1 INTRODUCTION

Feed constitutes a fundamental element in the poultry industry, significantly influencing the gastrointestinal tract and its overall health. The gastrointestinal tract is recognized as a sophisticated and dynamic organ critical for gut health. Numerous factors, including stressors, can potentially disturb the delicate balance of the gut ecosystem, leading to adverse effects on poultry health and productivity. Gastrointestinal dysbiosis poses significant challenges to the poultry sector by compromising gut health and elevating disease vulnerability. In contrast, a healthy gut in poultry facilitates efficient nutrient digestion and absorption, particularly in environments where these stressors are minimized (Diaz Carrasco et al., 2019).

Campylobacter jejuni, a gram-negative, non-spore-forming bacterium, is a widespread cause of gastroenteritis (Epps et al., 2013; Kaakoush et al., 2015; Moore et al., 2005). It is a significant pathogen responsible for human diarrheal disease. Poultry serves as a significant source of C. jejuni exposure, given that this bacterium thrives within the gastrointestinal tract, posing a major risk for foodborne illnesses in humans worldwide (Beier et al., 2019; Connerton et al., 2018). Its ability to colonize and suppress immune responses in chickens impacts broiler performance (Nooreh et al., 2021).

In the past, antibiotics were employed as growth promoters to combat gastrointestinal pathogens and mitigate the impact of stressors on gut function. Nevertheless, restrictions on antibiotic use in poultry production have been implemented due to rising consumer awareness about antibiotic-related health risks, bacterial resistance and food safety concerns (Ali et al., 2021). In light of the constraints imposed on antibiotic use in chicken production in recent years, the poultry industry has turned to alternative approaches, such as incorporating plant-derived feed additives. The rising interest in plant bioactive compounds, such as phytochemicals, within poultry diets stems from their promising capabilities as antioxidants and antimicrobial agents. As a result, these natural compounds are increasingly sought-after for their potential to support the well-being and health of poultry while aligning with the trend of reducing antibiotic usage (Ali et al., 2021).

Despite some reports on phytogenic additives as antibiotic alternatives for Campylobacter, only a few have been thoroughly studied. Inconsistent results emphasize the need for further research to understand their potential in poultry diets. Cinnamon, from the genus Cinnamomum (Lauraceae family), comprises over 250–300 aromatic evergreen shrubs and plant trees. However, only a few of these species hold substantial economic importance as a widely used spice globally (Ali et al., 2021). Cinnamon is rich in bioactive compounds, including volatile oils, flavonoids, curcuminoids, coumarins, tannins, alkaloids, xanthones, terpenoids, phenolics and more. These components contribute to its natural antioxidant, antimicrobial and anti-inflammatory properties (Liyanage et al., 2021; Ribeiro-Santos et al., 2017). Rosemary (Rosmarinus officinalis) is recognized for its medical benefits and aromatic properties, making it popular for various applications (Belenli et al., 2015). As a natural animal feed additive, its antimicrobial activity and antioxidant properties play a significant role (Abd El-Latif et al., 2013). These properties are attributed to the presence of bioactive compounds, including 1,8-cineole, rosemarinic acid and rosmarole, making it a valuable source of antioxidative compounds (Abd El-Latif et al., 2013; Franciosini et al., 2016; Jiang et al., 2011). Oregano (Origanum vulgare L.) is a highly aromatic herb with abundant active chemical compounds (Park et al., 2015). Oregano essential oil contains major components such as carvacrol and thymol, terpenoid compounds with antimicrobial properties (Teixeira et al., 2013). The polyphenols in oregano have also been studied for their antimicrobial activity and molecular mechanisms in suppressing microbes (Xu et al., 2008). Additionally, oregano extract serves as a natural antioxidant, effectively preventing lipid oxidation and contributing to improved meat quality and animal health (Lee et al., 2004).

Several studies have reported that these medicinal plants positively influence the performance, intestinal morphology, nutrient digestibility and gut microbiota of poultry (Abd El-Latif et al., 2013; Ashayerizadeh et al., 2009; Belenli et al., 2015; Franciosini et al., 2016; Mathlouthi et al., 2012; Ri et al., 2017; Saki et al., 2012). For example, the results of a study demonstrated that supplementing cinnamon led to significant improvements in body weight gain (BWG) and feed conversion ratio (FCR) in broilers, comparable to the group fed with oxytetracycline antibiotic (Singh et al., 2014). Moreover, Kanani et al. (2016) reported that including of 0.5% cinnamon in the feed significantly increased both feed intake (FI) and BWG compared to the control birds. Moreover, the use of rosemary in broiler chickens diets increased average daily gain, feed efficiency and European production efficiency factor (Petricevic et al., 2018). In another study, Ri et al. (2017) reported that the oregano supplementation increased broiler chickens' daily gain and daily FI during the grower period and the whole period.

Indeed, most of the previous research has primarily focused on investigating the effects of medicinal plants on poultry with normal health conditions. Therefore, this study sought to explore how cinnamon, rosemary and oregano powder affect broiler chickens challenged with C. jejuni regarding growth performance, excreta microbiota, intestinal morphology and nutrient digestibility.

2 MATERIALS AND METHODS

3 RESULTS

4 DISCUSSION

The results highlight several noteworthy findings that contribute to our understanding of the complex interactions between dietary treatments and C. jejuni challenge in broiler chickens’ production. The results revealed that the introduction of a C. jejuni challenge had a detrimental effect on birds’ performance, leading to reduced BWG and elevated FCR. These findings corroborate existing literature that establishes the negative impact of Campylobacter colonization on broiler growth and efficiency (Guyard-Nicodème et al., 2016). Importantly, treatments containing additives displayed a promising ability to mitigate the adverse effects of the Campylobacter challenge, resulting in performance resembling that of the NC group. These results emphasize the potential of medicinal plants in supporting broiler performance under pathogenic stressors. Notably, although the cinnamon-supplemented treatment exhibited relatively lower efficacy compared to other additives, it still demonstrated potential in alleviating the impact of Campylobacter challenge.

According to reports, antibiotics are known to enhance animal growth by altering bacterial metabolism (Ghunaim, 2009). Furthermore, exposure to antibiotics creates selective pressure within the digestive systems of animals, reducing the growth and spread of harmful bacterial strains such as Salmonella and Campylobacter (Ghunaim, 2009). As a result, the observed performance enhancement in the Erythromycin-fed group can be attributed to alterations in pathogen metabolism and reduced colonization. The FCR, representing the relationship between FI and weight gain, became more pronounced due to the decrease in growth rate induced by C. jejuni challenge in broilers. As the challenge primarily impacted growth rate without affecting FI, the resulting increase in FCR within the C. jejuni-infected group (PC) is readily apparent. Aligning with the present findings, existing literature highlights that broiler challenge with C. jejuni elevates the FCR (Gharib Naseri et al., 2012). However, this unfavourable effect can be mitigated through the incorporation of dietary supplements such as organic acids, probiotics and medicinal plants (Gharib Naseri et al., 2012).

Given that thymol and carvacrol are the principal and most potent constituents of oregano essential oil (Kırkpınar et al., 2011), and these two compounds are attributed to oregano's antimicrobial activity (Mathlouthi et al., 2012), the enhanced performance observed in the oregano-fed group can be linked to the antimicrobial effects, leading to a reduction in Campylobacter colonization. This reduction subsequently curtailed bacterial toxin production, facilitated the restoration of near-normal intestinal villi growth and improved nutrient digestion, like the characteristics observed in the NC group.

Verbenone, 1,8-cineole, camphor and borneol have been identified as the key compounds responsible for the antimicrobial properties of rosemary essential oil (Mathlouthi et al., 2012). Consequently, the improved performance observed in the rosemary-fed group can be attributed to the reduction in Campylobacter colonization. Furthermore, a separate study attributed enhanced growth performance in chickens fed with rosemary leaves to increased apparent dietary protein digestibility, along with improved nutrient availability in the intestines for efficient absorption, leading to accelerated growth (Botsoglou et al., 2007). This highlights yet another contributing factor to the improved performance of chickens receiving rosemary supplementation.

The inclusion of cinnamaldehyde in broiler diets has been cited as a promoter of growth performance attributed to its antimicrobial influence against detrimental bacteria and its enhancement of digestibility (Mehdipour et al., 2013). In our current experiment, the incorporation of cinnamon powder demonstrated performance improvement; however, this effect stood between the outcomes of the PC and NC treatments.

The reduction in Lactobacilli count within the PC group following the C. jejuni challenge could potentially be attributed to increased Campylobacter colonization in the digestive tract. This surge in Campylobacter colonization might lead to competitive inhibition, subsequently diminishing the presence of beneficial bacteria like Lactobacilli, thereby contributing to dysbiosis in the bird's gut. Notably, several Clostridium perfringens species have shown sensitivity to carvacrol, cinnamaldehyde, citral and thymol. Utilizing these compounds is considered an effective strategy for managing the colonization of pathogenic bacteria and preserving intestinal health (Ouwehand et al., 2010).

In the current study, the utilization of Erythromycin, despite its effectiveness in diminishing Campylobacter colonization, coincided with a reduction in Lactobacilli counts. This phenomenon can potentially be attributed to the broad-spectrum nature of the mentioned antibiotic. Beyond its impact on the reduction of gram-negative bacteria colonization, it also affects the population of gram-positive bacteria. In contrast, the utilization of phytogenic additives in this study ameliorated the decrease in Lactobacillus counts caused by the Campylobacter challenge, aligning their numbers more closely with those of the NC group. This beneficial effect of phytogenic additives could be attributed to their antibacterial properties against Campylobacter colonization. By curtailing the proliferation of Campylobacter, these compounds facilitate the establishment of a more balanced and stable intestinal microbial population, thereby mitigating the adverse consequences of the challenge.

In accordance with the findings of this study, previous research has indicated that incorporating antibiotics into broiler diets enhances growth performance while also leading to an increase in VH and VW and a decrease in CD (Marković et al., 2009). Phytogenic compounds, on the other hand, have been demonstrated to boost mucus production in the stomach and jejunum, signifying their protective role against gastrointestinal pathogens (Manzanilla et al., 2004). Further supporting these observations, the addition of a combination of thymol and carvacrol at 100 and 200 mg/kg, respectively, to broiler diets has been reported to elevate VH in the jejunum, although it showed no impact on VW (Hashemipour et al., 2013). The observed enhancement in intestinal morphology facilitated by the medicinal plants employed in this study can be attributed to their antibacterial compounds. By curbing Campylobacter colonization and preventing the decline in Lactobacilli count, these compounds contribute to optimal bacterial toxin concentrations. Consequently, they prevent villus damage and excessive CD, thus averting abnormal villus remodelling.

Plant essential oils serve as beneficial additives that enhance nutrient digestibility by elevating the activity of digestive enzymes and promoting the secretion of gastric and pancreatic juices. Their inherent antimicrobial properties contribute to the preservation of intestinal microvilli, consequently bolstering nutrients absorption. This effect is associated with an overall enhancement in digestibility coefficients (Fascina et al., 2012; Hernandez et al., 2004). Notably, an additional study underscores that the improved digestion and absorption in poultry's digestive systems when utilizing medicinal plants is attributed to the impact of their active components on the secretion of digestive enzymes such as proteases, lipase and amylase, coupled with the augmentation of intestinal villi count (Ponte et al., 2008). In this particular experiment, there was not a notable difference observed between the treatments concerning excreta DM and DM digestibility, contrary to what was mentioned in other cases. One potential reason for this disparity could be attributed to the type and quantity of herbal additives utilized in this research.

Biochemical parameters in the blood reflect the state of body organs influenced by various internal and external factors. According to the findings of this study, as well as research conducted by Lee et al. (2004) and Bampidis et al. (2005), which involved the inclusion of carvacrol, thymol and cinnamaldehyde in the diet of broilers, and oregano along with a commercial product containing essential oils in the diet of turkeys, it was revealed that these potent plant substances could potentially have positive effects on reducing blood cholesterol levels. This effect might occur through the inhibition of the activity of the regulatory enzyme responsible for cholesterol production, namely 3-hydroxy 3-methylglutaryl coenzyme A reductase. Additional research conducted by Shamlo et al. (2014) demonstrated that incorporating medicinal plants like oregano into the diet of broilers stimulates the proliferation of beneficial bacteria such as Lactobacillus and Bifidobacterium within the intestine. This proliferation contributes to a reduction in blood cholesterol levels. These specific bacteria disrupt the intestinal-hepatic cycle of bile acids through deconjugation, consequently diminishing cholesterol absorption by reducing micelle formation. Traesel et al. (2011) supported our findings by indicating that using levels of 50, 100 and 150 mg/kg of a blend of oregano and rosemary essential oils in broiler diets did not affect HDL, triglyceride levels, total protein, albumin, globulin and the albumin-to-globulin ratio. However, in contrast to the outcomes of the current study, the same researchers reported that incorporating oregano and rosemary essential oils in the diet of broiler chickens did not significantly impact serum cholesterol levels. This discrepancy might be attributed to the relatively low levels of essential oils used in their research. Additionally, due to the volatile nature of essential oils leading to rapid evaporation, it might be necessary to prepare rations containing essential oils at least every other day to minimize evaporation and potentially observe their positive effects.

The calculation formulas for atherogenic indices indicate that lower values are indicative of hypocholesterolemic and hypolipidemic effects. Although treatments containing herbal additives did exhibit a reduction in atherogenic indices to some degree, their overall impact was not considered significant. The experimental treatments notably influenced total cholesterol levels but did not sufficiently impact the other components within the atherogenic indices formulas, thereby resulting in minimal alteration of these indicators.

The assessment of poultry health relies heavily on analysing serum enzyme activities. Enzymes like ALT and AST, found predominantly in the liver, serve as specific indicators of hepatic cellular damage and inflammation (Selim et al., 2021). LDH isoenzymes, existing in various bird tissues, including liver, muscle, kidney and blood cells, decrease notably during liver diseases (Viveros et al., 2002). ALP, produced in all tissue types, aids in dephosphorylation and functions optimally in alkaline conditions (Selim et al., 2021). The findings from our study revealed that C. jejuni resulted in elevated serum ALT levels, suggesting a potential link to increased tissue damage and the disruption of liver functions. This connection is supported by previous reports associating higher levels of these markers with liver damage and inflammation (Selim et al., 2021; Viveros et al., 2002). Our findings align with reports stating that carvacrol usage inhibits blood biochemical changes induced by acrylamide, such as heightened ALT enzyme concentrations, consequently reducing liver damage (Cerrah et al., 2023). Additionally, thymol administration has been noted to alleviate liver damage characterized by decreased ALT levels (Dou et al., 2022). The observed reduction in ALT due to the medicinal plants used in this study can be credited to their antioxidant, antimicrobial and anti-inflammatory properties (Geyikoglu et al., 2019).

5 CONCLUSIONS

The findings from this study lead to the conclusion that both oregano and rosemary can mitigate the pathogenic impact of C. jejuni. These additives offer a viable alternative to Erythromycin in broiler feeding strategies. Nonetheless, employing higher concentrations is recommended if cinnamon is chosen for more robust outcomes. However, further experimentation is necessary to determine optimal cinnamon for achieving desired effects.

AUTHOR CONTRIBUTIONS

Zahra Alimohammadi: Methodology; data curation; project administration. Hassan Shirzadi: Supervision; conceptualization; investigation; methodology; data curation; data analysis; funding acquisition; Writing — original draft. Kamran Taherpour: Conceptualization; investigation; methodology; data curation. Enayat Rahmatnejad: Investigation; methodology; writing; review and editing. Ali Khatibjoo: Investigation; methodology; validation; data analysis.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.