Ultrastructural Changes in The Ileal Mucosa of Broilers Exposed to Necrotic Enteritis and Bacillus Amyloliquefaciens H57

The villi of the intestinal mucosa are lined by a single layer of columnar cells, comprising enterocytes, goblet and enteroendocrine cells, and various types of immune cells, each with a distinct function. Chicken gut mucosa is exposed to an enormous number of feed antigens, and pathogenic bacteria that often impair the intestinal barrier function. Ultrastructural examination of enterocytes, their organelles and other features, such as mitochondria, microvilli, and tight junctions sealing adjacent epithelial cells, can help to reveal the state of gut health. In chickens, disruption of the epithelium and disintegration of villi occur following gut infections, including necrotic enteritis (NE), which can cause severe damage. Birds with subclinical NE do not display clinical signs of disease, but can experience malabsorption and poor growth. Probiotics are being investigated as alternatives to antimicrobials, and research with the probiotic Bacillus amyloliquefaciens strain H57 (H57) has shown significant effects on performance and feed utilization. The aim of this study was to examine ultrastructural events occurring on ileal epithelium in broilers exposed to subclinical NE and fed H57 (2x10⁸ spores/g feed). Subclinical NE was induced in chicks exposed to a high dose of Eimeria vaccine via drinking water, and Clostridium perfringens (CP) culture mixed with feed as described by Shini et al. (2020). On day 21, six birds per treatment were sampled and, ileal mucosa was evaluated using scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

The exposure of broilers to coinfection with Eimeria and CP caused subclinical NE as shown by lesions on the intestinal mucosa. Ultramicroscopic examination of ileal mucosa revealed significant villus damage, such as focal erosions of epithelial cells, exposure of lamina propria, and villous atrophy. The degree of damage was found to be higher (P < 0.001) in NE birds when compared to other treatments, and it was graded as 2.6 vs. 0.8 in NE vs. NE-H57 birds, respectively. There was a higher (P < 0.05) abundance of segmented filamentous bacteria attached to villi in birds exposed to NE. TEM revealed severe enterocyte damage, and loss of cellular integrity in NE birds. Mitochondria, in particular, were undergoing morphological alterations. In NE birds, mitochondria were irregular in form, containing electron-lucent regions of matrix, swollen or damaged cristae, most probably due to oxidative stress, resulting in increased ROS exposure and lowered ATP production. In control, H57, and NE-H57 birds, most mitochondria were round or elongated, with mild or no structural damage. Tight junctions were longer (P < 0.001) and more regular in the control and H57 treated birds when compared to NE birds. Tight junctions are crucial for the maintenance of epithelial barrier integrity. The results suggest that H57 contributes to the maintenance of intestinal barrier integrity and function. Establishing the links between the mucosa and probiotics will enable the development of new strategies to maintain gut health in the antibiotic-free era in commercial poultry.

ACKNOWLEDGMENT: Funding was provided by an Advance Queensland Industry Partnership grant.