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Page 8 of 19 Son et al. Microbiome Res Rep. 2025;4:29 https://dx.doi.org/10.20517/mrr.2025.14

reaches a plateau has varied across studies [10,14] . Some studies have opted to inoculate pigs 3-5 days post-
weaning to capitalize on the stress experienced immediately after weaning [20,24,26] . However, this approach
may reduce the effectiveness of the challenge due to the lingering passive immunity provided by sow’s milk.
Conversely, inoculating too late may reduce susceptibility due to the pigs’ adaptation to post-weaning stress.
Therefore, inoculation at 7 days post-weaning (based on an average age of 21 days at weaning) is considered
+
optimal in the F18 E. coli challenge model.
Inoculation method and dosage are also key variables affecting the success of F18 E. coli infection. To
+
improve consistency, it is recommended to divide the inoculation into multiple doses while monitoring pig
responses, rather than administering a single high dose. This approach accounts for individual variation in
susceptibility. A single high dose may not reliably induce infection across all pigs. To mitigate this issue, 3-4
separate inoculations are often employed. Previous studies have used between 1 and 4 inoculations to
ensure successful infection [9,27-29] . If F18 E. coli colonization is successful, clinical signs such as diarrhea may
+
appear within 24 h [18,36] . Additional symptoms include feed refusal, lethargy, sunken eyes, droopy ears, and
elevated body temperature, often occurring before the onset of diarrhea. These indicators can be used to
assess infection. Based on observed responses, additional inoculations may be necessary to ensure consistent
infection and to avoid false-negative results in intervention studies. Regarding dosage, no universally
accepted standard exists for inducing diarrhea. Reported dosages to induce PWD have ranged from 1.9 ×
8
10
10 to 3 × 10 CFU [24,25,34,35] . Thus, administering multiple moderate doses rather than a single high dose is
recommended to reduce variability in infection outcomes.
THE IMPORTANCE OF RESEARCH ON JEJUNAL MUCOSA-ASSOCIATED MICROBIOTA
AND IMMUNITY IN PIGS
Intestinal health is crucial for pig production, as it directly influences nutrient utilization and growth
performance. When intestinal health is compromised, nutrients that would otherwise support growth are
redirected toward tissue repair and immune responses, ultimately reducing growth efficiency and
productivity. Historically, antimicrobial growth promoters (AGPs) were widely used to maintain intestinal
[77]
health in pigs . However, growing concerns over antimicrobial resistance have led to a decline in AGP
usage, exacerbating intestinal health challenges. Consequently, increasing research attention has focused on
maintaining intestinal health without the use of AGPs [2,39,40] .

Both the composition of the microbiota and intestinal immunity are critical determinants of intestinal
health and must be understood to improve pig health and performance. The intestinal microbiota not only
affects nutrient digestion but also modulates immune responses. Moreover, the intestine is the largest
immune organ in pigs and plays a key role in protecting against pathogens. Considering the essential roles
of both microbiota and immunity, selecting the appropriate intestinal region for research is crucial. Among
the various intestinal segments, the jejunum is particularly important for investigating intestinal immunity
in pigs. It is the first site where the immune system interacts with ingested materials, including pathogens.
Additionally, the jejunum contains both isolated and distributed Peyer’s patches, indicating consistent
immune activity along its length . Several immune-related genes, including C-C chemokine receptor type
[3]
10 and IL-15, are more highly expressed in the jejunum than in the ileum in 28-day-old pigs . As the
[78]
longest portion of the small intestine, the jejunum is also the primary location for nutrient digestion and
absorption in pigs [2,79] . It has more villi and microvilli than the large intestine, resulting in a greater surface
area. Most nutrients are absorbed in the jejunum, with only the remaining portion passed to the ileum and
large intestine. Additionally, the small intestine harbors a lower concentration of microbiota compared to
+
the large intestine, making it more vulnerable to disturbances, such as those caused by F18 E. coli in
challenge models.