The increasing global demand for poultry meat has been attributed, in part, to the ongoing increase in the human population (Tabashsum et al., 2023). The poultry industry has grown considerably in recent decades, and the population of poultry, particularly chickens, has increased more than five-fold in the last 50 years (Mottet and Tempio, 2019). At the same time, poultry farms have adopted various techniques, such as genetic selection and the use of antibiotics, to maximize both productivity and quality while minimizing costs and production time (Siegel, 2014; Gadde et al., 2017; Carney et al., 2022).

In this context, a tight relationship has been observed between poultry growth efficiency, expressed as the feed conversion ratio (FCR), and the microbiota of the gastrointestinal tracts (GIT) of these animals (Stanley et al., 2012). Notably, it has been reported that the diversity of the GIT microbiome is inversely related to the FCR value (Diaz Carrasco et al., 2019). Antibiotics have been widely used as growth promoters in livestock agriculture (Gadde et al., 2017). However, excessive use of these agents has promoted the spread of antibiotic-resistant pathogenic bacteria, with concomitant concerns that these organisms may be transmitted to humans as zoonoses (García et al., 2020). As a result, the use of antibiotics as growth promoters has been restricted or even banned in many countries. Consequently, there has been increasing interest in exploring alternatives to the use of antibiotics in promoting livestock growth (Bahaddad et al., 2023).

Another issue in avian agriculture is infection by Campylobacter jejuni, a bacterium that is infamous for causing foodborne illness in humans. Infection by C. jejuni is primarily associated with the handling and consumption of raw or undercooked meat, and typically results in symptoms such as intense (and sometimes bloody) diarrhea, fever, and abdominal pain that can last from 7 to 10 days (Kemper and Hensel, 2023). According to the World Health Organization (WHO), the global burden of Campylobacter infection is 7.5 million cases per day (World Health Organization (WHO), 2013). A key strategy for reducing clinical cases of C. jejuni infection is to control and decrease the prevalence of this bacterium at the farm level. Studies have shown that an approximately 10-fold reduction in C. jejuni populations on chicken carcasses can significantly decrease the incidence of campylobacteriosis in humans (Rosenquist et al., 2003; Iwata et al., 2023).

Probiotics, defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (Hill et al., 2014), constitute an attractive alternative to antibiotics and a valid approach to decreasing the occurrence of C. jejuni. Some probiotics have been shown to improve feed intake and digestion while stimulating the immune system, promoting growth in livestock, including poultry (Krysiak et al., 2021). In addition, several probiotics are known to be beneficial in countering C. jejuni (Arsi et al., 2015; Mohan, 2015; Nishiyama et al., 2015). In Arsi et al. (2015), several bacterial strains were isolated from the ceca of birds and tested in an in vivo challenge in poultry against C. jejuni. They demonstrated a reduction of approximately 1- to 2-log units in C. jejuni abundance following oral administration of Bacillus spp. and Ligilactobacillus salivarius subsp. salivarius and L. s. salicinius.

The probiotic bacterium Limosilactobacillus ingluviei (formerly Lactobacillus ingluviei), a heterofermentative member of the lactic acid bacteria, was first isolated from the crop of a pigeon (Baele et al., 2003; Zheng et al., 2020). Previous studies have investigated L. ingluviei for its potential anti-Salmonella activity (Thomas et al., 2019) and growth-promoting effects (Angelakis and Raoult, 2010; Angelakis et al., 2012). The C37 strain of L. ingluviei (LIC37) was independently isolated in our laboratory from the intestines of broiler chickens, and we previously have reported the in vitro characteristics of this isolate, including its growth characteristics and potential utility as a probiotic agent (Tsukagoshi et al., 2020; Sirisopapong et al., 2023). However, the in vivo activity of LIC37 and its effects on host health have not yet been clarified.

In the present study, we sought to evaluate the potential application of LIC37 as a probiotic agent in chicks. Specifically, we observed the inhibitory impacts of this isolate on colonization of the chick GIT by C. jejuni, and clarified the associated changes in the cecal microbiota of chicks administered LIC37. We demonstrated that C. jejuni infection in these animals was strongly suppressed and that primary effect of LIC37 administration on the GIT microflora was an increase in the cecal abundance of LIC37, with no other significant changes in the bacterial diversity in this organ. In addition, we observed that LIC37 administration may induce the growth of a beneficial bacterium belonging to the genus Blautia. The results suggested that LIC37 may have potential as a probiotic agent protecting against C. jejuni on poultry farms.

L. ingluviei, a novel probiotic lactic acid bacteria, has been shown to have beneficial effects on chickens, including anti-Salmonella activity (Thomas et al., 2019) and growth-promoting effects (Angelakis and Raoult, 2010; Angelakis et al., 2012). LIC37, a strain of L. ingluviei isolated from broiler chickens, has demonstrated immunological effects in vitro (Tsukagoshi et al., 2020; Sirisopapong et al., 2023). In this study, we examined the potential utility of LIC37 in maintaining livestock. Specifically, we investigated the in vivo effects of LIC37 administration on C. jejuni infection and the associated changes in the cecal microbiota of chickens.

We first tested the effects of LIC37 on growth promotion, a common criterion for assessing probiotics (Krysiak et al., 2021). In contrast to previous findings with other strains of L. ingluviei (Angelakis and Raoult, 2010; Angelakis et al., 2012), we observed that LIC37 administration did not significantly enhance chicken weight gain over the course of the study (Figure 1). However, no adverse effects, such as attenuation of weight gain, were detected in the present study. Moreover, there were no observed signs or symptoms of morbidity attributable to the administration of LIC37 alone. In fact, the growth rate in the LIC37 group was comparable to or slightly better than that in the control group. These findings not only underscore the potential for LIC37 as a probiotic, but they also suggest that this strain is safe for use.

Campylobacter jejuni is well known for its role in foodborne illnesses. This pathogen colonizes the GITs of both wild and domestic warm-blooded animals, with poultry acting as the primary reservoir (Skarp et al., 2016). Historically, antibiotics have been employed to control C. jejuni in the poultry industry, but there is growing concern about the overuse of antimicrobials, particularly given the increased prevalence of drug-resistant bacteria (Mingmongkolchai and Panbangred, 2018). Consequently, the poultry industry is striving to reduce or eliminate antibiotic use, both for therapeutic applications and as growth promotors, and is exploring alternative strategies for controlling C. jejuni in poultry production (Al Hakeem et al., 2022).

Several studies have investigated probiotics with the potential to reduce C. jejuni colonization and infection (Eeckhaut et al., 2016; Saint-Cyr et al., 2017; Smialek et al., 2018; Massacci et al., 2019). For example, Smialek et al. (2018) demonstrated that the supplementation of chicken feed with Lavipan, a probiotic consisting of several Lactobacillus strains, reduced C. jejuni invasion in the GIT of broiler chickens. In addition, the use of bacilli as probiotics in host species other than those from which the bacteria were originally derived has become increasingly common (Mingmongkolchai and Panbangred, 2018), despite evidence suggesting that strains with the same ecological origin may be more effective (Krysiak et al., 2021). Therefore, evaluating the safety of various bacilli for consumption by livestock remains crucial (Hong et al., 2005). In this study, we used LIC37, a strain that was originally isolated from broiler chickens (Tsukagoshi et al., 2020). We sought to increase the proportion of this bacterium in the gut of recipient chickens by oral administration alone, without any additional supplementation with any chemicals or bacteria derived from other host species. We propose that this strategy is likely the most effective for preserving the health of poultry, humans, and the surrounding environment. This strategy significantly reduced the number of C. jejuni not only in the cecum, but also in the ileum and the jejunum (data not shown). Furthermore, we evaluated the balance of the cecal microbiota in animals infected with C. jejuni, both with and without prophylactic administration of LIC37. Our findings showed that there were no significant changes in the composition of the cecal microbiota of infected animals, except for the proportion of C. jejuni (Figure 3). The observed changes did not affect the α-diversity of the cecal microbiota, which measures species diversity within a community (Figure 3C). However, the β-diversity, which assess the degree of diversity between groups, showed that the cecal microbiome in both the LIC37 and C. jejuni + LIC37 groups resembled that observed in the Ctrl group, while the C. jejuni group formed a distinct cluster (Figure 3). Our results indicate that increasing the abundance of LIC37, itself a chicken isolate, to approximately 1% of the cecal microbiota efficiently prevents C. jejuni infection.

Other studies have shown that individual strains of Lactobacilli are no longer detectable in the cecum of inoculated chicks at 1 week after a single oral inoculation at hatching (Kubasova et al., 2019). We therefore considered that the ability of LIC37 to attach to intestinal epithelial cells and persist in the GIT for extended periods should be investigated. Considering that LIC37 is a native component of the chicken gut microbiome, we expected that this bacterium would persist in the GIT; however, distinguishing between administered bacteria and native bacteria was initially considered to be challenging. Despite this, the cecal abundance of L. ingluviei remained elevated in chicks administered LIC37, even more than a week after oral administration. Analysis of the LDA scores revealed that three classes of bacteria showed significant differences in abundance between the cecal contents of the C. jejuni and C. jejuni + LIC37 groups, with L. ingluviei being one of the most abundant bacteria in the cecal content of the C. jejuni + LIC37 group (Figure 2).

Members of the Limosilactobacilli, including L. ingluviei, are known to be more abundant in the ileum compared to the cecum as the ileal microbiome has a higher prevalence of facultative anaerobes (Sengupta et al., 2013; Kubasova et al., 2021). We therefore expected that LIC37 would show excellent adhesion to the intestinal wall, including the cecum in chickens, thereby enhancing the potential of LIC37 as a novel probiotic against C. jejuni infection. Indeed, our in vivo results supported this expectation and corroborated our earlier in vitro analyses (Sirisopapong et al., 2023), which showed that LIC37 was capable of adhering to Caco-2 cells. Consequently, the present study clarified the potential application of LIC37 as an in vivo probiotic agent.

Separately, Saint-Cyr et al. (2017) demonstrated that oral treatment with L. salivarius SMXD51 every other day (Q2D) for 35 days exhibited anti-C. jejuni activity in vivo. In contrast, our study employed a simpler regimen of only two Q2D doses (on Days 4 and 6) to prevent C. jejuni colonization. However, our study did not follow the chicks to market age, when C. jejuni can reach densities of 1 × 10⁹ CFU per gram of cecal content, suggesting that the dosing schedule in our study was not optimized for long-term efficiency under field conditions (Al Hakeem et al., 2022). Therefore, further studies are necessary to evaluate the long-term impacts of LIC37 exposure on the gut microbiota in both chicks and adult chickens. Despite these limitations, previous studies have shown that a 2-log decrease in the number of C. jejuni on chicken carcasses can result in a 30-fold decrease in the incidence of C. jejuni infection in humans (Rosenquist et al., 2003; Hermans et al., 2011). Given that this pathogen typically colonizes animals at between 2 and 3 weeks of age (Pielsticker et al., 2012; Al Hakeem et al., 2022), we consider that our dosing regimen, involving LIC37 administration on Days 4 and 6, is likely to be beneficial and effective in preventing C. jejuni infection.

Although, C. jejuni colonization was mitigated by prophylactic dosing with LIC37, the underlying mechanism of this effect remains unknown. In general, there exist four possible (non-mutually exclusive) mechanisms whereby probiotics may prevent C. jejuni infection: (A) stimulation of cellular responses, (B) interactions between pathogens and pre-treated intestinal cells, (C) immunomodulatory effects, and (D) attenuation of intestinal inflammatory processes (Balta et al., 2022). In addition, bacteriocin activity has also been noted as a possible mechanism (Messaoudi et al., 2012; Zommiti et al., 2016). The findings of our previous study suggested that some component of LIC37 exhibited in vitro anti-inflammatory properties, which resulted in the suppression of the production of pro-inflammatory cytokines such as interleukin 6 and tumor necrosis factor α (Tsukagoshi et al., 2020). Therefore, we hypothesize that the in vivo efficacy of LIC37 in this study may involve mechanisms (B) and (C), although further analyses will be needed to confirm this.

On the other hand, we observed that only a limited number of bacterial species exhibited increased abundance in the LIC37-dosed group compared to the C. jejuni group, as assessed by LDA scores (Figure 2). Notably, the genus Blautia, which includes bacteria associated with metabolic and inflammatory diseases as well as biotransformation processes, accumulated in the cecal microbiota of the C. jejuni + LIC37 group (Liu et al., 2021). Blautia has shown potential as a probiotic; for example, mice administered Blautia wexlerae orally have demonstrated attenuation of both high-fat diet–induced obesity and diabetes, an effect that is linked to the unique amino-acid metabolism profile of this bacterium (Hosomi et al., 2022). Furthermore, it should be noted that members of the genus Blautia typically produce bacteriocins as secondary metabolites. Hatziioanou et al. (2017) showed that Blautia obeum A2-162 produces a novel antibiotic, nisin O, which exhibits activity against the enteric poultry pathogen Clostridium perfringens in the presence of trypsin (Sirisopapong et al., 2023). Additionally, oral administration of Limosilactobacillus reuteri and L. fermentum, which are related to L. ingluviei, has been reported to enhance the abundance of Blautia in the gut and fecal microbiota of humans, piglets, and mice (Burakova et al., 2022; Wang et al., 2022; Lin et al., 2023). In this study, the administration of LIC37 may have resulted in increases in the abundance of such beneficial bacteria, which might be advantageous for host health, but the mechanism of this effect remains unclear. Further studies will be needed to clarify the role of Blautia in the gut microbiota and to explore the relationship between the host microbiota and any bacterial pathogens, including C. jejuni.

In conclusion, the findings of the present study suggest that LIC37 may have utility as a probiotic in chicks, demonstrating preventive activity against C. jejuni infection. LIC37 demonstrated excellent adhesion to the cecal wall of chickens in vivo, persisting in the cecum for more than 1 week. Further, dosing with LIC37 led to an increase in the abundance of beneficial microorganisms, including Blautia. These findings indicate that that LIC37 could be a practical and effective probiotic agent against C. jejuni on poultry farms. However, the practical application of LIC37 as a probiotic will require further research to optimize the LIC37 exposure regimen, including the feeding method, timing, and dosage.