Efficacy of a Phytogenic Feed Additive in Broilers Challenged with Subclinical Necrotic Enteritis

Necrotic enteritis (NE) in broiler chickens is an economically important disease which can be controlled by antibiotics (Timbermont, et al., 2011). The occurrence of necrotic lesions in the small intestine is associated with proliferation of Clostridium perfringens, which leads to lower growth rate in subclinically infected chickens. Increasing concerns regarding antibiotic resistance and the presence of drug residues in animal products have led many countries to ban the use of antibiotic growth promoters which has therefore triggered a search for viable alternatives to antibiotics in the animal. Phytogenic feed additives (PFAs) are plant derived extracts that have been investigated extensively as alternatives to antibiotic growth promoters due to their strong antibacterial activity, antioxidant capacity and their beneficial impact on health and performance in broiler production systems (Delaquis, et al., 2002; Paraskeuas, et al., 2017). PFAs are categorized as sensory and flavoring compounds, which consist mainly of plant extracts (essential oils, oleoresins, and flavonoids) and their active principles. Essential oils present in PFA, which contain most of the active substances of the plant, have been shown to improve gut health by modulating the intestinal microbiota and effecting intestinal gene expression and animal performance (Paraskeuas, et al., 2017, Paraskeuas and Mountzouris, 2019). Supplementation of essential oils to poultry has been shown to stimulate the production of endogenous enzymes such as lipase and amylase (Platel and Srinivasan, 2000). The PFA used in this study is a blend of essential oils including carvacrol. Carvacrol has strong antiinflammatory and antibacterial activity and has previously shown to reduce C. perfringens load in NE infected broilers (Liu, et al., 2016). We aimed to evaluate and compare the effects of a PFA product, a blend of essential oil (EOs) on growth performance and intestinal gene expression in broiler chickens subjected to a subclinical necrotic enteritis challenge.

A total of 384 d-old Cobb 500 chicks (as hatched) were vent sexed, and 16 birds (8 males and 8 females) were randomly allocated to floor pens covered in wood shavings. The experiment consisted of three treatments, with eight replicates per treatment. The three treatments were: 1) non-challenged birds - no additive, 2) NE challenged birds - no additive, 3) NE challenged - PFA. The dietary dose of PFA (Activo®, supplied by EW Nutrition) was 100 g/T. All diets were formulated based on Cobb 500 nutrient specifications (Cobb-Vantress, 2018). Diets were based on wheat and soybean meal and were fed ad libitum for the duration of the trial period. The PFA product used in this study is a mixture of different phytomolecules, including carvacrol. The experiment was performed with three periods: starter (d 0-10), grower (d 11- 24) and finisher (d 25-35). On d 9, birds in the challenged groups were subjected to an oral gavage of 1 ml Eimeria species (E. acervulina at 5,000 oocytes/mL, E. maxima at 5,000 oocytes/mL, E. brunetti at 2,500 oocytes/mL). The birds in the non-challenged group were inoculated with 1 ml of PBS. On days 14 and 15, birds in the challenged treatments were inoculated with approximately 108 CFU of Clostridium perfringens NE18 strain, and control birds were inoculated with 1mL of sterile thioglycollate broth. Average body weight gain (WG), feed intake (FI) and feed conversion ratio (FCR) were calculated considering the mortality. Performance data were analysed for the treatment effect with male percentage (corrected to dead birds) as a covariate. On d 16, two birds from each pen were humanely euthanised and jejunum tissue was collected for gene expression analysis. For each sample, total RNA was extracted after homogenization in TRIsureTM (Bioline, Sydney, Australia) following the manufacturer’s instructions. The quantity and purity of the samples were measured with NanoDrop ND-8000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA), and the RNA Nano 6000 kit was used to measure RNA integrity on the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Waldron, Germany). The extracted RNA of each sample was reverse transcribed with the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Amplification and detection were carried out in duplicates using an SYBR Green kit SensiFAST SYBR No-ROX (Bioline, Sydney, Australia) with Rotorgene 6000 real-time PCR machine (Corbett Research, Sydney, Australia). The data were analysed with SPSS software Version 28 using oneway-ANOVA and the significant differences between treatments were examined with Tukey’s HSD post hoc test.

As illustrated in Table 1, during day 0-35 non-challenged birds presented significantly higher WG and lower FCR compared to challenged groups. However PFA supplementation improved FCR and WG significantly (P < 0.001) compared to the challenged birds fed with nonsupplemented diets.

Jejunal gene expression is presented in Table 2. Challenged birds fed with nonsupplemented diets showed a significantly higher expression (P= 0.002) of CASP3 compared to other groups. Addition of PFAs in the diet reduced the expression of CASP3 in the challenged birds indicating alleviated damage to the intestine. Furthermore, the nonsupplemented challenged birds presented a significantly lower expression of OCLN (P < 0.001), SOD1 (P = 0.018) and APN (P= 002) compared to the non-challenged birds.

This study investigated the responses of NE challenged broilers supplemented with a PFA product, and compared their responses to non-challenged and NE challenged birds which were fed with basal diet or diets supplemented with PFAs. In the present study, a successful subclinical NE infection was introduced in challenged groups, as typical signs such as impaired FCR and BW were observed in the challenged birds (M'Sadeq, et al., 2015). The effect of the challenge can also be observed with the downregulated expression of OCLN, an important protein in the tight junction complex, implying compromised gut health by the challenge. The PFA supplemented group showed an improved FCR compared to the challenged birds fed with control diets. Furthermore, weight gain and expression of genes related to cell death (CASP3) and antioxidant capacity (SOD1) in the PFA supplemented birds did not differ from the nonchallenged treatments suggesting a positive effect of PFA in maintaining the health of the intestinal epithelial cells. Phytogenic feed additives are plant compounds that can stimulate appetite and endogenous secretions, and have antimicrobial and coccidiostatic activities in monogastric animals (Wenk, 2003). Previous studies have shown the positive effect of carvacrol on the upregulation of tight junction proteins in broilers under lipopolysaccharide challenged (Liu, et al., 2020). Although we have not observed a significantly higher expression of these genes in the PFA supplementation groups, the numerically higher expression of the tight junction gene (occludin, OCLN) could be a sign of a moderately improved gut environment and lower damage to the epithelium cells creating a stronger tight junction bond between cells. The downregulation of genes related to cell death (caspase 3, CASP3) observed in the PFA treated birds, could be an indication of a healthier epithelial lining. Similar results have been reported by Mousavi, et al. (2020), where carvacrol, reduced CASP3 expression in the intestine of Campylobacter infected mouse. The beneficial effects of the PFA on broiler performance observed in the study could be attributable to improved gut health conditions. It is not yet clear if this PFA is affecting C. perfringens, Eimeria or both of these disease agents. However, it can be concluded that supplementation of this PFA in diets can help improve performance through modulation of gut health in broilers under subclinical NE challenges.

ACKNOWLEDGMENTS: We acknowledge EW Nutrition GmbH© for funding this project, Bioproperties for providing Eimeria, and Prof. Robert Moore for providing Clostridium perfringens EHE-18 strain. This project was co-funded by AgriFutures and managed through Poultry Hub Australia.