0.2914
27	5.60^a	6.15^a	2.97^a	5.47^b	2.92^b	0.80	0.0273
28	7.32^a	6.72^a	2.55^b	2.87^b	2.12^b	0.33	0.0001
29	15.07^a	13.05^a	6.95^b	7.60^b	4.02^b	1.43	0.0003
30	10.95^a	13.95^a	6.92^b	2.55^c	4.15^bc	1.11	0.0001
31	14.97^a	8.82^b	8.20^b	7.37^b	8.45^b	1.41	0.0116
32	4.90^a	4.20^ab	3.42^ab	1.15^c	2.70^bc	0.63	0.0079
33	5.50^a	4.45^ab	2.25^c	1.37^c	2.52^bc	0.66	0.0029
34	2.90^a	3.42^a	1.02^b	0.97^b	1.37^b	0.31	0.0001
Mean	11.75^a	10.00^a	6.15^b	6.24^b	4.94^b	0.83	0.0001

CNT, control; MON, monensin sodium; VAC, vaccinated against coccidiosis; EOB, essential oil blend;

a,b,c means in the same row with a different superscript letter show significant difference (P<0.05).

Lesion scores of challenged broilers were not affected by dietary and vaccine treatments (data not shown).

Serum antibody titers of IBD, analyzed before and after coccidial infection, are shown in Table 6. Blood analysis showed that IBD titers in cocci-vaccinated and EOB-treated birds were lower than in all the other treatment groups two days before infection (17 days of age). A similar pattern was observed 11 days after exposure to coccidial infection (30 days of age) but this similarity was not statistically significant (P>0.05). None of the anticoccidial procedures applied in this study influenced serum IBD titers at Day 42. However, the drastic decrease in infected, untreated birds is noteworthy.

Table 6 Serum IBD titers of birds subjected to different oral anticoccidial procedures.

Age of chickens, days	Uninfected	Infected					SEM	P
	CNT	CNT	MON	VAC	EOB	VAC + EOB
17	8905^a	7902^a	8713^a	6672^ab	5328^b	8107^a	981	0.0483
30	9355	7881	8942	7534	5988	9183	1310	0.2072
42	8678^a	4269^b	9672^a	9721^a	8073^a	8305^a	1020	0.0498

CNT, control; MON, monensin sodium; VAC, vaccinated against coccidiosis; EOB, essential oil blend;

a,b,c means in the same row with a different superscript letter show significant difference (P<0.05).

Data relevant to carcass yield and percentage weight of some internal organs, with total intestine length, are shown in Table 6. Neither coccidial infection nor the anticoccidial programs applied influenced carcass yield or the relative weights of spleens and livers following infection with mixed Eimeria spp. Nevertheless, oral administration with MON, EOB or VAC led to significant decreases in the relative weights of cecum and intestinal length compared to the infected, untreated control program (P<0.01). However, there was no difference between these groups themselves (Table 7). The total intestine lengths of uninfected, untreated birds were significantly lower (21 cm) than those of their infected counterparts.

Table 7 Carcass yield, relative weight of liver, spleen and caecum, and intestinal length of birds treated with monensin sodium, live attenuated vaccine or essential oil blend after an experimental coccidial infection.

Groups	Carcass weight, g	Carcass yield, %	Liver, %	Spleen, %	Caecum weight, %	Intestinal length°, cm
Uninfected control	2072	79.13	2.39	0.111	0.333^c	233^bc
Infected control	2053	78.40	2.39	0.105	0.443^a	254^a
Infected MON	2051	77.85	2.22	0.107	0.375^bc	240^bc
Infected VAC	2010	77.74	2.19	0.115	0.380^b	233^bc
Infected EOB	2032	79.12	2.39	0.095	0.371^bc	227^c
Infected VAC+EOB	2068	78.35	2.35	0.113	0.384^b	238^bc
SEM	27.06	0.60	0.08	0.008	0.015	6.06
P	0.5953	0.4386	0.4083	0.5248	0.0004	0.0164

MON, monensin sodium; VAC, vaccinated against coccidiosis; EOB, essential oil blend;

° duedonum+jejunum+ileum+colon;

a,b,c means in the same row with a different superscript letter show significant difference (P<0.05).

Discussion

As we know, coccidial infections may present in poultry as acute clinical or subclinical infections. However, as they occur at the subclinical level, where there is no great increase in mortality but clear signs of intestinal disorder are present, inflammation of the gastrointestinal tract or disruption of gastrointestinal balance are likely to be observed (Cook, 1998; Chapman et al., 2008). Hence, in the case of subclinical forms of coccidial infection, damage to the intestinal mucosa, caused by both protozoans and pathogenic bacteria, leads to decreased digestion and absorption of nutrients, reduced weight gain and a deterioration in feed conversion (McDougald, 2003; Girgis, 2007; Chapman et al., 2008).

Like earlier studies describing the typical signs of coccidiosis at the subclinical level (McDougald, 2003), the experimental infection scheme in our study depressed body weight gain and impaired feed conversion ratio within similar ranges (7.4-14.3; 7.6-14.3 points, respectively), while showing minimal mortality. However, neither of the anticoccidial procedural treatments employed in this study was very successful at alleviating the deleterious effects of coccidial infection on broiler growth rate and feed conversion efficiency. The reduction in broiler performance that resulted from mixed Eimeria infection highlights the detrimental effects of infection by this parasite on bird performance, even in subclinical cases. This emphasizes the fact that broilers, once infected with coccidia, remain smaller than their non-infected counterparts, although feed consumption may be comparable.

Several preliminary studies have confirmed that the main active compounds of oregano oil, carvacrol and thymol exert anticoccidial effects against E. tenella (Giannenas et al., 2003; Williams, 1997), E. acervulina (Ibrir et al., 2009) and a mixed infective agent composed of Eimeria spp. (Saini et al., 2003; Christaki et al., 2004; Oviedo-Rondón et al., 2005; 2006a,^b). The herbal additives examined in these studies have been shown to be effective tools in the prevention or alleviation of the destructive effects of coccidial infection on the growth performance of broilers, thereby ensuring compensatory growth during the recovery phase. However, a relatively lesser degree of protection from infection and a relatively slower recovery rate of growth were seen with herbal additives than with chemical anticoccidials.

Unlike previous observations suggesting that dietary treatment with the essential oils of some herbs in some way improves the ability of infected birds to recover from coccidial infection, the specific blend of essential oils used here led to a deterioration in the overall live performance of broiler chickens throughout the growth period. We supposed that the depressed live performance of chickens receiving EOB could be due to the possibly toxic effects on chicken health of the phenolic constituents of the essential oils used. In fact, former in vivo and in vitro tests have shown that phenols can be specifically used as oocysticides against E. tenella (Allen et al., 1997; Williams, 1997). Nevertheless, Giannenas et al. (2003) have asserted that the major components of the oregano essential oils carvacrol and thymol may have a toxic effect on the upper layer of mature enterocytes of the intestinal mucosa due to the hydrophobic character of carvacrol (Sikkema et al., 1995). Therefore, Christaki et al. (2004) have stressed that, due to concerns about the toxicity and side effects of medicinal herbs, their levels of administration, mechanisms of action and clinical effects should be extensively investigated before they are approved for wide-spread use in animal diets. It is interesting to observe that despite the fact that the carvacrol content of our essential oil blend (19 ppm) was much lower than that declared by Giannenas et al. (2003) (300 ppm) and Ibrir et al. (2009) (250 ppm), a noticeable deterioration in the overall live performance of birds receiving EOB was clearly seen, contrasting with corresponding works.

We also speculated that the main active phenolic components of the EOB used in this study had interacted with each other; the combinations of carvacrol, thymol, 1,8 cineol and camphor may have led to unexpected toxicity, even when administered at lower doses, which was not seen in the single dietary treatments of carvacrol in previous studies. Moreover, the type of experimental infection observed (mixed Eimeria spp. vs E. tenella or E. acervulina) may have produced results which may be inconsistent with those of previous studies regarding dietary inclusion of essential oils. Indeed, it is helpful to consider that essential oils vary considerably with respect to their phenolic compound contents (Marino et al., 1999; Faleirio et al., 2003). Such variation may adversely affect the performance, metabolism and immunity of broilers (Lee et al., 2004; Brenes and Roura, 2010).

Vaccinating chickens against coccidiosis on Day 1 induced significant reductions in feed intake and body weight, and impaired the feed conversion ratio during the pre-infection period (Days 0 to 18). Consistent with our findings, cocci vaccination has frequently been linked to temporary lower performance in young birds (Williams and Gobbi, 2002; Oviedo-Rondón et al., 2005) which, in turn, is associated with bacterial enteritis (Chapman et al., 2002; McDougald, 2003; De Gussem et al., 2008). Unlike the depressed growth pattern observed during the starter period, birds showed compensatory growth and recovery throughout the finisher period. The relative improvement in body weight gain of vaccinated birds during the whole post-infection period differed by 3–7% from that of the infected control group and was comparable to the MON-treated group. Similar levels of benefit were also observed in the feed conversion ratio of the vaccinated groups. This highlights the fact that birds vaccinated at one day of age managed to recover from stress and maintain immuno-competence during the relatively short post-infection period (Days 19 to 31) and also after. Confirming our observations, some preliminary studies and experiences have shown that birds correctly vaccinated with cocci vaccines perform just as well or better than birds receiving traditional in-feed anticoccidals, with regard to reduction of intestinal lesions and oocyst shedding (Oviedo-Rondón et al., 2005; 2006a; Garcia and Bolis, 2005). In fact, Waldenstedt (2003) has reported that vaccinated broilers reared in organic production systems exhibit similar performances to those of non-vaccinated groups until Day 55.

In our study, the combination of a cocci-vaccine with an EOB (VAC+EOB) was scheduled so as to alleviate the reported temporary depressed growth of live vaccines, which was anticipated to help modulate gut micro flora. The combination concept was also based on principles of synergy or additive effect, and took into account the complementary mechanisms of both compounds. However, in contrast to our expectations, combining VAC with EOB did not produce real synergistic or additive benefits compared to single use of alternative anticoccidial programs. Nevertheless, we are not able to give any obvious scientific explanation of how the depressed growth effect of treatment with EOB alone disappeared when combined with the vaccine and even demonstrated results comparable to those achieved with only vaccination. This is in agreement with a previous study (Waldenstedt, 2003) that indicated that vaccination against coccidiosis, in combination with administration of an oregano essential oil preparation, may be an alternative method of controlling intestinal health in organically-produced chickens.

It is important to note that mortality was considerably lower than the mortality due to coccidial infection reported in earlier works (Giannenas et al., 2003; Christaki et al., 2004). Indeed, the infection dose of mixed Eimeria spp. used in this investigation is likely to be markedly lower than the levels used with commercial flocks. Therefore, we hypothesize that the coccidial or bacterial infection was not severe enough for us to observe significant mortality.

Broilers infected with coccidiosis and treated with no anticoccidial had significantly lower antibody titers against IBD at Day 42 compared to uninfected broilers, reflecting a reduction in the responsiveness of the immune system to coccidial exposure. Both VAC and EOB treatments adversely affected the immune response of treated compared to untreated birds during the initiation period, but this negative effect disappeared entirely thereafter. Furthermore, vaccinated birds rapidly compensated for the inadequacies of their IBD titers until the end of the experiment. Due to a lack of information on the immune modulator effects of novel coccidiosis control programs, we are not able to compare our results with those of previous studies.

Overall means indicated that dietary supplementation with EOB, either alone or in combination with the vaccine, and also individual application of the vaccine, resulted in significant reductions in fecal oocyst count following infection with mixed Eimeria spp. Nevertheless, contrary to our general expectation, MON, being a traditional ionophore anticoccidial, did not reduce oocyst count at any age compared with the untreated control program.

In fact, counting the number of oocysts in feces in the laboratory is one of the principal ways of diagnosing coccidiosis. However, the link between number of oocysts in feaces and bird performance is not so clear, as was the case in the present study. Therefore, further studies are necessary to better understand the underlying mechanism responsible for this relationship. In confirmation of our own observations, several earlier works have proven that extracts and essential oils of selected herbs effectively reduce fecal oocyst shedding when broiler chickens are experimentally infected with coccidiosis (Giannenas et al., 2003; Christaki et al., 2004; Oviedo-Rondón et al., 2006a; Ibrir et al., 2009; Lee et al., 2010).

Coccidial infection considerably modified intestinal morphology, significantly increasing the cecum weight and overall intestine length of infected birds compared with uninfected birds. However, all of the anticoccidial procedures examined in this study decreased intestinal measurements. Our results imply that anticoccidial feed additives, such as MON and EOB, or VAC, may have tackled the inflammation that occurred in the intestinal segments as the result of mixed Eimeria spp. infection. There is a lack of scientific consensus on this and consequently future studies on the considerable influence of anticoccidial procedures on intestinal morphology in cases of coccidial infection would be of value.

Conclusions

In conclusion, the number of oocysts excreted, as well as marked compensatory growth performance, showed that a commercial coccivaccine was as effective as MON in reducing the severity of coccidiosis. It did, however, result in an immediate reduction in performance, though this was only temporary. The data indicated that combining vaccination with the administration of a specific essential oil blend could be nearly as effective when it comes to reducing oocyst shedding after mixed coccidia infection as administering either of these separately. Therefore, in the future, the long-term sustainability of coccidiosis control in broiler chickens may be facilitated by combination strategies that involve the alternate use of vaccines and the essential oils of some selected herbs with successive flocks.