IMMUNOLOGY, HEALTH, AND DISEASE Comparative evaluation of probiotic and salinomycin effects on performance and coccidiosis control in broiler chickens Wael Abdelrahman,*† michaela mohnl,* Klaus Teichmann,‡ Barbara Doupovec,* Gerd Schatzmayr,‡ Brett Lumpkins,§ and Greg mathis§ *BIOMIN Holding GmbH, Herzogenburg, Austria 3130; †Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt 41522; ‡BIOMIN Research Center, Technopark 1, Tulln, Austria 3430; and §Southern Poultry Research, 96 Roquemore Road, Athens, GA 30607 ratio and mortality were recorded throughout the experiment. On d 21 and 42, intestinal lesions and litter conditions were scored. On d 7, 14, 21, 28, 35, and 42, oocyst counts were determined from 10 freshly collected fecal samples per pen. The results showed that mortality, litter, and lesion scores at d 21 and 42, and oocyst shedding at d 21 did not differ significantly between the Prob mix and the Sal groups. However on d 28, oocyst shedding was significantly lower in the Sal group than in the PC group but insignificantly lower than the Prob mix group. Body weights of the Prob mix group at d 42 were significantly lower than the Sal group; however, the feed conversion ratio values were similar between the 2 groups. The results of this study showed that probiotics supplementation could be considered as a potential strategy to control coccidiosis in broiler chickens.
Key words: probiotic, salinomycin, coccidiosis, broiler, performance 2014 Poultry Science 93:1–7 http://dx.doi.org/10.3382/ps.2014-04212
INTRODUCTION
increased susceptibility to other diseases such as necrotic enteritis (Burridge et al., 1979; mcDougald and Fitz-Coy, 2008). Coccidia are ubiquitous protozoa that are present wherever chicken are raised, which exclude the possibility of controlling the disease by quarantine or disinfection. Control of coccidia is greatly dependent on the use of chemotherapeutic agents. The use of chemotherapeutics to control these protozoa has proven to be successful in many parts of the world due to their ease of use and the ability to provide uniform treatment and prevention. Nevertheless, these drugs, on some occasions, can be toxic to birds due to poor feed mixing, incorrect identification of the premix or the feed, or cross-contamination. They also have to be rotated from time to time to minimize any possible resistance developed against them by Eimeria species. Despite the global acceptance and success of these drugs in controlling a costly avian disease, the poultry
Coccidiosis is a significant enteric disease in poultry with the potential to inflict a considerable economic effect on farm profitability. The financial loss to the poultry industry as a result of coccidiosis worldwide has been estimated annually at US $3 billion, which is mainly due to prophylactic or therapeutic in-feed medications and also as a result of the effect of the disease on birds’ health (Williams, 1999; Dalloul and Lillehoj, 2006). Eimeria species multiply in the bird’s intestinal tract, causing considerable tissue damage and subsequently impaired nutrient absorption and blood loss. In addition, it can cause diarrhea, dehydration, mortality, transient drop in egg production in laying flocks and ©2014 Poultry Science Association Inc. Received may 27, 2014. Accepted September 10, 2014. 1 Corresponding author: [email protected]
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ABSTRACT The annual financial loss to the poultry industry as a result of coccidiosis has been estimated at about US $3 billion. The objective of this study was to evaluate and compare the effects of probiotics and salinomycin as feed additives on performance and coccidiosis control in male broilers raised to 42 d of age. The study consisted of 360 Cobb male broiler chickens randomly allocated to 4 groups each with 3 replicates. Group 1: untreated, unchallenged negative control group (NC); group 2: untreated, challenged positive control group (PC); group 3: negative control supplemented with salinomycin 66 mg/kg, challenged group (Sal); and group 4: negative control supplemented with probiotics, challenged (Prob mix). On d 15, all birds (except group 1) were challenged with approximately 75,000, 25,000, and 75,000 of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocytes, respectively, that were mixed into the feed. Feed conversion
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MATERIALS AND METHODS The experiment was conducted at Southern Poultry Research facilities, Athens, Georgia. The experimental house was divided into 12 pens of equal size, each hav-
ing an area of 3.72 m2, with fresh wood shavings as bedding with a thickness of approximately 10 cm on a concrete floor. Each pen has 1.5-m-high side walls with a 0.5-m bottom of solid wood to prevent bird migration. The temperature of the building was monitored daily and was optimum for the age of the birds according to the breed requirements. Illumination was provided by fluorescent bulbs placed above the pens. The experiment was conducted in accordance with the principles and specific guidelines presented in the Federation of Animal Science Societies (FASS, 2010).
Experimental Design and Dietary Treatments The birds were obtained from Cobb-Vantress hatchery, Cleveland, Georgia. Bird sexing and vaccination were done at the hatchery without administration of any coccidia vaccine. A total of 360 Cobb male broiler chickens were randomly allocated into 4 equal groups, 90 birds per group, each with 3 replicates. The pen was the experimental unit, all pens were numbered consecutively, and treatment numbers were identified on pen cards. The groups consisted of 1) negative control group (NC): commercial feed (Table 1), no additives, untreated, unchallenged; 2) positive control group (PC): commercial feed, no additives, untreated, challenged with a mixture of Eimeria species; 3) negative control supplemented with salinomycin group (Sal): 66 mg of salinomycin/kg of feed in the commercial feed, challenged with a mixture of Eimeria species; 4) negative control supplemented with probiotics mix group (Prob mix); commercial feed with probiotics and challenged with a mixture of Eimeria species.
Trial Diet and Water Supply The diets were provided ad libitum in one tube-type feeder per pen. From d 1 until 7, feed was also supplied on trays, directly placed on the litter. All feeds were fed as mash and were mixed and issued fresh weekly. Diet composition was appropriate for the birds’ growing stage (Table 1) and meets the NRC (1994) requirements. Unconsumed feed was weighed and discarded weekly. Each batch of feed was mixed and bagged separately. Each bag was identified with the study number, date of mixing, type of feed, and the correct treatment number. Complete records of feed mixing and test article inventories were maintained. Water was provided ad libitum from one Plasson-type automatic watering fount per pen.
Coccidia Species Challenge On d 15, all birds except NC were challenged with approximately 75,000, 25,000, and 75,000 of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oo-
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industry has been under constant pressure to reduce its dependence on antimicrobials including anticoccidial drugs. In addition, the development of drug resistance by the Eimeria species and also the public health concern about drug residues in poultry meat, eggs, and by-products are forcing the industry to look for alternatives. The development of the resistance or decreased sensitivity of the Eimeria species to chemotherapeutic agents was reported many years ago from different parts of the world including Europe and the Americas (Fallis et al., 1973; Duszynski and Box, 1978; Cawthorn et al., 1981), and it can cause a significant reduction in the effectiveness of these drugs. Some coccidia species develop lower resistance to certain drugs, but long-term exposure leads eventually to a loss of sensitivity and resistance. The main strategy to reduce developing such resistance is to use less intensive shuttle and rotation programs and incorporate other methods in controlling the disease. Relevant success is achieved with vaccines in controlling coccidiosis especially in broiler breeders. Nevertheless, it has not yet achieved satisfactory levels particularly in broilers because usage is limited by the possibility of adverse effects on feed efficiency and high production costs, especially when these vaccines include more than one Eimeria species. Yet another limiting factor for the use of vaccines against coccidia is that the inclusion of several species of Eimeria in one vaccine can cause further depression in weight gain and feed conversion, and a potential vaccine failure (Dalloul and Lillehoj, 2006). To facilitate the development of novel control strategies for coccidiosis, it is crucial to learn more about the interaction between Eimeria species and birds with a comprehensive understanding of the immune system of birds. Gut microflora play an important role in the bird immune system and are considered one of the first lines of defense against pathogens. Probiotic supplementation of gut microflora in humans and animals has been shown to increase gut defensive mechanisms against enteric pathogens. Probiotics have been studied and used extensively in the poultry industry for their benefits on birds’ performance, immunity, and protection against enteric diseases (Lutful-Kabir, 2009). However, there is still a paucity of literature discussing its use in controlling parasitic diseases such as coccidiosis (Tierney et al., 2004; Dalloul et al., 2005; Lee et al., 2007b, 2010a,b; Hume et al., 2011; Ritzi et al., 2014). The aim of this study was to evaluate and compare the potential protective effects of probiotics and salinomycin as feed additives on broiler performance and coccidiosis control under experimental settings.
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PROBIOTICS AND COCCIDIOSIS CONTROL IN BROILERS Table 1. Composition of the diets (as-fed basis): starter (d 0–20), grower (d 21–34), and finisher (d 35–42) Starter
Grower
Finisher
Ingredient, % Corn, yellow, ground Soybean meal (with 48% CP) Poultry fat Dicalcium phosphate Limestone Salt Vitamin premix1 dl-Methionine Trace mineral premix2 Contents by calculation ME, kcal/kg CP, % Digestible Lys, % Digestible Met, % Met + Cys, %
56.12 37.50 3.00 1.75 0.80 0.30 0.25 0.20 0.08 3,096 22.3 1.18 0.53 0.89
60.80 32.61 3.43 1.56 0.78 0.32 0.25 0.17 0.08 3,140 20.6 1.01 0.48 0.76
68.00 26.22 2.99 1.32 0.62 0.35 0.25 0.17 0.08 3,191 18.1 0.85 0.45 0.70
1Vitamin mix provided the following (per kg of diet): 2.4 mg of thiamine mononitrate; 44 mg of nicotinic acid; 4.4 mg of riboflavin; 12 mg of d-Ca pantothenate; 12.0 µg of vitamin B12 (cobalamin); 4.7 mg of pyridoxine∙HCl; 0.11 mg of d-biotin; 5.5 mg of folic acid; 3.34 mg of menadione sodium bisulfite complex; 220 mg of choline chloride; 27.5 µg of cholecalciferol; 6,306.6 IU of trans-retinyl acetate; 11 IU of all-rac α-tocopheryl acetate; 125 mg of ethoxyquin. 2Trace mineral mix provides the following (per kg of diet): 60 mg of manganese (MnSO ∙H O); 30 mg of iron 4 2 (FeSO4∙7H2O); 50 mg of zinc (ZnO); 5 mg of copper (CuSO4∙5H2O); 0.15 mg of iodine (ethylene diamine dihydroiodide); 0.3 mg of selenium (NaSeO3).
cytes, respectively. All coccidia used in this trial were contemporaneous and isolated from US commercial production farms in 2012. For each species, the dose was selected to produce a moderate coccidiosis infection. The oocysts were mixed together giving a total volume of 550 mL. Each inoculum had 30 mL and was mixed into the feed found at the base of the tube feeder of the challenged pens where each pen had 30 birds.
Feed Samples
Probiotics Mixture Preparation
Performance Parameters
The probiotic product that was used in the experiment is a multi-species, host-specific probiotic (PoultryStar-BIOMIN GmbH, Herzogenburg, Austria) and included Bifidobacterium animalis subspecies animalis DSM 16284, Lactobacillus salivarius subspecies salivarius DSM 16351, and Enterococcus faecium DSM 21913 with a cfu content of 5.00 × 1011 per kg. The product was mixed into the finished feed at an inclusion rate of 1 g per kg of feed, giving a final concentration of 5.00 × 108 cfu per kg of feed.
All birds were kept under observation for 42 d where performance parameters including feed intake, live BW, and feed conversion ratio (FCR), and health status were determined throughout the experiment on a weekly basis. Bird weights by pen were recorded on d 0 and weekly.
Trial Monitoring Birds and housing facilities were inspected twice daily. During the inspections, the following were checked and recorded: general health status, constant feed and water supply, temperature, removal of all dead birds, and observation of any unexpected events. The number of birds found dead during the study was noted on the daily mortality record, and the birds were not replaced. Pen number, mortality date, sex, weight, and diagnosis were recorded.
Feeds samples from the beginning (d 1), middle (d 14), and end (d 42) of each treatment were mixed to form a composite sample. Samples were taken from the composite for each treatment to ensure the minimum count (cfu) of the included probiotic mixture in group 4 (Prob mix).
Coccidiosis Lesion Scoring On d 21 and 42, 3 birds from each pen were selected, euthanized, and examined for the presence and degree of coccidiosis lesions. The upper, middle, and cecal regions of the intestinal tract were scored, using the system of Johnson and Reid (1970), where 0 is normal and 1, 2, 3, or 4 indicate increasing severity of infection.
Oocyst Counting On d 21, 28, 35, and 42, 10 fresh fecal samples were collected per pen, pooled, and kept in separate airtight plastic bags. After homogenization, samples were stored at 4°C until assessed for oocyst counts, which were de-
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Item
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Abdelrahman et al. Table 2. Feed intake (kg/bird), final BW (kg/bird), and feed conversion ratio (FCR) values on d 421 Item
NC
Feed intake* Live BW* FCR*
4.07 ± 2.31 ± 1.76 ±
PC
0.08a 0.08a 0.03b
3.91 ± 1.91 ± 2.04 ±
Sal
0.08a 0.03b 0.01a
3.93 ± 2.19 ± 1.79 ±
Prob mix
0.12a 0.03a 0.02b
3.65 ± 0.05b 2.01 ± 0.01b 1.81 ± 0.02b
a–cMeans
with no common superscript within a row are significantly different. = negative control group, PC = positive control group, Sal = salinomycin group, and Prob mix = probiotics mix group. *P < 0.003. 1NC
termined by dilution and counts via microscope using a McMaster counting chamber and stated as oocysts per gram of excreta.
ferences in the ANOVA were compared using the Tukey difference procedure (Snedecor and Cochran, 1967).
Performance Parameters
On d 21 and 42, litter conditions were graded. The following point scale was used to grade the quality of the litter/bedding: 0 = dry, friable material throughout the pen; 1 = predominantly dry material but with some evidence of crusting around drinkers and feeders; 2 = litter material is mostly acceptable but with some areas of wet shavings or capped material; 3 = poor quality litter material with a large proportion of wet areas and capping of the litter; 4 = unacceptable litter quality—wet and capped but with a few areas of dry material remaining; 5 = all litter is wet and soggy, no dry areas left.
The results of this study showed that the overall FCR values of NC, Sal, and Prob mix groups were significantly lower than the PC group, whereas the overall FCR values of the Prob mix and the Sal groups were similar (P ≥ 0.05). The results of this study showed that BW of birds in the Prob mix group at d 42 were similar to the PC group but significantly lower than the Sal and NC groups, whereas BW in the Sal group were significantly higher than the PC and Prob mix groups. Feed intake in the Prob mix group was significantly lower than in all other groups, whereas feed intake for birds in the Sal group was equal to the feed intake of PC birds (Table 2).
Disposal of Birds and Feed
Oocyst Count, Litter Scoring, Coccidia Lesions, and Mortality
All birds and feed were buried in the Southern Poultry Research’s pit as described in the Southern Poultry Research standard operating procedure.
Oocyst shedding at d 21 and 28, and the overall cumulative count were similar in the Prob mix and the Sal groups. On d 28, oocyst shedding in the Sal group was significantly lower than the PC group. On d 35 and 42, oocyst shedding was similar among all the groups (Table 3). Litter scores at d 21 and 42 were not different between PC, Sal, and Prob mix groups (Figure 1).
Statistical Analysis Data were analyzed by ANOVA using the GLM procedure of SPSS (IBM SPSS 19, IBM Corp., Armonk, NY). Pen was the experimental unit for feed intake and FCR, for live BW each animal was the experimental unit. The means for treatments showing significant dif-
Table 3. Oocyst count per gram of fecal material, lesion scores, and cumulative mortality1 Item Oocyst count d 21 d 28 d 35 d 42 Cumulative Lesion score at d 21 Upper IT Middle IT Ceca Mortality (birds/group) a,bMeans
NC
1,340 9,380 6,164 8,844 25,728
± ± ± ± ±
1,674b 5,092ab 3,091 4,254 6,034b
0.00b 0.00b 0.22 0
PC
± 38,603a ± 139,610a ± 1,608 ± 2,214 ± 164,358a 2.11a 2.11a 2.22 7
153,832 203,680 1,608 2,814 361,934
Sal
± 3,481ab ± 879b ± 1,608 ± 4,294 ± 1,360ab 0.78ab 1.67ab 0.89 9
30,150 3,082 1,608 5,896 40,736
Prob mix
32,160 ± 13,381ab 15,276 ± 5,428ab 0.00 402 ± 232 47,838 ± 15,079ab 1.33ab 1.56ab 2.22 9
with no common superscript within a row are significantly different (P ≤ 0.05). oocyst count per gram of fecal material (pooled per pen) on d 21, 28, 35, 42 and cumulative in the 4 groups of the trial. Lesion scores on d 21 at 3 different parts of the intestinal tract (IT). NC = negative control group, PC = positive control group, Sal = salinomycin group, and Prob mix = probiotics mix group. 1Weekly
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RESULTS
Litter Condition Scoring
PROBIOTICS AND COCCIDIOSIS CONTROL IN BROILERS
Intestinal lesion scores were similar at d 21 in PC, Sal, and Prob mix groups in upper and middle intestinal tract, and ceca. On d 42, no apparent intestinal lesions were recorded. Similarly, there were no significant differences in bird mortalities between the PC, Sal, and Prob mix groups (Table 3).
DISCUSSION The current work investigated the protective effect of the Enterococcus faecium, Bifidobacterium animalis, and Lactobacillus salivarius-based probiotic as a feed additive against the experimental challenge of broiler chickens with different Eimeria species in comparison with salinomycin. The FCR of the birds treated with probiotics was significantly lower than the challenged birds that did not receive probiotics or salinomycin but similar to the birds that were challenged and had received salinomycin. Similarly, the overall cumulative oocyst shedding in the birds treated with probiotics did not differ from the birds that received salinomycin. Moreover, intestinal lesion scores at d 21 were similar in both groups that were challenged and received probiotics or salinomycin,
which was also reflected in the birds’ mortality; there were no significant differences between the groups that received probiotics or salinomycin. The main finding of this study was that feeding probiotics or salinomycin to birds gave similar effects, where birds in both group shed less oocysts and had fewer intestinal lesions, which are indicative of healthier intestine. Intact intestine is crucial for proper nutrient absorption and utilization resulting in optimum health and performance. This could be attributed to the fact that Eimeria species are intracellular parasites that must invade the host epithelial cells to replicate. Probiotic bacteria may compete for attachment sites and occupy common receptors on the epithelial cells. This would reduce infiltration by motile Eimeria parasitic stages, and consequently, their replication and shedding. Another possible explanation is the well-documented immune modulation effect of probiotics (Dalloul et al., 2003a,b, 2005), which enhances the birds’ immune response against Eimeria, resulting in the protective effect of probiotics against the pathological effect of coccidiosis. Other researchers recorded similar results to our findings, where several experiments were conducted to study the effect of lactic acid-producing probiotic bacteria on reducing the clinical signs of coccidiosis was investigated by few authors. For instance, Pediococcus acidilactici improved weight gains significantly in birds challenged with E. acervulina or E. tenella in comparison with the positive control group. Eimeria-specific antibodies were higher in birds infected with E. tenella and treated with Pediococcus in comparison with other groups, although oocyst shedding and weight gains were not improved (Lee et al., 2007a). Similar to the results of this study, Dalloul and others investigated the effect of Lactobacillus-based probiotics in birds challenged experimentally with E. acervulina. They reported that feeding Lactobacillus-based probiotics reduced the oocyst shedding in infected birds in 3 different experiments by 20, 14, and 24%, which were significantly lower than the control groups. They also noticed that the Lactobacillus-fed birds had better immune responses to coccidia infection as shown by lower intestinal invasion by E. acervulina, higher interleukin-2 production, an increase in intestinal intraepithelial lymphocyte subpopulation (Dalloul et al., 2003a,b), and higher cytokine levels in comparison with control groups (Dalloul et al., 2005), which might explain the protective effect of probiotics against coccidiosis in our results. Not only in vivo, but also in vitro studies confirmed the protective action of lactic acid-producing probiotic bacteria such as Lactobacillus acidophilus and Lactobacillus salivarius against Eimeria species. Both Lactobacillus species inhibited the invasion of Eimeria tenella into Madin-Darby bovine kidney cells in vitro, which was explained due to their extracellular metabolic factors (Tierney et al., 2004). These findings highlight the positive effect of Lactobacillus-
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Figure 1. Litter scores on d 21 and 42. No significance differences were noted between probiotics mix and salinomycin groups on both days. NC = negative control group, PC = positive control group, Sal = salinomycin group, and Prob mix = probiotics mix group. a,bMeans with no common superscript are significantly different (P ≤ 0.05).
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and route, dose, and method of application of probiotics and also the possible interaction with Eimeria vaccines. To conclude, the results of this study showed that broilers that received the multi-species host-specific probiotic (PoultryStar) in their diet performed very similar to broilers that received salinomycin in terms of overall FCR, oocyst shedding, mortality, and intestinal lesions. The multi-species nature of the probiotic fosters activity in a wider range of conditions, similar to other multi-species probiotics, resulting in greater efficacy (Fuller, 1989; Timmerman et al., 2006). In the light of these results, it could be suggested that probiotics might be used as a complementary approach in coccidiostat shuttle and rotation programs to reduce coccidiosis incidence and severity, and to reduce the use of coccidiostats and subsequently the prevalence of Eimeria species resistance toward these compounds.
REFERENCES Burridge, M. J., W. J. Bigler, D. J. Forrester, and J. M. Hennemann. 1979. Serologic survey for Toxoplasma gondii in wild animals in Florida. J. Am. Vet. Med. Assoc. 175:964–967. Cawthorn, R. J., D. Rainnie, and G. Wobeser. 1981. Experimental transmission of Sarcocystis sp. (Protozoa: Sarcocystidae) between the shoveler (Anas clypeata) duck and the striped skunk (Mephitis mephitis). J. Wildl. Dis. 17:389–394. Dalloul, R. A., and H. S. Lillehoj. 2006. Poultry coccidiosis: recent advancements in control measures and vaccine development. Expert Rev. Vaccines 5:143–163. Dalloul, R. A., H. S. Lillehoj, T. A. Shellem, and J. A. Doerr. 2003a. Enhanced mucosal immunity against Eimeria acervulina in broilers fed a Lactobacillus-based probiotic. Poult. Sci. 82:62–66. Dalloul, R. A., H. S. Lillehoj, T. A. Shellem, and J. A. Doerr. 2003b. Intestinal immunomodulation by vitamin A deficiency and Lactobacillus-based probiotic in Eimeria acervulina-infected broiler chickens. Avian Dis. 47:1313–1320. Dalloul, R. A., H. S. Lillehoj, N. M. Tamim, T. A. Shellem, and J. A. Doerr. 2005. Induction of local protective immunity to Eimeria acervulina by a Lactobacillus-based probiotic. Comp. Immunol. Microbiol. Infect. Dis. 28:351–361. Duszynski, D. W., and E. D. Box. 1978. The opossum (Didelphis virginiana) as a host for Sarcocystis debonei from cowbirds (Molothrus ater) and grackles (Cassidix mexicanus, Quiscalus quiscula). J. Parasitol. 64:326–329. Fallis, A. M., R. L. Jacobson, and J. N. Raybould. 1973. Haematozoa in domestic chickens and guinea fowl in Tanzania and transmission of Leucocytozoon neavei and Leucocytozoon schoutedeni. J. Protozool. 20:438–442. FASS. 2010. Guide for the Care and Use of Agricultural Animals in Research and Teaching. 3rd ed. Fed. Anim. Sci. Soc., Champaign, IL. Fuller, R. 1989. Probiotics in man and animals. J. Appl. Bacteriol. 66:365–378. Giannenas, I., E. Papadopoulos, E. Tsalie, E. Triantafillou, S. Henikl, K. Teichmann, and D. Tontis. 2012. Assessment of dietary supplementation with probiotics on performance, intestinal morphology and microflora of chickens infected with Eimeria tenella. Vet. Parasitol. 188:31–40. Giannenas, I., E. Tsalie, E. Triantafillou, S. Hessenberger, K. Teichmann, M. Mohnl, and D. Tontis. 2014. 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. Avian Pathol. 43:209–216. Graat, E. A., A. M. Henken, H. W. Ploeger, J. P. Noordhuizen, and M. H. Vertommen. 1994. Rate and course of sporulation
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based probiotics on infected birds’ immune response leading to increased resistance to Eimeria species. Giannenas et al. (2012) evaluated the effect of feed dietary supplementation with different probiotics preparation given to experimentally challenged broilers with E. tenella sporulated oocysts. Birds were challenged with E. tenella and given the anticoccidial lasalocid at the level of 60 mg/kg of feed. The other groups were all challenged with E. tenella and were given the basal diet supplemented with different probiotic preparations including the multi-species probiotic mix (PoultryStar) at low (5.00 × 108 cfu/kg of diet) or high (5.00 × 109 cfu/kg of feed) inclusion rates. The overall performance of birds fed the multi-species probiotic mix at both inclusion rates was significantly higher than the infected control groups, whereas overall oocyst shedding was significantly lower in the lasalocid-treated group. Concurrent with our data, the latter group also showed significantly lower cecal lesion scores compared with the untreated infected group but did not differ from the probiotic mix group. Similarly, the same research group looked further at the effect of PoultryStar given via different routes (drinking water and feed) on the performance of broiler chickens experimentally challenged with sporulated oocysts of E. acervulina (5.00 × 104), E. maxima (2.00 × 104), and E. tenella (2.00 × 104) in comparison with lasalocid at 75 mg/kg. Our data came in agreement with what they found, where the inclusion of probiotics in feed resulted in considerable improvement in both growth performance and intestinal health in comparison with infected control birds and similar performance to that of the lasalocid group. On contrary to our findings, the probiotic groups had lesion scores and oocyst numbers higher than the lasalocid group. Interestingly, they found that birds in groups treated with probiotics had the highest villous height values in comparison with the other groups, which could not be done in this current work (Giannenas et al., 2014). One of the limitations of this study is the presence of oocysts in the fecal material of the negative control group. Every possible measure was taken into consideration to prevent this contamination and to keep the negative control group free from coccidia. However, this proved to be difficult due to the ubiquitous nature of Eimeria species and the possibility of mechanical transmission of Eimeria species (Graat et al., 1994), which highlights the importance of using an effective method to control the disease. Based on current knowledge, advances in probiotics methods, in line with the previously mentioned studies and our data, using probiotics as alternatives to conventional chemotherapeutics to control coccidiosis appears to be a possibility. However, it is more sensible to use probiotics as a complementary approach in shuttle and rotation programs to reduce disease incidences and the prevalence of Eimeria species resistance to anticoccidial drugs (Travers et al., 2011). In addition, more research is needed on the effect of administration time
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Lutful-Kabir, S. M. 2009. The role of probiotics in the poultry industry. Int. J. Mol. Sci. 10:3531–3546. McDougald, L. R., and S. H. Fitz-Coy. 2008. Protozoal infections. Pages 1067–1117 in Diseases of Poultry. Y. M. Saif, ed. Blackwell Publishing, Ames, IA. NRC. 1994. Nutrient Requirements of Poultry. 9th rev. ed. National Academy Press, Washington, DC. Ritzi, M. M., W. Abdelrahman, M. Mohnl, and R. A. Dalloul. 2014. Effects of probiotics and application methods on performance and response of broiler chickens to an Eimeria challenge. Poult. Sci. 93:2772–2778. http://dx.doi.org/10.3382/ps.2014-04207. Snedecor, G. W., and W. G. Cochran. 1967. Statistical Methods, 6th ed. Iowa State University Press, Ames. Tierney, J., H. Gowing, D. Van Sinderen, S. Flynn, L. Stanley, N. McHardy, S. Hallahan, and G. Mulcahy. 2004. In vitro inhibition of Eimeria tenella invasion by indigenous chicken Lactobacillus species. Vet. Parasitol. 122:171–182. Timmerman, H. M., A. Veldman, E. van den Elsen, F. M. Rombouts, and A. C. Beynen. 2006. Mortality and growth performance of broilers given drinking water supplemented with chicken-specific probiotics. Poult. Sci. 85:1383–1388. Travers, M. A., I. Florent, L. Kohl, and P. Grellier. 2011. Probiotics for the control of parasites: An overview. J. Parasitol. Res. 2011:610769. Williams, R. B. 1999. A compartmentalised model for the estimation of the cost of coccidiosis to the world’s chicken production industry. Int. J. Parasitol. 29:1209–1229.
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of oocysts of Eimeria acervulina under different environmental conditions. Parasitology 108:497–502. Hume, M. E., N. A. Barbosa, S. E. Dowd, N. K. Sakomura, A. G. Nalian, A. Martynova-Van Kley, and E. O. Oviedo-Rondon. 2011. Use of pyrosequencing and denaturing gradient gel electrophoresis to examine the effects of probiotics and essential oil blends on digestive microflora in broilers under mixed Eimeria infection. Foodborne Pathog. Dis. 8:1159–1167. Johnson, J., and W. M. Reid. 1970. Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments with chickens. Exp. Parasitol. 28:30–36. Lee, K. W., S. H. Lee, H. S. Lillehoj, G. X. Li, S. I. Jang, U. S. Babu, M. S. Park, D. K. Kim, E. P. Lillehoj, A. P. Neumann, T. G. Rehberger, and G. R. Siragusa. 2010a. Effects of direct-fed microbials on growth performance, gut morphometry, and immune characteristics in broiler chickens. Poult. Sci. 89:203–216. Lee, K. W., H. S. Lillehoj, S. I. Jang, G. Li, S. H. Lee, E. P. Lillehoj, and G. R. Siragusa. 2010b. Effect of Bacillus-based direct-fed microbials on Eimeria maxima infection in broiler chickens. Comp. Immunol. Microbiol. Infect. Dis. 33:e105–e110. Lee, S., H. S. Lillehoj, D. W. Park, Y. H. Hong, and J. J. Lin. 2007a. Effects of Pediococcus- and Saccharomyces-based probiotic (MitoMax) on coccidiosis in broiler chickens. Comp. Immunol. Microbiol. Infect. Dis. 30:261–268. Lee, S. H., H. S. Lillehoj, R. A. Dalloul, D. W. Park, Y. H. Hong, and J. J. Lin. 2007b. Influence of Pediococcus-based probiotic on coccidiosis in broiler chickens. Poult. Sci. 86:63–66.
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Key words: probiotic, salinomycin, coccidiosis, broiler, performance 2014 Poultry Science 93:1–7 http://dx.doi.org/10.3382/ps.2014-04212
INTRODUCTION
increased susceptibility to other diseases such as necrotic enteritis (Burridge et al., 1979; mcDougald and Fitz-Coy, 2008). Coccidia are ubiquitous protozoa that are present wherever chicken are raised, which exclude the possibility of controlling the disease by quarantine or disinfection. Control of coccidia is greatly dependent on the use of chemotherapeutic agents. The use of chemotherapeutics to control these protozoa has proven to be successful in many parts of the world due to their ease of use and the ability to provide uniform treatment and prevention. Nevertheless, these drugs, on some occasions, can be toxic to birds due to poor feed mixing, incorrect identification of the premix or the feed, or cross-contamination. They also have to be rotated from time to time to minimize any possible resistance developed against them by Eimeria species. Despite the global acceptance and success of these drugs in controlling a costly avian disease, the poultry
Coccidiosis is a significant enteric disease in poultry with the potential to inflict a considerable economic effect on farm profitability. The financial loss to the poultry industry as a result of coccidiosis worldwide has been estimated annually at US $3 billion, which is mainly due to prophylactic or therapeutic in-feed medications and also as a result of the effect of the disease on birds’ health (Williams, 1999; Dalloul and Lillehoj, 2006). Eimeria species multiply in the bird’s intestinal tract, causing considerable tissue damage and subsequently impaired nutrient absorption and blood loss. In addition, it can cause diarrhea, dehydration, mortality, transient drop in egg production in laying flocks and ©2014 Poultry Science Association Inc. Received may 27, 2014. Accepted September 10, 2014. 1 Corresponding author: [email protected]
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ABSTRACT The annual financial loss to the poultry industry as a result of coccidiosis has been estimated at about US $3 billion. The objective of this study was to evaluate and compare the effects of probiotics and salinomycin as feed additives on performance and coccidiosis control in male broilers raised to 42 d of age. The study consisted of 360 Cobb male broiler chickens randomly allocated to 4 groups each with 3 replicates. Group 1: untreated, unchallenged negative control group (NC); group 2: untreated, challenged positive control group (PC); group 3: negative control supplemented with salinomycin 66 mg/kg, challenged group (Sal); and group 4: negative control supplemented with probiotics, challenged (Prob mix). On d 15, all birds (except group 1) were challenged with approximately 75,000, 25,000, and 75,000 of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocytes, respectively, that were mixed into the feed. Feed conversion
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MATERIALS AND METHODS The experiment was conducted at Southern Poultry Research facilities, Athens, Georgia. The experimental house was divided into 12 pens of equal size, each hav-
ing an area of 3.72 m2, with fresh wood shavings as bedding with a thickness of approximately 10 cm on a concrete floor. Each pen has 1.5-m-high side walls with a 0.5-m bottom of solid wood to prevent bird migration. The temperature of the building was monitored daily and was optimum for the age of the birds according to the breed requirements. Illumination was provided by fluorescent bulbs placed above the pens. The experiment was conducted in accordance with the principles and specific guidelines presented in the Federation of Animal Science Societies (FASS, 2010).
Experimental Design and Dietary Treatments The birds were obtained from Cobb-Vantress hatchery, Cleveland, Georgia. Bird sexing and vaccination were done at the hatchery without administration of any coccidia vaccine. A total of 360 Cobb male broiler chickens were randomly allocated into 4 equal groups, 90 birds per group, each with 3 replicates. The pen was the experimental unit, all pens were numbered consecutively, and treatment numbers were identified on pen cards. The groups consisted of 1) negative control group (NC): commercial feed (Table 1), no additives, untreated, unchallenged; 2) positive control group (PC): commercial feed, no additives, untreated, challenged with a mixture of Eimeria species; 3) negative control supplemented with salinomycin group (Sal): 66 mg of salinomycin/kg of feed in the commercial feed, challenged with a mixture of Eimeria species; 4) negative control supplemented with probiotics mix group (Prob mix); commercial feed with probiotics and challenged with a mixture of Eimeria species.
Trial Diet and Water Supply The diets were provided ad libitum in one tube-type feeder per pen. From d 1 until 7, feed was also supplied on trays, directly placed on the litter. All feeds were fed as mash and were mixed and issued fresh weekly. Diet composition was appropriate for the birds’ growing stage (Table 1) and meets the NRC (1994) requirements. Unconsumed feed was weighed and discarded weekly. Each batch of feed was mixed and bagged separately. Each bag was identified with the study number, date of mixing, type of feed, and the correct treatment number. Complete records of feed mixing and test article inventories were maintained. Water was provided ad libitum from one Plasson-type automatic watering fount per pen.
Coccidia Species Challenge On d 15, all birds except NC were challenged with approximately 75,000, 25,000, and 75,000 of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oo-
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industry has been under constant pressure to reduce its dependence on antimicrobials including anticoccidial drugs. In addition, the development of drug resistance by the Eimeria species and also the public health concern about drug residues in poultry meat, eggs, and by-products are forcing the industry to look for alternatives. The development of the resistance or decreased sensitivity of the Eimeria species to chemotherapeutic agents was reported many years ago from different parts of the world including Europe and the Americas (Fallis et al., 1973; Duszynski and Box, 1978; Cawthorn et al., 1981), and it can cause a significant reduction in the effectiveness of these drugs. Some coccidia species develop lower resistance to certain drugs, but long-term exposure leads eventually to a loss of sensitivity and resistance. The main strategy to reduce developing such resistance is to use less intensive shuttle and rotation programs and incorporate other methods in controlling the disease. Relevant success is achieved with vaccines in controlling coccidiosis especially in broiler breeders. Nevertheless, it has not yet achieved satisfactory levels particularly in broilers because usage is limited by the possibility of adverse effects on feed efficiency and high production costs, especially when these vaccines include more than one Eimeria species. Yet another limiting factor for the use of vaccines against coccidia is that the inclusion of several species of Eimeria in one vaccine can cause further depression in weight gain and feed conversion, and a potential vaccine failure (Dalloul and Lillehoj, 2006). To facilitate the development of novel control strategies for coccidiosis, it is crucial to learn more about the interaction between Eimeria species and birds with a comprehensive understanding of the immune system of birds. Gut microflora play an important role in the bird immune system and are considered one of the first lines of defense against pathogens. Probiotic supplementation of gut microflora in humans and animals has been shown to increase gut defensive mechanisms against enteric pathogens. Probiotics have been studied and used extensively in the poultry industry for their benefits on birds’ performance, immunity, and protection against enteric diseases (Lutful-Kabir, 2009). However, there is still a paucity of literature discussing its use in controlling parasitic diseases such as coccidiosis (Tierney et al., 2004; Dalloul et al., 2005; Lee et al., 2007b, 2010a,b; Hume et al., 2011; Ritzi et al., 2014). The aim of this study was to evaluate and compare the potential protective effects of probiotics and salinomycin as feed additives on broiler performance and coccidiosis control under experimental settings.
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PROBIOTICS AND COCCIDIOSIS CONTROL IN BROILERS Table 1. Composition of the diets (as-fed basis): starter (d 0–20), grower (d 21–34), and finisher (d 35–42) Starter
Grower
Finisher
Ingredient, % Corn, yellow, ground Soybean meal (with 48% CP) Poultry fat Dicalcium phosphate Limestone Salt Vitamin premix1 dl-Methionine Trace mineral premix2 Contents by calculation ME, kcal/kg CP, % Digestible Lys, % Digestible Met, % Met + Cys, %
56.12 37.50 3.00 1.75 0.80 0.30 0.25 0.20 0.08 3,096 22.3 1.18 0.53 0.89
60.80 32.61 3.43 1.56 0.78 0.32 0.25 0.17 0.08 3,140 20.6 1.01 0.48 0.76
68.00 26.22 2.99 1.32 0.62 0.35 0.25 0.17 0.08 3,191 18.1 0.85 0.45 0.70
1Vitamin mix provided the following (per kg of diet): 2.4 mg of thiamine mononitrate; 44 mg of nicotinic acid; 4.4 mg of riboflavin; 12 mg of d-Ca pantothenate; 12.0 µg of vitamin B12 (cobalamin); 4.7 mg of pyridoxine∙HCl; 0.11 mg of d-biotin; 5.5 mg of folic acid; 3.34 mg of menadione sodium bisulfite complex; 220 mg of choline chloride; 27.5 µg of cholecalciferol; 6,306.6 IU of trans-retinyl acetate; 11 IU of all-rac α-tocopheryl acetate; 125 mg of ethoxyquin. 2Trace mineral mix provides the following (per kg of diet): 60 mg of manganese (MnSO ∙H O); 30 mg of iron 4 2 (FeSO4∙7H2O); 50 mg of zinc (ZnO); 5 mg of copper (CuSO4∙5H2O); 0.15 mg of iodine (ethylene diamine dihydroiodide); 0.3 mg of selenium (NaSeO3).
cytes, respectively. All coccidia used in this trial were contemporaneous and isolated from US commercial production farms in 2012. For each species, the dose was selected to produce a moderate coccidiosis infection. The oocysts were mixed together giving a total volume of 550 mL. Each inoculum had 30 mL and was mixed into the feed found at the base of the tube feeder of the challenged pens where each pen had 30 birds.
Feed Samples
Probiotics Mixture Preparation
Performance Parameters
The probiotic product that was used in the experiment is a multi-species, host-specific probiotic (PoultryStar-BIOMIN GmbH, Herzogenburg, Austria) and included Bifidobacterium animalis subspecies animalis DSM 16284, Lactobacillus salivarius subspecies salivarius DSM 16351, and Enterococcus faecium DSM 21913 with a cfu content of 5.00 × 1011 per kg. The product was mixed into the finished feed at an inclusion rate of 1 g per kg of feed, giving a final concentration of 5.00 × 108 cfu per kg of feed.
All birds were kept under observation for 42 d where performance parameters including feed intake, live BW, and feed conversion ratio (FCR), and health status were determined throughout the experiment on a weekly basis. Bird weights by pen were recorded on d 0 and weekly.
Trial Monitoring Birds and housing facilities were inspected twice daily. During the inspections, the following were checked and recorded: general health status, constant feed and water supply, temperature, removal of all dead birds, and observation of any unexpected events. The number of birds found dead during the study was noted on the daily mortality record, and the birds were not replaced. Pen number, mortality date, sex, weight, and diagnosis were recorded.
Feeds samples from the beginning (d 1), middle (d 14), and end (d 42) of each treatment were mixed to form a composite sample. Samples were taken from the composite for each treatment to ensure the minimum count (cfu) of the included probiotic mixture in group 4 (Prob mix).
Coccidiosis Lesion Scoring On d 21 and 42, 3 birds from each pen were selected, euthanized, and examined for the presence and degree of coccidiosis lesions. The upper, middle, and cecal regions of the intestinal tract were scored, using the system of Johnson and Reid (1970), where 0 is normal and 1, 2, 3, or 4 indicate increasing severity of infection.
Oocyst Counting On d 21, 28, 35, and 42, 10 fresh fecal samples were collected per pen, pooled, and kept in separate airtight plastic bags. After homogenization, samples were stored at 4°C until assessed for oocyst counts, which were de-
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Item
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Abdelrahman et al. Table 2. Feed intake (kg/bird), final BW (kg/bird), and feed conversion ratio (FCR) values on d 421 Item
NC
Feed intake* Live BW* FCR*
4.07 ± 2.31 ± 1.76 ±
PC
0.08a 0.08a 0.03b
3.91 ± 1.91 ± 2.04 ±
Sal
0.08a 0.03b 0.01a
3.93 ± 2.19 ± 1.79 ±
Prob mix
0.12a 0.03a 0.02b
3.65 ± 0.05b 2.01 ± 0.01b 1.81 ± 0.02b
a–cMeans
with no common superscript within a row are significantly different. = negative control group, PC = positive control group, Sal = salinomycin group, and Prob mix = probiotics mix group. *P < 0.003. 1NC
termined by dilution and counts via microscope using a McMaster counting chamber and stated as oocysts per gram of excreta.
ferences in the ANOVA were compared using the Tukey difference procedure (Snedecor and Cochran, 1967).
Performance Parameters
On d 21 and 42, litter conditions were graded. The following point scale was used to grade the quality of the litter/bedding: 0 = dry, friable material throughout the pen; 1 = predominantly dry material but with some evidence of crusting around drinkers and feeders; 2 = litter material is mostly acceptable but with some areas of wet shavings or capped material; 3 = poor quality litter material with a large proportion of wet areas and capping of the litter; 4 = unacceptable litter quality—wet and capped but with a few areas of dry material remaining; 5 = all litter is wet and soggy, no dry areas left.
The results of this study showed that the overall FCR values of NC, Sal, and Prob mix groups were significantly lower than the PC group, whereas the overall FCR values of the Prob mix and the Sal groups were similar (P ≥ 0.05). The results of this study showed that BW of birds in the Prob mix group at d 42 were similar to the PC group but significantly lower than the Sal and NC groups, whereas BW in the Sal group were significantly higher than the PC and Prob mix groups. Feed intake in the Prob mix group was significantly lower than in all other groups, whereas feed intake for birds in the Sal group was equal to the feed intake of PC birds (Table 2).
Disposal of Birds and Feed
Oocyst Count, Litter Scoring, Coccidia Lesions, and Mortality
All birds and feed were buried in the Southern Poultry Research’s pit as described in the Southern Poultry Research standard operating procedure.
Oocyst shedding at d 21 and 28, and the overall cumulative count were similar in the Prob mix and the Sal groups. On d 28, oocyst shedding in the Sal group was significantly lower than the PC group. On d 35 and 42, oocyst shedding was similar among all the groups (Table 3). Litter scores at d 21 and 42 were not different between PC, Sal, and Prob mix groups (Figure 1).
Statistical Analysis Data were analyzed by ANOVA using the GLM procedure of SPSS (IBM SPSS 19, IBM Corp., Armonk, NY). Pen was the experimental unit for feed intake and FCR, for live BW each animal was the experimental unit. The means for treatments showing significant dif-
Table 3. Oocyst count per gram of fecal material, lesion scores, and cumulative mortality1 Item Oocyst count d 21 d 28 d 35 d 42 Cumulative Lesion score at d 21 Upper IT Middle IT Ceca Mortality (birds/group) a,bMeans
NC
1,340 9,380 6,164 8,844 25,728
± ± ± ± ±
1,674b 5,092ab 3,091 4,254 6,034b
0.00b 0.00b 0.22 0
PC
± 38,603a ± 139,610a ± 1,608 ± 2,214 ± 164,358a 2.11a 2.11a 2.22 7
153,832 203,680 1,608 2,814 361,934
Sal
± 3,481ab ± 879b ± 1,608 ± 4,294 ± 1,360ab 0.78ab 1.67ab 0.89 9
30,150 3,082 1,608 5,896 40,736
Prob mix
32,160 ± 13,381ab 15,276 ± 5,428ab 0.00 402 ± 232 47,838 ± 15,079ab 1.33ab 1.56ab 2.22 9
with no common superscript within a row are significantly different (P ≤ 0.05). oocyst count per gram of fecal material (pooled per pen) on d 21, 28, 35, 42 and cumulative in the 4 groups of the trial. Lesion scores on d 21 at 3 different parts of the intestinal tract (IT). NC = negative control group, PC = positive control group, Sal = salinomycin group, and Prob mix = probiotics mix group. 1Weekly
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RESULTS
Litter Condition Scoring
PROBIOTICS AND COCCIDIOSIS CONTROL IN BROILERS
Intestinal lesion scores were similar at d 21 in PC, Sal, and Prob mix groups in upper and middle intestinal tract, and ceca. On d 42, no apparent intestinal lesions were recorded. Similarly, there were no significant differences in bird mortalities between the PC, Sal, and Prob mix groups (Table 3).
DISCUSSION The current work investigated the protective effect of the Enterococcus faecium, Bifidobacterium animalis, and Lactobacillus salivarius-based probiotic as a feed additive against the experimental challenge of broiler chickens with different Eimeria species in comparison with salinomycin. The FCR of the birds treated with probiotics was significantly lower than the challenged birds that did not receive probiotics or salinomycin but similar to the birds that were challenged and had received salinomycin. Similarly, the overall cumulative oocyst shedding in the birds treated with probiotics did not differ from the birds that received salinomycin. Moreover, intestinal lesion scores at d 21 were similar in both groups that were challenged and received probiotics or salinomycin,
which was also reflected in the birds’ mortality; there were no significant differences between the groups that received probiotics or salinomycin. The main finding of this study was that feeding probiotics or salinomycin to birds gave similar effects, where birds in both group shed less oocysts and had fewer intestinal lesions, which are indicative of healthier intestine. Intact intestine is crucial for proper nutrient absorption and utilization resulting in optimum health and performance. This could be attributed to the fact that Eimeria species are intracellular parasites that must invade the host epithelial cells to replicate. Probiotic bacteria may compete for attachment sites and occupy common receptors on the epithelial cells. This would reduce infiltration by motile Eimeria parasitic stages, and consequently, their replication and shedding. Another possible explanation is the well-documented immune modulation effect of probiotics (Dalloul et al., 2003a,b, 2005), which enhances the birds’ immune response against Eimeria, resulting in the protective effect of probiotics against the pathological effect of coccidiosis. Other researchers recorded similar results to our findings, where several experiments were conducted to study the effect of lactic acid-producing probiotic bacteria on reducing the clinical signs of coccidiosis was investigated by few authors. For instance, Pediococcus acidilactici improved weight gains significantly in birds challenged with E. acervulina or E. tenella in comparison with the positive control group. Eimeria-specific antibodies were higher in birds infected with E. tenella and treated with Pediococcus in comparison with other groups, although oocyst shedding and weight gains were not improved (Lee et al., 2007a). Similar to the results of this study, Dalloul and others investigated the effect of Lactobacillus-based probiotics in birds challenged experimentally with E. acervulina. They reported that feeding Lactobacillus-based probiotics reduced the oocyst shedding in infected birds in 3 different experiments by 20, 14, and 24%, which were significantly lower than the control groups. They also noticed that the Lactobacillus-fed birds had better immune responses to coccidia infection as shown by lower intestinal invasion by E. acervulina, higher interleukin-2 production, an increase in intestinal intraepithelial lymphocyte subpopulation (Dalloul et al., 2003a,b), and higher cytokine levels in comparison with control groups (Dalloul et al., 2005), which might explain the protective effect of probiotics against coccidiosis in our results. Not only in vivo, but also in vitro studies confirmed the protective action of lactic acid-producing probiotic bacteria such as Lactobacillus acidophilus and Lactobacillus salivarius against Eimeria species. Both Lactobacillus species inhibited the invasion of Eimeria tenella into Madin-Darby bovine kidney cells in vitro, which was explained due to their extracellular metabolic factors (Tierney et al., 2004). These findings highlight the positive effect of Lactobacillus-
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Figure 1. Litter scores on d 21 and 42. No significance differences were noted between probiotics mix and salinomycin groups on both days. NC = negative control group, PC = positive control group, Sal = salinomycin group, and Prob mix = probiotics mix group. a,bMeans with no common superscript are significantly different (P ≤ 0.05).
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and route, dose, and method of application of probiotics and also the possible interaction with Eimeria vaccines. To conclude, the results of this study showed that broilers that received the multi-species host-specific probiotic (PoultryStar) in their diet performed very similar to broilers that received salinomycin in terms of overall FCR, oocyst shedding, mortality, and intestinal lesions. The multi-species nature of the probiotic fosters activity in a wider range of conditions, similar to other multi-species probiotics, resulting in greater efficacy (Fuller, 1989; Timmerman et al., 2006). In the light of these results, it could be suggested that probiotics might be used as a complementary approach in coccidiostat shuttle and rotation programs to reduce coccidiosis incidence and severity, and to reduce the use of coccidiostats and subsequently the prevalence of Eimeria species resistance toward these compounds.
REFERENCES Burridge, M. J., W. J. Bigler, D. J. Forrester, and J. M. Hennemann. 1979. Serologic survey for Toxoplasma gondii in wild animals in Florida. J. Am. Vet. Med. Assoc. 175:964–967. Cawthorn, R. J., D. Rainnie, and G. Wobeser. 1981. Experimental transmission of Sarcocystis sp. (Protozoa: Sarcocystidae) between the shoveler (Anas clypeata) duck and the striped skunk (Mephitis mephitis). J. Wildl. Dis. 17:389–394. Dalloul, R. A., and H. S. Lillehoj. 2006. Poultry coccidiosis: recent advancements in control measures and vaccine development. Expert Rev. Vaccines 5:143–163. Dalloul, R. A., H. S. Lillehoj, T. A. Shellem, and J. A. Doerr. 2003a. Enhanced mucosal immunity against Eimeria acervulina in broilers fed a Lactobacillus-based probiotic. Poult. Sci. 82:62–66. Dalloul, R. A., H. S. Lillehoj, T. A. Shellem, and J. A. Doerr. 2003b. Intestinal immunomodulation by vitamin A deficiency and Lactobacillus-based probiotic in Eimeria acervulina-infected broiler chickens. Avian Dis. 47:1313–1320. Dalloul, R. A., H. S. Lillehoj, N. M. Tamim, T. A. Shellem, and J. A. Doerr. 2005. Induction of local protective immunity to Eimeria acervulina by a Lactobacillus-based probiotic. Comp. Immunol. Microbiol. Infect. Dis. 28:351–361. Duszynski, D. W., and E. D. Box. 1978. The opossum (Didelphis virginiana) as a host for Sarcocystis debonei from cowbirds (Molothrus ater) and grackles (Cassidix mexicanus, Quiscalus quiscula). J. Parasitol. 64:326–329. Fallis, A. M., R. L. Jacobson, and J. N. Raybould. 1973. Haematozoa in domestic chickens and guinea fowl in Tanzania and transmission of Leucocytozoon neavei and Leucocytozoon schoutedeni. J. Protozool. 20:438–442. FASS. 2010. Guide for the Care and Use of Agricultural Animals in Research and Teaching. 3rd ed. Fed. Anim. Sci. Soc., Champaign, IL. Fuller, R. 1989. Probiotics in man and animals. J. Appl. Bacteriol. 66:365–378. Giannenas, I., E. Papadopoulos, E. Tsalie, E. Triantafillou, S. Henikl, K. Teichmann, and D. Tontis. 2012. Assessment of dietary supplementation with probiotics on performance, intestinal morphology and microflora of chickens infected with Eimeria tenella. Vet. Parasitol. 188:31–40. Giannenas, I., E. Tsalie, E. Triantafillou, S. Hessenberger, K. Teichmann, M. Mohnl, and D. Tontis. 2014. 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. Avian Pathol. 43:209–216. Graat, E. A., A. M. Henken, H. W. Ploeger, J. P. Noordhuizen, and M. H. Vertommen. 1994. Rate and course of sporulation
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based probiotics on infected birds’ immune response leading to increased resistance to Eimeria species. Giannenas et al. (2012) evaluated the effect of feed dietary supplementation with different probiotics preparation given to experimentally challenged broilers with E. tenella sporulated oocysts. Birds were challenged with E. tenella and given the anticoccidial lasalocid at the level of 60 mg/kg of feed. The other groups were all challenged with E. tenella and were given the basal diet supplemented with different probiotic preparations including the multi-species probiotic mix (PoultryStar) at low (5.00 × 108 cfu/kg of diet) or high (5.00 × 109 cfu/kg of feed) inclusion rates. The overall performance of birds fed the multi-species probiotic mix at both inclusion rates was significantly higher than the infected control groups, whereas overall oocyst shedding was significantly lower in the lasalocid-treated group. Concurrent with our data, the latter group also showed significantly lower cecal lesion scores compared with the untreated infected group but did not differ from the probiotic mix group. Similarly, the same research group looked further at the effect of PoultryStar given via different routes (drinking water and feed) on the performance of broiler chickens experimentally challenged with sporulated oocysts of E. acervulina (5.00 × 104), E. maxima (2.00 × 104), and E. tenella (2.00 × 104) in comparison with lasalocid at 75 mg/kg. Our data came in agreement with what they found, where the inclusion of probiotics in feed resulted in considerable improvement in both growth performance and intestinal health in comparison with infected control birds and similar performance to that of the lasalocid group. On contrary to our findings, the probiotic groups had lesion scores and oocyst numbers higher than the lasalocid group. Interestingly, they found that birds in groups treated with probiotics had the highest villous height values in comparison with the other groups, which could not be done in this current work (Giannenas et al., 2014). One of the limitations of this study is the presence of oocysts in the fecal material of the negative control group. Every possible measure was taken into consideration to prevent this contamination and to keep the negative control group free from coccidia. However, this proved to be difficult due to the ubiquitous nature of Eimeria species and the possibility of mechanical transmission of Eimeria species (Graat et al., 1994), which highlights the importance of using an effective method to control the disease. Based on current knowledge, advances in probiotics methods, in line with the previously mentioned studies and our data, using probiotics as alternatives to conventional chemotherapeutics to control coccidiosis appears to be a possibility. However, it is more sensible to use probiotics as a complementary approach in shuttle and rotation programs to reduce disease incidences and the prevalence of Eimeria species resistance to anticoccidial drugs (Travers et al., 2011). In addition, more research is needed on the effect of administration time
PROBIOTICS AND COCCIDIOSIS CONTROL IN BROILERS
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