Control of eight predominant Eimeria spp. involved in economic coccidiosis of broiler chicken by a chemically characterized essential oil

O R I G I N A L A R T I C L E

Control of eight predominant Eimeria spp. involved in economic coccidiosis of broiler chicken by a chemically characterized essential oil

E.K. Barbour¹, R.R. Bragg², G. Karrouf^3,4, A. Iyer⁵, E. Azhar⁶, S. Harakeh⁶and T. Kumosani⁵

1 Animal and Veterinary Sciences Department, Faculty of Agricultural and Food Sciences, American University of Beirut, Beirut, Lebanon adjunct to Biochemistry Department, Faculty of Science and Experimental Biochemistry Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia

2 Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, South Africa 3 King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia

4 Surgery, Anaesthesiology and Radiology Department, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt 5 Biochemistry Department, Faculty of Science and Experimental Biochemistry Unit, King Fahd Medical Research Center, King Abdulaziz

University, Jeddah, Saudi Arabia

6 Special Infectious Agents Unit–Biosafety Level 3, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia

Keywords

chicken broiler, coccidiosis, essential oil, eucalyptus, peppermint, terpenes.

Correspondence

Elie K. Barbour, Department of Animal and Veterinary Sciences, Faculty of Agricultural and Food Sciences, American University of Beirut, P.O. Box 11-0236, Beirut, Lebanon.

E-mail: eb01@aub.edu.lb

2015/0095: received 16 January 2014, revised 12 December 2014 and accepted 13 Decem- ber 2014

doi:10.1111/jam.12731

Abstract

Aim: To control eight most predominant Eimeria spp. involved in the economic disease of coccidiosis in broiler chicken, by a chemically characterized essential oil of eucalyptus and peppermint.

Methods and Results: The experimental design consisted of 160 day-old- broiler chicks, divided into four equal groups (G1, G2, G3 and G4), with 40 birds per group. Each group was divided into four equal subgroups. Birds in G1 were deprived of essential oil treatment and of Eimeria challenge. Birds in G2 were unchallenged, and administered the essential oil in drinking water at 069 ml kg ¹ body weight. Birds in G3 were untreated with essential oil, and each of its four subgroups was challenged at a different age (14, 21, 28 and 35 days). Birds in G4 were treated with essential oil, and challenged in the same manner as for G3. Equal number of birds from all subgroups (n =10) were sacrificed at the sixth day after the time allocated for each challenge. The 6 day incubation period post challenge resulted in respective mean per cent weight increase in G2 and G1 birds equivalent to 578 and 531% (P<005). In addition, the essential oil improved the per cent weight increase in challenged birds (546%) compared to the challenged-untreated birds (186%) (P<005). The mean feed conversion, mortality, intestinal lesion scores and oocyst counts were significantly reduced in the challenged-treated birds compared to the challenged-untreated birds (P<005).

Conclusions: The results support the hypothesis of using the essential oils of eucalyptus and peppermint to control the most prevalentEimeriaspp. involved in coccidiosis of broiler chicken, helping in improvement of their production, alleviation of lesions and reduction in intestinal oocyst counts.

Significance and Impact of the Study: This study provides information about the possibility of using this blend of essential oil as a coccidiostat for the protection of broiler chickens against the prevalent eight Eimeria spp. of coccidiosis.

Introduction

Eimeria spp. infection in poultry is an economically important disease requiring a continuous supplementa- tion of coccidiostat in their feed. The continual and large-scale use of coccidiostats in the feed is causing the emergence of serious resistance in these protozoa to the drugs used during production (Chapman 1997; Stephen et al. 1997). In addition, most broiler operations in developing countries are not following the proper with- drawal periods of coccidiostats from the feed before slaughter, resulting in significant residues in chicken car- casses, even in operations respecting the recommended withdrawal periods (Mortier et al.2005).

The most common coccidial infections in broilers are caused by eight species ofEimeria(Leeet al.2011). These are Eimeria acervulina (Assis et al. 2010), Eimeria bru- netti(Hein 1974),Eimeria maxima(Schnitzler and Shirley 1999), Eimeria mivati (Vrba et al.2011),Eimeria necatrix (Conway and Mckenzie 2007), Eimeria hagani (Joyner and Long 1974), Eimeria praecox (Reperant et al. 2012) andEimeria tenella(Railliet and Lucet 1891).

The optimization of reproduction of the intestinal pathogenesis by a controlled challenge with the eight spe- cies of Eimeria is of paramount importance, before the evaluation of any drug or immunopotentiator against these protozoa (Elmusharaf et al.2010). In addition, the multiplication and pathogenesis of Eimeriaspp. is related to innate immunity of different chicken breeds (Lillehoj 1994); thus, the inclusion of a certain breed in the Eime- ria spp. challenge requires a detailed optimization to reproduce the pathogenesis of the challenging organisms in its birds. The establishment of a chicken model for achieving Koch’s postulate, using mixed infection by the eight species of Eimeria, was recently documented (Bar- bour et al.2013a,b).

The anti-parasitic activity of essential oils is sporadi- cally reported in the literature (Fatani and Hilali 1994;

Kamsuk et al. 2007; Grabensteiner et al. 2008; Khater 2014). The coccidiostat activity of terpenes present in other essential oils against Eimeria spp. in poultry has a paucity of documentations (Giannenas et al. 2003; Ovie- do-Rondon et al.2006). A blend of eucalyptus and pep- permint has been evaluated with regard to its protection abilities against different pathogens in poultry including Mycoplasma gallisepticum, infectious bronchitis virus and avian influenza virus (Barbour et al. 2006, 2011). This blend showed acceptable degrees of protection and improvement in production. Other workers reported an improvement in broiler performance when essential oils were administered as a dietary supplement (Suk et al.

2003; Hernandez et al. 2004; Crosset al. 2007). Timber- mont et al. (2010) showed that broiler diets supple-

mented with essential oil of eucalyptus, containing terpenes as the main active ingredients, contributed to the prevention of necrotic enteritis induced by interaction betweenClostridium perfringens and Paracox-5^TM anticoc- cidial vaccine. The active ingredients of eucalyptus oil enabled the stimulation of the immune system response by enhancing the phagocytic activity of monocytes (Seraf- inoet al.2008). In addition, the cineole active ingredient in the eucalyptus essential oil has been proved to control the secretions of mucus in the epithelial layer of the respiratory system air passages (Juergenset al.2004). The in vivo andin vitro virucidal activity of terpenes in euca- lyptus essential oils against poultry viruses was docu- mented by Barbour et al. (2006, 2010). The antiviral effect of terpenes in peppermint essential oil has been also studied (Siddiqui et al. 1996; Sivropoulou et al.

1997; Schuhmacheret al.2003; Ocaket al.2008).

To our knowledge, this is the first research investigat- ing the coccidiostat activity of quantified terpenes, pres- ent in an essential oil blend of eucalyptus and peppermint 1/1 (v/v), against eightEimeriaspecies which are frequently involved in coccidiosis of chicken world- wide.

Methods and materials

Experimental design Birds and vaccination

A total of 160 day-old Ross 308 birds were divided into four groups (G1, G2, G3 and G4), with 40 birds per group. The birds were fed according to the nutrient requirements recommended by NRC 1992. The feed did not contain any coccidiostat. A bivalent live Newcastle and Infectious Bronchitis vaccine was administered intra- occularly at 1 and 14 days of age, while a live Gumboro vaccine was administered in the drinking water at 14 and 21 days of age.

Chemically characterized essential oil blend

The essential oil blend constituted of equal volumes of eucalyptus and peppermint oils, provided by Chemist Werner Krull, EWABO Chemikalien GmbH & Co., Kolpingstraße 4, 49835 Wietmarschen, Germany. The chemically characterized blend revealed the following per- centages of terpenes: cineol (422%), limonene (35%),

L-menthol (487%), phellandrene (05%), a-pinene (10%), b-pinene (03%) and terpineol (03%). The dilu- tion of the essential oil blend in drinking water, adminis- tered ad libitum to birds in groups G3 and G4, was 002%, providing 069 ml of the blend/kg body weight.

The safety of this essential oil administration protocol in chicken was concluded in a previous research, targeting

the alleviation of a highly pathogenic velogenic Newcastle Disease injuries in broilers (Barbouret al.2013a,b).

Description of the different treatments

The description of the different treatments is presented in Table 1. The birds were divided into four major groups (G1, G2, G3and G4), with 40 birds per group. Each group was divided into four subgroups. The assignment of essential oil treatment and challenge by the eightEimeria spp. is indicated in Table 1.

The challenge was with a total of 176910⁵ oocysts per bird of equal number of sporulated oocysts of all eight species ofEimeria, namelyE. acervulina, E. brunetti, E. hagani, E. maxima, E. mivati, E. necatrix, E. praecox and E. tenella. The sporulated oocysts were sourced from the nonattenuated strains provided by Intervet Inc. (Sum- mit, NJ). The oocyst counts were accomplished according to the protocol of Conway and Mckenzie (2007).

Evaluation of the coccidiostat activity in the essential oils The evaluation of the coccidiostat activity of the terpenes in the essential oils against the mixed infection by the eightEimeriaspp. in broilers required quantitative assess- ment of five parameters, observed during and/or at the end of the 6-day period following each of the four challenge dates. This quantitative assessment was in accordance with previously established protocols for Anti- coccidial Susceptibility Testing (AST) (Holdsworth et al.

2004; USDHHS 2012). The measured parameters were:

i the mean live body weight and per cent of weight increase in each broiler group,

ii the mean feed conversion ratio, iii the mean per cent mortality,

iv the mean sore of lesions (0–3 scores) in each of the four intestinal organs (duodenum, jejunum, ileum and caecum), and

v the determination of the mean of log10ofEimeriaoo- cysts counts per gram of each of the four intestinal organs, observed at the end of the 6-day period fol- lowing each of the four challenges.

The weight gain was calculated by subtraction of the chicken weight at the end of the sixth day following the challenge from the weight at challenge time. The feed con- version was calculated by the division of the consumed weight of feed by each group of chicken, during the 6 days following the challenge, divided by the bird’s live weight gain during that period. The mortality frequency was cal- culated during the 6-day period following challenge. The scoring of the lesions was accomplished by a trained researcher, scoring ‘0’, ‘1’, ‘2’ and ‘3’ to respective intesti- nal lesions namely, absence of gross lesions, mild, moder- ate and severe congestion (Johnson and Reid, 1970).

Briefly, the score of ‘0’ was given for the part of the intes- tine showing no lesions. The score of ‘1’ was given for the part of intestine with few scattered petichiae and white spots located on the serosa. The score of ‘2’ was given for ballooning of the intestinal part, whereas the score of ‘3’

was given for the presence of pin point haemorrhages in

Table 1 Description of the different treatments Groups and

subgroups

Number of birds

Treatment with essential oil

Treatment at age (days)

Eimeriaspp.

challenge

Age at challenge (days)

Age at sacrifice (days)

G1 40 No – No –

G1a 10 No – No – 20

G1b 10 No – No – 27

G1c 10 No – No – 34

G1d 10 No – No – 41

G2 40 Yes No –

G2a 10 Yes 4–7, 16–18 No – 20

G2b 10 Yes 4–7, 16–18, 25–27 No – 27

G2c 10 Yes 4–7, 16–18, 25–34 No – 34

G2d 10 Yes 4–7, 16–18, 25–39 No – 41

G3 40 No –

G3a 10 No – Yes 14 20

G3b 10 No – Yes 21 27

G3c 10 No – Yes 28 34

G3d 10 No – Yes 35 41

G4

G4a 10 Yes 4–7, 16–18 Yes 14 20

G4b 10 Yes 4–7, 16–18, 25–27 Yes 21 27

G4c 10 Yes 4–7, 16–18, 25–34 Yes 28 34

G4d 10 Yes 4–7, 16–18, 25–39 Yes 35 41

the serosa associated with ballooning of the intestinal part.

The determination of the oocysts count per gram of each of the four intestinal organs was according to the protocol documented by Haug et al. (2006) and Conway and Mckenzie (2007). Briefly, the intestine was rinsed in a sal- ine solution to remove the faecal material, and 4 g of the rinsed organ was collected. The weighed intestinal organ was homogenized and put in 56 ml of 35% NaCl solution to induce floatation of the oocysts. The floating oocysts were put in a two-compartment McMaster chamber, allowing for the debris to settle and the oocyst to float after a period of 5 min. The oocysts were counted within the grid of the two chambers, under a compound micro- scope and at 1009 magnification. The obtained total count was multiplied by 50 to conclude the count of the oocyst per gram of the intestinal part. The calculated count was transformed to log to the base 10 (log10).

Statistics

The mean values of the above measured parameters were compared among the different treatments by ^ANOVA fol- lowed by the Tukey’s test, reporting significant differences in means at P<005. The mortality frequencies were compared among the different treatments by the chi- squared method.

Results

Chicken growth

Table 2 presents the mean live body weight and the per cent weight increase in birds during the 6-day period fol- lowing each of the four challenges, administered at four different ages. The mean live body weight of the unchal- lenged birds administered the essential oil (13168 g) was

improved compared to that of the control-unchallenged birds deprived of the essential oils (12446 g) (P>005).

The essential oils were also able to improve the mean body weight in challenged birds (12876 g) compared to that in challenged birds deprived of the essential oils (10309 g) (P>005). However, the mean per cent weight increase was significantly less in the challenged group deprived of essential oil (186%) compared to the similarly challenged group administered the essential oil (546%) (P<005). In addition, the essential oil in the unchallenged group had a growth promotion effect with a mean per cent weight increase of 578% compared to the similarly unchallenged group deprived of the oil (531%) (P<005).

Feed conversion

The improvement in weight gain in both the challenged and unchallenged groups of birds which were treated with the essential oils when compared to the untreated groups (Table 3) was also associated with improvements in mean feed conversion. In the 6 day period post chal- lenge, a feed conversion rate (FCR) of 146 was recorded in the treated, unchallenged group of birds (G2). In com- parison, a FCR of 153 was found in the nonchallenged, nontreated group of birds (G1). This difference in FCR was found to be statistically significant (P< 005) (Table 3). Similarly, FCR was found to improve in the challenged birds which were treated with the essential oils (all G4 subgroups) when compared with the challenged and untreated groups of birds (all G3 subgroups). The mean feed conversion ratio for the challenged and treated birds was 150 compared to a FCR of 243 obtained in similarly challenged but nontreated birds. This difference was also found to be statistically significant (P<005).

Table 2 Mean weight (g) and per cent weight increase of birds* at 6 days post challenge Age at

challenge (days)

Weight in grams (per cent weight increase) at 6 days post challenge

G1 G2 G3 G4 SEM†

14 6451 (792) 6620 (870) 5973 (215) 6501 (826)

21 9471 (457) 9824 (493) 7297 (177) 9676 (475)

28 14666 (552) 15378 (577) 1123 (194) 14661 (524)

35 19198 (324) 2085 (372) 16739 (156) 20667 (357)

Mean live body weight Mean %weight increase

12446^a (531)^a

13168^a (578)^b

10310^a (186)^c

12876^a (546)^a

1320 12 Means of the body weight in a row, followed by the same Greek letter are not significantly different atP>005.

Means of the per cent weight increase in a row, followed by different alphabets are significantly different atP<005.

*Each group started with 10 birds. G1has control birds deprived of essential oils andEimeriaspp. challenge. Birds in G2group were administered the essential oils, but deprived of challenge. Birds in G3 group were deprived of essential oils and challenged. Birds in G4 group were administered the essential oil in drinking water and challenged.

†SEM=standard error of mean body weight and of per cent weight increase.

Mortality

No mortalities were recorded in the unchallenged groups of birds (G1 and G2). Mortalities were recorded in the challenged group of birds during the 6 days after each challenge date. It was found that the treatment of chal- lenged birds with the essential oil resulted in a statistically significant lower mortality rate (25%) when compared to that of the challenged and untreated group of birds (200%) (P<005) (Table 4).

Intestinal lesion scores

The mean lesion scores in the duodenum, jejunum, ileum and caecum at 6 days post challenge with the Eimeria spp. cocktail were similar in G1, G2 and G4 birds (P>005) (Table 5) compared to a higher significant mean lesion score in challenged and untreated birds (G3

birds) (P<005).

Oocyst counts in intestinal organs

The oocyst counts per gram of each of the four intestinal organs were always absent in the unchallenged birds of G1 and G2 groups (Table 6). There was a consistent sig- nificant reduction (P< 005) in the oocyst counts of the four intestinal organs of challenged and treated birds (all G4 subgroups) compared to those obtained in challenged and untreated birds (all G3 subgroups). The average reduction in log10 of the oocyst counts in G4birds com- pared to that of G3birds was equivalent to 376.

Discussion

The growth-promoting effect of treatment with the terp- enes of the essential oil blend was clearly demonstrated in the group of birds which were not challenged (G2) when compared with the untreated, nonchallenged birds (G1).

The data obtained from G1 and G2demonstrate that the

Table 3 Mean feed conversion of birds in the different treatments

Age at challenge (days)

Feed conversion of the four groups* in the 6-day period following each challenge

G1 G2 G3 G4 SEM†

14 168 158 250 165

21 141 140 210 145

28 147 142 295 144

35 154 143 217 147

Mean feed conversion 153^a 146^a 243^b 150^a 016

Means of feed conversion in a row followed by different superscripts are significantly different (P<005).

*Each group started with 10 birds. G1has control birds deprived of essential oils andEimeriaspp. challenge. Birds in G2group were administered the essential oils, but deprived of challenge. Birds in G3group were deprived of essential oils and challenged. Birds in G4group were adminis- tered the essential oil in drinking water and challenged.

†SEM=standard error of the means in the same row.

Table 4 Number and per cent mortality of birds in the different treatments

Age at challenge (days)

No. (%) mortality in the four groups* during the 6-day period following challenges administered at different ages

G1 G2 G3 G4 SEM†(no./%)

14 00 (00) 00 (00) 3 (300) 20 (100)

21 00 (00) 00 (00) 2 (200) 00 (00)

28 00 (00) 00 (00) 2 (200) 00 (00)

35 00 (00) 00 (00) 1 (100) 00 (00)

Mean no. (%) mortality 00 (00)^a 00 (00)^a 2 (200)^b 05 (25)^a 025/17 Mean no. (%) mortality in a row followed by different superscripts are significantly different (P<005).

*Each group started with 10 birds. G1has control birds deprived of essential oils andEimeriaspp. challenge. Birds in G2group were administered the essential oils, but deprived of challenge. Birds in G3group were deprived of essential oils and challenged. Birds in G4group were adminis- tered the essential oil in drinking water and challenged.

†SEM=standard error of means in the same row.

treatment of birds with this blend is safe and has no neg- ative impact on production parameters. This growth pro- motion is most likely due to the inhibitory effect of

active ingredients of the terpenes against a wide spectrum of micro-organisms existing in the digestive tract of the unchallenged birds.

Table 5 Mean score of lesions in four intestinal organs of birds for the different treatments

Age at challenge (days) Intestinal organ

Mean score of lesions in the four groups*at the 6-day period following each challenge

G1 G2 G3 G4 SEM†

14 Duodenum 01^a 00^a 24^b 01^a 013

Jejunum 00^a 00^a 25^b 01^a 013

Ileum 00^a 00^a 17^b 02^a 012

Caecum 00^a 00^a 19^b 01^a 010

21 Duodenum 02^a 01^a 24^b 01^a 012

Jejunum 01^a 00^a 19^b 00^a 010

Ileum 00^a 00^a 15^b 01^a 011

Caecum 01^a 00^a 15^b 00^a 009

28 Duodenum 02^a 01^a 21^b 02^a 014

Jejunum 00^a 00^a 16^b 01^a 010

Ileum 01^a 01^a 25^b 00^a 013

Caecum 00^a 00^a 25^b 02^a 010

35 Duodenum 01^a 02^a 24^b 01^a 013

Jejunum 01^a 00^a 24^b 00^a 014

Ileum 00^a 00^a 17^b 00^a 010

Caecum 00^a 00^a 15^b 01^a 010

Means of lesion scores in a row followed by different superscripts are significantly different (P<005). Lesion scores range between 0 and 3.

*Each group started with 10 birds. G1has control birds deprived of essential oils andEimeriaspp. challenge. Birds in G2group were administered the essential oils, but deprived of challenge. Birds in G3group were deprived of essential oils and challenged. Birds in G4group were adminis- tered the essential oil in drinking water and challenged.

†SEM=standard error of means in the same row.

Table 6 Log10of the mean oocysts count per gram of intestinal organs* in birds for the different treatments*

Age at challenge (days) Intestinal organ

Mean log10of oocysts count per gram of intestinal organ at end of 6-days period following challenges administered at different ages

G1 G2 G3 G4 SEM†

14 Duodenum 00^a 00^a 37^b 11^c 002

Jejunum 00^a 00^a 52^b 14^c 001

Ileum 00^a 00^a 57^b 06^c 003

Caecum 00^a 00^a 63^b 22^c 001

21 Duodenum 00^a 00^a 41^b 12^c 005

Jejunum 00^a 00^a 54^b 13^c 003

Ileum 00^a 00^a 45^b 11^c 003

Caecum 00^a 00^a 67^b 24^c 001

28 Duodenum 00^a 00^a 31^b 12^c 001

Jejunum 00^a 00^a 54^b 11^c 003

Ileum 00^a 00^a 52^b 11^c 001

Caecum 00^a 00^a 67^b 23^c 003

35 Duodenum 00^a 00^a 43^b 12^c 003

Jejunum 00^a 00^a 52^b 13^c 001

Ileum 00^a 00^a 54^b 11^c 002

Caecum 00^a 00^a 62^b 23^c 002

Oocysts count in a row followed by different superscripts are significantly different (P<005).

*Each group started with 10 birds. G1has control birds deprived of essential oils andEimeriaspp. challenge. Birds in G2group were administered the essential oils, but deprived of challenge. Birds in G3group were deprived of essential oils and challenged. Birds in G4group were adminis- tered the essential oil in drinking water and challenged.

†SEM=standard error of means in the same row.

Similar growth-promoting effects were also seen when the birds were treated with the blend and challenged (G4

subgroups) compared to similarly challenged birds that were deprived of the blend (G3 subgroups). It was also established that this growth-promoting effect was statisti- cally significant (Table 2).These findings clearly demon- strate that treatment with this blend has a clear inhibitory effect on the eight Eimeria spp. used in the challenge, which can be seen from the improved growth rate (Table 2), improved FCR (Table 3), reduced mortal- ity rate (Table 4) as well as the reduction in oocyst counts (Table 5). Previous works documented the inhibi- tory effects of terpenes present in essential oils of euca- lyptus and peppermint against avian bacteria (Suk et al.

2003; Barbouret al.2006; Crosset al.2007; Timbermont et al. 2010) and avian viruses (Schuhmacher et al. 2003;

Barbour et al. 2010). This work clearly demonstrates an inhibitory effect of the essential oils against the challenge by the eight species ofEimeria.It is worth noting that no documents exist in the literature about the coccidiostat activity of terpenes present in eucalyptus and peppermint.

It is also worth noting that the growth-promoting effects of essential oils in broilers have been previously reported by Ocaket al.2008.

The mean conversion of feed to live body weight in essential oil-treated birds was significantly improved in both the unchallenged and challenged birds compared to their respective nonessential oil-treated groups, during the 6 days post challenge with the cocktail of Eimeria spp.

used in this experiment. This improvement is most likely due to the impact of terpenes on inhibition of multiplica- tion of environmental micro-organisms in unchallenged birds, in addition to the inhibition ofEimeriaspp. multi- plication in challenged birds (Table 6). Previous literature showed the benefits of control ofEimeriaspp. infection in broilers on feed conversion ratios (Min et al.2004). The standard addition of coccidiostats in the feed of broilers for 80% of the life of the bird with the withdrawal of the coccidiostats before slaughter is a globally adopted proto- col in the broiler industry (NRC 1999; EFSA 2011).

Improvements in growth and feed conversion by the terpenes of eucalyptus and peppermint were also associ- ated with a significant reduction in the mean frequency of mortality, during the 6 days post challenge with the cocktail of eight Eimeria spp. These data confirm the beneficial characteristics of the terpenes-active ingredients in alleviation of the injuries that result from the known pathogenicity of theEimeriaspp. used in this experiment (McDougald 2003; DeGussem 2011). A previous histo- pathological study demonstrated a reduction in micro- scopic lesions produced by avian pathogens, as a result of treatment with the same essential oil blend (Barbour et al.2006).

This reduction in mortality in challenged birds by the terpenes was associated with a significant reduction in the mean score lesions in all the four organs of the intes- tine (Table 5) compared to the mean scores in challenged birds that were deprived of these ingredients. These data support the hypothesis of the alleviation of injuries of the eight Eimeria spp. in broilers by the terpenes of these oils, due mainly to the impediment of their multiplica- tion (Table 5). There was a consistent significant reduc- tion in the oocyst counts present in the four intestinal organs ofEimeria spp.-challenged birds that were treated with the terpenes of these essential oils compared to challenged birds that were nontreated, averaging in reduction to 376 of log10 of the counts. This significant reduction in the oocyst count due to terpenes is confir- mative of thein vivoinhibitory effect of such essential oil ingredients against the eightEimeria spp. that are preva- lent in poultry infections (McDougald 2003; DeGussem 2011).

In conclusion, the terpenes of eucalyptus and pepper- mint are able to act as growth promoters with a coccidio- stat effect against the Eimeria spp. challenge, as confirmed by the significant improvements in feed con- version, reduction in mortality, lower intestinal lesion scores and drop in the intestinal oocyst counts. Future investigations will experiment on the addition of other terpenes present in other essential oils, in an attempt to search for higherin vivoinhibition ofEimeriaspp.

Acknowledgements

This work was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant no. 32/67459. The authors acknowledge with thanks the technical and financial support of DSR.

Conflicts of Interest

No conflicts of interests exist among authors of this manuscript.

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