https://orcid.org/0000-0002-2513-8538Abstract
Background: Besides the anticoccidial drug resistance problem, increasing consumer concerns about food safety and residues have propelled the quest for alternative prevention and control strategies amongst which phytotherapy has gained appeal due to a renewed interest in natural medicine.Objective: The objective was in vivo screening of four phytochemicals/extracts and a fungal immunomodulatory protein (FIP) against an Eimeria acervulina infection in broilers.Animals and methods: Four phytochemicals/extracts (extract from Echinacea purpurea, betaine (Betain™), curcumin, carvacrol (two different doses)), and a recombinant FIP from Ganoderma lucidum cloned and expressed in Escherichia coli were investigated for their anticoccidial potential. The experiment was conducted in a battery cage trial with 54 cages of eight birds each. Broilers infected with E. acervulina (a low and high infection dose of 104 and 105 sporulated oocysts, respectively) and treated with the phytochemicals/extracts or the FIP were compared with broilers treated with the anticoccidial salinomycin sodium (Sacox®) and with an untreated uninfected and an untreated infected control group. Coccidiosis lesion scores, body weight gains and oocyst shedding were used as parameters.Results: The results showed a coccidiosis infection dose effect on the mean coccidiosis lesion scores. The phytochemicals/extracts and the FIP failed to reduce coccidiosis lesion scores and oocyst shedding, while salinomycin efficiently controlled the E. acervulina infection and enabled significantly higher body weight gains.Conclusion: In conclusion, the selected phytochemicals/extracts and the FIP did not reduce the lesions of an experimentally induced E. acervulina infection.
1. Introduction
Coccidiosis is a disease of poultry with great economic impact, which is generally controlled using anticoccidial drugs. However, the efficacy of these compounds is jeopardized due to the worldwide occurrence of resistance (Chapman Citation1997; Peek & Landman Citation2003, Citation2004, Citation2006) which will most probably further increase in the future if the large scale use of anticoccidial drugs is continued. The losses inflicted by coccidiosis (higher feed conversion, growth depression and increased mortality) to commercial poultry worldwide have been estimated at €2 billion per annum, stressing the urgent need for more efficient strategies to control this parasite (Williams Citation1999; Shirley et al. Citation2005).
Besides the anticoccidial drug resistance problem, increasing consumer concerns about food safety and residues have propelled the quest for alternative prevention and control strategies amongst which phytotherapy has gained appeal due to a renewed interest in natural medicine. However, although numerous studies have shown antiparasitic efficacy in vitro, in vivo evidence is scarce (Windisch et al. Citation2008; Cravotto et al. Citation2009; Burt et al. Citation2013). Therefore, based on a recent literature study (Peek & Landman Citation2011), an in vivo screening of four phytochemicals/extracts and a fungal immunomodulatory protein (FIP) (Guo et al. Citation2004, Citation2005; Dalloul et al. Citation2006) against coccidiosis was performed. Betaine from Beta vulgaris (Augustine et al. Citation1997; Matthews et al. Citation1997; Waldenstedt et al. Citation1999; Matthews & Southern Citation2000; Klasing et al. Citation2002; Fetterer et al. Citation2003; Eklund et al. Citation2005), curcumin from Curcuma longa (Allen et al. Citation1998; Khalafalla et al. Citation2011), root extract from Echinacea purpurea (Allen Citation2003) or carvacrol from Origanum vulgare (Giannenas et al. Citation2003; Oviedo-Rondón et al. Citation2006) were chosen as candidate preventive anticoccidiosis treatment. Echinacea (Allen et al. Citation1998; Allen Citation2003) and betaine (Augustine et al. Citation1997; Matthews et al. Citation1997; Matthews & Southern Citation2000; Waldenstedt et al. Citation1999; Klasing et al. Citation2002; Fetterer et al. Citation2003) were previously reported to be active against coccidiosis, while curcumin (Allen et al. Citation1998) and carvacrol (Giannenas et al. Citation2003; Oviedo-Rondón et al. Citation2006) have been reported to induce immunomodulatory stress proteins. Anticoccidial effects of mushroom extracts were demonstrated earlier also (Guo et al. Citation2004, Citation2005; Dalloul et al. Citation2006) of which some might be ascribed to FIPs, which have shown immunomodulating properties in in vitro and in vivo studies (Liu et al. Citation2003; Lin et al. Citation2009; Wichers Citation2009).
The phytochemicals/extracts and the FIPs were studied using a battery cage trial in which their effect on an experimentally induced Eimeria acervulina infection was compared to a traditional prevention anticoccidial product (i.e. salinomycin sodium, Sacox®).
2. Material and methods
2.1. Phytochemicals/extracts, FIP and anticoccidial drug
2.1.1. Phytochemicals/extracts
Standardized extract of E. purpurea root (E. purpurea radix, 4:1, standardized to 6% total polyphenols, NatuurApotheek, Pijnacker, the Netherlands), betaine (Betain™ 96, batch nr: 812040000, Trouw Nutrition, Putten, the Netherlands), curcumin (Curcuma longa (turmeric) powder (batch nr: C1386, Sigma-Aldrich Chemie B.V., Zwijndrecht, the Netherlands)) and carvacrol (carvacrol ≥98%, FCC, Kosher, batch nr: W224502, Sigma-Aldrich Chemie B.V., Zwijndrecht, the Netherlands) were used.
2.1.2. Fungal immunomodulatory protein
Cloning of the FIP LZ9 and production details have been described previously (Peek et al. Citation2013). Briefly, based on degenerated primers designed towards the LZ8 gene, encoding an FIP of Ganoderma lucidum (Kino et al. Citation1989), a fragment was amplified, cloned and sequenced. The cloned FIP sequence had 88% homology to the LZ8 gene and was named LZ9. The gene was cloned in pET101 TOP expression vector and fused in frame to the V5/HIS tag. Escherichia coli BL21Star cells were used for expression. A crude lysate of recombinant bacteria was used to add to the feed. This crude lysate will mostly contain E. coli derived compounds that might have an influence on the E. acervulina infection. Therefore, a control feed sample was prepared using a transgenic E. coli BL21 strain expressing an FIP with a point mutation in the start of the gene introducing a frame shift and a premature stop of the protein. This sample could be used as a control for the FIP responses and was named FIP control.
2.1.3. Anticoccidial drug
The anticoccidial product salinomycin was mixed as a commercial premix (Sacox® 200 microgranulate, batch nr: 041036046, Huvepharma AD, Sofia, Bulgaria) with the chicken feed in order to produce the desired concentration of 70 mg/kg ration in the treated ration.
2.2. Addition of phytochemicals/extracts, the FIP and the anticoccidial drug to the feeds and assessment of their concentration
A complete formulated broiler mash free of antibiotic/anticoccidial drugs () was mixed with phytochemicals/extracts, the FIP or the anticoccidial drug to produce the desired concentration of the treated rations. The required amount of phytochemicals/extracts, FIP or anticoccidial product was first mixed with approximately 2.5 kg of mash to safeguard adequate mixing. Thereafter, it was further mixed in a blender with the remainder of the feed (Research Diet Services B.V., Wijk bij Duurstede, the Netherlands). To assess the mixing procedure of the phytochemicals/extracts, the FIP and the anticoccidial product in the samples of feed, charges were subjected to chemical analyses. The four phytochemicals/extracts (E. purpurea, betaine, curcumin and carvacrol), the FIP and the anticoccidial drug (salinomycin) were analyzed as described in detail by Peek et al. Citation2013 and Peek & Landman Citation2003. Briefly, the content of E. purpurea in the feed was determined by HPLC analysis of cichoric acid and by semiquantitative thin-layer chromatographic analysis of alkylamides, betaine was determined by a colorimetric assay on the periodide reaction of quaternary nitrogen compounds, curcumin was determined by spectrophotometric analysis and carvacrol by semiquantitative thin-layer chromatographic analysis. The amount of FIP in the crude sample that was mixed in the feed was determined by semiquantitative immunoblot using an anti-V5 antibody (V5 mouse mAb, AP conjugate; Invitrogen, Burlington, Canada) and a secondary antibody (anti-mouse IgG; Sigma-Aldrich St Louis, MO, USA) followed by colorimetric detection. Positope, a protein with a V5 epitope, was used as a positive control and was used as a calibration protein. It was spiked in unsupplemented feed to which no FIP-protein was added and subsequently treated the same as the other samples. The concentration of the ionophore (salinomycin) in prepared feed samples was determined by an HPLC system.
Table 1. Ingredients and chemical composition of the unsupplemented control feed used in the experiment to examine the effect of phytochemicals/extracts, the FIP and the anticoccidial drug salinomycin in broilers against an experimentally induced E. acervulina infection.
In , an overview of the intended and obtained concentrations of the phytochemicals/extracts, FIP and anticoccidial drug in supplemented feeds is given.
Table 2. Intended and obtained concentrations of phytochemicals/extracts, the FIP and the anticoccidial drug salinomycin in the feeds provided in the experiment to examine the effect of phytochemicals/extracts, the FIP and the anticoccidial drug salinomycin in broilers against an experimentally induced E. acervulina infection.
2.3. Experimental design, chickens, inocula and parameters
2.3.1. Experimental design
Eight groups of experimental broilers (groups 1 to 8) received in feed treatments from 1 day of age until the end of the experiment at 13 days of age. Per group the following in feed products were given: group 1, E. purpurea (1250 mg/kg); group 2, FIP control (given as 860 mg crude sample/kg, which was equal to 0 mg FIP/kg); group 3, FIP LZ9 (given as 840 mg crude lysate/kg, which was equal to 10 mg FIP/kg); group 4, betaine (1500 mg/kg); group 5, curcumin (500 mg/kg); group 6, carvacrol low dose (200 mg/kg); group 7, carvacrol high dose (1000 mg/kg) and group 8, an anticoccidial drug (salinomycin; 70 mg/kg). Group 9 received unsupplemented control feed. All groups, each consisting of 48 broilers, were divided in three equally sized subgroups (16 broilers per subgroup) that were inoculated with 104.0 or 105.0 sporulated E. acervulina oocysts or left non-inoculated (subgroups A, B and C). Each subgroup was housed in two cages with eight birds per cage. The subgroups 9A and 9B infected with the low and high E. acervulina doses, respectively, represented the positive control birds, while the non-infected subgroup 9C represented the negative control birds (). Subgroup sizes of 16 birds were used as these would enable the detection of differences of approximately 10% in body weight gain and a difference of 0.5 in lesion scores on the scale of Johnson and Reid Citation(1970) with 95% level of confidence and a power of 80% (Thrusfield et al. Citation2001).
Table 3. Experimental design.
One-day-old broiler chickens were tagged with Swiftack® (Heartland Animal Health Inc. Fair Play, MO, USA) weighed, divided in weight classes of 30–31, 32–33, 34–35 and 36–37 g and thereafter distributed proportionally to the battery cages in order to avoid differences in average weight between groups at start. At the age of 8 days, broilers were individually weighed and inoculated with the prepared E. acervulina oocyst suspensions. At the day of the postmortem examination (day 5 post-inoculation (p.i.)) the broilers were individually weighed and the mean body weight gain per cage between 8 and 13 days was calculated. The coccidial lesion scores were determined as described by Johnson and Reid Citation(1970). Oocyst shedding was assessed in one homogenized sample of fresh droppings per cage collected from day 4 until day 5 p.i. (OPG) (Peek & Landman Citation2003).
2.3.2. Chickens, housing and ethics
Specified Pathogen Free (SPF) male broiler chickens (n = 8 per cage) (GD – Animal Health Service, Deventer, the Netherlands) were housed in stainless steel wire cages (height × width × depth = 30 × 40 × 50 cm) from the start (day 0) until the end of the experiment (day 13). Water and feed were provided ad libitum. The housing temperature was 34 ºC at 1 day of age followed by a gradual decline to 24 ºC at 13 days of age. A scheme of 22 h light per 24 h was applied.
The study was approved by the Institutional Experimental Committee, DEC-Consult Foundation according to Dutch law on experimental animals (Wet op de dierproeven = Animal Research Act).
2.3.3. Inocula
The E. acervulina reference laboratory strain (Weybridge W119, Animal Health and Veterinary Laboratories Agency, Weybridge, UK) sensitive to salinomycin (Peek & Landman Citation2003) kept at GD since 1990 and rejuvenated approximately every half year, was used. The strain was stored as an oocyst suspension at 2–8 ºC in a 2.5% (w/v) potassium dichromate solution with a concentration of 107.3 oocysts per ml and a sporulation percentage of 83%. The high and low inoculation doses were each prepared for 200 birds using 1.10 and 0.11 ml of this oocyst suspension, respectively. The potassium dichromate was removed by centrifugation (2643 × g; 15 min) and assessment of the oocysts concentrations was performed using a Fuchs–Rosenthal haemocytometer counting chamber. The inoculation dose was adjusted to 104.0 or 105.0 sporulated oocysts per bird for the low and high dose, respectively. The inocula were applied into the crop using a 1-ml syringe without needle and a volume of 1 ml tap water. Negative control chickens were placebo inoculated with 1 ml tapwater.
2.3.4. Parameters
Birds were weighed at 0, 8 and 13 days of age. The mean body weight gain (± SEM) per cage and subgroup was calculated for the period from 8 to 13 days of age (0 to 5 days p.i.).
Postmortem examination was performed at the age of 13 days (day 5 p.i.) in order to determine coccidiosis lesions. The individual lesion scores caused by the E. acervulina infection were determined following the method of Johnson and Reid Citation(1970). Briefly, E. acervulina lesion score 1 features ladder-like white streaks in the duodenal loop (≤5/cm2); in lesion score 2 the lesion density is higher but not coalescent; in lesion score 3 the lesions are more numerous and coalesce, while thickening of the intestinal wall is visible, and finally in lesion score 4 the mucosal wall is grayish with lesions completely coalesced. Thereafter, the mean lesion score of E. acervulina per subgroup was calculated. The scoring of coccidiosis lesion scores was performed double blind.
Oocyst shedding per cage was expressed as the number of oocysts per gram droppings. Counts were performed by means of a modification of the McMaster counting chamber technique as described by Peek and Landman Citation(2003).
Birds were stunned with a mixture of CO2 and O2 and debleeded by an incision of the vena jugularis before postmortem examination.
2.4. Statistical analyses
Lesion scores and body weight gains were not found to be significantly different between cages, therefore these parameters were analyzed together for two cages (one subgroup). Subsequently, a one-way ANOVA test was done if residuals showed a normal distribution and homogenous variance. If that was not the case, a Kruskal–Wallis test was performed.
The statistical analysis of the coccidiosis lesion scores (the animal being the experimental unit) and the oocyst shedding of E. acervulina (the cage being the experimental unit) was done with the Kruskal–Wallis test. Mean body weight gains (the animal being the experimental unit) from 8 to 13 days of age were analyzed with the Bonferroni all-pair comparison one-way ANOVA test. Differences were considered significant when p < 0.05 (User's manual Statistix 8.2 for Windows® Citation2010).
3. Results
3.1. Concentration of phytochemicals/extracts, FIP and the anticoccidial drug in the feeds
The results of the chemical analysis of phytochemicals/extracts, FIPs and the anticoccidial drug in feed samples yielded concentrations close to the aimed doses except for E. purpurea and are presented in .
3.2. Inocula
Oocyst counting of the inocula yielded doses of 105.0 and 104.0 sporulated E. acervulina oocysts per bird for the high and low infection dose, respectively.
3.3. Animal experiment
Mean body weight gain (8 to 13 days of age), mean coccidiosis lesion score (at 13 days of age) and mean number of oocysts per gram droppings (OPG) per subgroup ± SEM are outlined in .
Table 4. Effect of in feed treatment with phytochemicals/extracts, the FIP or the anticoccidial drug salinomycin on the body weight gain, the mean coccidiosis lesion score and number of oocysts per gram droppings (OPG) of broilers inoculated with sporulated E. acervulina oocysts at 8 days of age. Values are the mean of n = 16 (birds), except for OPG where n = 2 (cages).
The mean body weight gain of the uninfected treated and uninfected non-treated (negative control) subgroups were not significantly different from each other and averaged 119.3 ± 1.8 g.
There was no significant difference between the body weight gains of the birds infected with 104 sporulated oocysts per bird and the negative control, except for broilers fed curcumin which showed a significant lower body weight gain compared to the salinomycin treated subgroup. A significant difference between the mean weight gain of infected treated subgroups given 105 sporulated oocysts per bird and the infected non-treated (positive) control subgroup was not found. The group fed salinomycin and the uninfected non-treated group (negative control group) had significantly higher body weight gains and were not significantly different from each other.
In the uninfected treated and uninfected non-treated (negative control) subgroups, no coccidiosis lesions and oocysts excretion were observed. No significant difference was observed between the mean lesion score of the treated chickens infected with 104 sporulated oocysts and the infected non-treated (positive) control subgroup, except for the birds fed salinomycin, which showed a significantly lower lesion score. The coccidiosis lesion scores of the subgroups given 105 sporulated oocysts per bird did not differ significantly from each other, except for the group given salinomycin, which differed significantly from the FIP LZ9 and the positive control subgroup but not from the other subgroups. The salinomycin treated birds had the lowest lesion scores.
Analysis of the OPGs showed no significant difference between treatments per infection dose although the oocyst excretion of the subgroups given salinomycin was the lowest for both inoculated doses (). Lower oocyst counts of droppings were found in the subgroups given the higher inoculation dose except in the salinomycin treated birds. Log10 transformation did not result in a normal distribution of the OPGs, while analysis of the transformed data using the Kruskal–Wallis test yielded exactly the same results as analysis of non-transformed data.
4. Discussion
The analysis of feed samples showed that the obtained concentrations phytochemicals/extracts (except for E. purpurea), FIP and salinomycin were close to the desired doses ().
The inability to detect standardized E. purpurea root extract in chicken feed samples by HPLC analysis of cichoric acid was not related to insufficient sensitivity of the analytical method. Since cichoric acid was neither detectable in extracts of the unsupplemented control feed spiked with 1250 mg/kg Echinacea (with a theoretical amount of 250 ng of cichoric acid which exceeds the detection limit by 10 to 25 times), it was concluded that cichoric acid (but probably also other polyphenolic constituents present in the standardized E. purpurea root extract) was bound to the feed matrix and could not be extracted. Most likely, the high content of proteins and/or polysaccharides in the feed samples play a role in this respect as it is well known that these macromolecules can interact with and precipitate polyphenols (see for instance McManus et al. Citation1985). The ineffectiveness of Echinacea-supplemented feeds as demonstrated in this study may, at least in part, be attributed to this limited bioavailability of polyphenolic constituents from the feed matrix since cichoric acid (and related constituents) is generally considered as one of the active principles in this plant (Barnes et al. Citation2005).
On the other hand, the relative low concentration of standardized E. purpurea root extract found in the Echinacea-supplemented feed samples may also have been due to the late time of analysis – after the expiry date of the samples (in some feed samples, fungal growth was observed which might have affected feed composition) – and possible degradation of the alkylamide components. The relative high concentration of betaine in the feed samples is probably due to the low specificity of the colorimetric reaction used; in addition to betaine, other quaternary ammonium compounds (amongst which choline) also react with potassium triiodide and result in a high background signal (Wall et al. Citation1960; Storey & Wyn Jones Citation1977). The relative low concentration of carvacrol observed for the quantitative analysis of the chicken feeds supplemented with high carvacrol concentrations is related to the incidental and partial overlap of the carvacrol spots with spots of other constituents in the feed.
Coccidiosis was successfully induced in all E. acervulina inoculated birds, while negative controls remained free of the parasite. The lower mean lesion scores of birds infected with the high infection dose is probably the result of the so-called ‘crowding effect’, which was confirmed by the lower OPG counts found in almost all infected groups except for the birds given salinomycin. A ‘crowding effect’ may occur when the number of available functional intestinal target host cells, needed for the development of the parasite and production of oocysts (gametogony), is reduced by sloughing of epithelium cells during the development of an infection (enormous number of schizogony generations) and is associated with high infection doses of the parasite (Tyzzer et al. Citation1932; Williams Citation2001).
No statistically significant difference was observed between the weight gains of the uninfected treated and uninfected non-treated birds, indicating that there was no growth promoting or growth retarding effect of the compounds used. Although a statistically significant lower body weight gain was found in curcumin treated broilers infected with 104 sporulated oocysts of E. acervulina compared to salinomycin medicated birds, this difference was very small; birds treated with Echinacea showed 1 g more body weight gain than curcumin treated birds, which was not significantly lower than that of salinomycin treated broilers. In birds inoculated with 105 sporulated oocysts of E. acervulina, salinomycin treated broilers showed significantly higher body weight gain compared to all groups except the negative controls. This indicated that the coccidiosis infection was well controlled despite the high inoculation dose. Considering the high inoculation dose (105 sporulated oocysts per bird), lower coccidiosis lesion scores were obtained compared to the low inoculation dose (104 sporulated oocysts per bird) and this prevented the unambiguous detection of significant differences in mean lesion scores between salinomycin treated birds and the other treated subgroups. No significant differences in OPGs were found between coccidiosis infected groups, although OPGs of salinomycin treated birds were always lower. Generally, lower OPGs were found in the groups given the higher inoculation dose, which was likely also the result of ‘crowding effect’. The lack of significant differences between coccidiosis infected groups was explained by the large variation in variance between cages.
The phytochemicals/extracts and FIP failed to reduce coccidiosis lesion scores and oocyst shedding, while salinomycin efficiently controlled the E. acervulina infection and enabled significantly higher body weight gains, at both low and high infection doses.
In conclusion, the selected phytochemicals/extracts and FIP failed to significantly reduce coccidiosis lesions in broilers experimentally inoculated with E. acervulina. Therefore, they cannot be used to replace the anticoccidial drug (salinomycin) at the concentrations tested.
Acknowledgements
This study was funded by the Dutch Ministry of Economic Affairs, Agriculture and Innovation, coordinated by Immuno Valley foundation and embedded in the research program ALTernatives to ANTibiotics (ALTANT) ‘Modulation with immune stimulating phytochemicals (MODIPHY) as an alternative for antimicrobial treatment’.
ORCID
W.J.M. Landman https://orcid.org/0000-0002-2513-8538
Related Research Data
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