Gross Morphometry, Histomorphometry, and Immunohistochemistry Confirm Early and Persistent Jejunal Crypt Hyperplasia in Poults with Enteritis and Depressed Growth

Abstract

Phosphorylated histone 3 (PH3) and cleaved caspase 3 (CCASP3) were used to detect proliferating and apoptotic cells, respectively, in the jejunums of female sibling poults, with and without enteritis and depressed growth, from hatch to day 35. Poults that developed enteritis and depressed growth (SIB flock) were raised on a commercial farm in eastern North Carolina, whereas poults with normal growth and no enteritis (TAU flock) were raised in the Teaching Animal Unit at North Carolina State University College of Veterinary Medicine. Beginning on day 5 through day 35 and at processing, TAU poults were significantly heavier than SIB poults. Jejunal weights, relative jejunal weights, and jejunal densities were greater in SIB poults from day 10 through 35. Jejunal efficiency (body weight /jejunal length) was higher in TAU poults at day 5 and days 10 through 35. Mucosal thickness was greater in SIB poults between days 7 and 21 but greater in TAU poults at days 28 and 35. From day 7 to 35, villus-to-crypt ratios were higher for TAU poults and lower for SIB poults because hyperplastic crypts formed a greater percentage of the mucosa in SIB poults. By day 7, PH3- and CCASP3-positive cells were increased in SIB poults, showing that mucosal changes resulted from combined crypt epithelial hyperplasia and increased apoptosis of villous enterocytes. Findings in this study confirm that enteritis, in the absence of clinical signs, and depressed growth in turkey poults begins by day 7, can be identified microscopically, persists for at least 35 days, is associated with lower processing weights, and has a profound negative effect on turkey growth.

Keywords: crypt hyperplasia; gross morphometry; growth; gut health; histomorphometry; immunohistochemistry; poult enteritis; turkey.

Fig. 1.

Poult (A) and jejunal (B) weights for sampled SIB and TAU poults from day 0 to 35. Box plots show minimum, first quartile, median, third quartile, and maximum values. Significantly different values (p ≤ 0.05) are enclosed in rectangles. (A) TAU poults at 5, 10, 12, and day 21 to 35 were significantly heavier than SIB poults. (B) Except for day 28, jejunal weights of SIB poults were significantly heavier than TAU poults between days 10 and 35. Differences in poult and jejunal weights were greatest at day 35.

Fig. 2.

Relative jejunal weight (A), jejunal length (B), jejunal density (C), and jejunal efficiency (D) for sampled SIB and TAU poults from day 0 to 35. Box plots show minimum, first quartile, median, third quartile, and maximum values. Significantly different values (p ≤ 0.05) are enclosed in rectangles. (A) Relative jejunal weight was greater in TAU poults at day 3 and greater in SIB poults at days 10 to 35, except for day 28 when the relative jejunal weight was high in TAU poults. (B) Jejunal length was similar in both SIB and TAU poults except for day 14 when the jejunum of SIB poults was significantly longer. (C) At day 3, jejunal density was greater in TAU poults. Jejunal density was significantly greater in SIB poults at days 10 to 35, except for day 28. Median values at day 28 were similar for both SIB and TAU poults. (D) Jejunal efficiency was significantly greater in TAU poults at day 5 and days 10 to 35.

Fig. 3.

Median mucosal thickness (A), villous height (B), crypt depth (C), and V:C (D) in sampled SIB and TAU poults from day 0 to 35. Box plots show minimum, first quartile, median, third quartile, and maximum values. Significantly different values (p < 0.05) are enclosed in rectangles. (A) Compared to TAU poults, mucosal thickness was significantly greater in SIB poults at days 7 and 12 to 21, but significantly lower at days 28 and 35. (B) Villous height was greater in SIB poults at day 14, but greater in TAU poults at days 10, 12, and 21 to 35. (C) Crypt depth was greater in SIB poults at days 7 to 35. (D) V:Cs were greater in TAU poults at days 7 to 35.

Fig. 4.

Day 7 TAU and SIB poults, jejunums. (A–C, TAU poult, H&E). (A) Normal villi, crypts, and portion of muscularis; (B) relationship of crypts to base of villi; (C) organized arrangement of crypt enterocytes with occasional mitotic figures. (D–F, SIB poult, H&E, compare with A–C). (D) Mild to moderate crypt hyperplasia; (E) early crypt hyperplasia with dysplasia of crypt enterocytes; (F) hyperplasia and dysplasia of crypt enterocytes. (G–I, TAU poult, PH3). (G) PH3-positive cells in the crypts, brown staining at the tips of villi is not specific; (H) cells positive for PH3 expression in the crypts; (I) higher magnification showing PH3-positive cells in crypts. (J–L, SIB poult, PH3, compare with G–I). (J) PH3-positive cells in the crypts; (K) PH3-positive cells in the crypts; (L) higher magnification showing PH3-positive cells in crypts. (M–O, TAU poult, CCASP3). (M) Relatively few positive cells at villous tips; (N) CCASP3-positive cell at the villus tip; (O) higher magnification showing CCASP3-positive cell at the villous tip. Compare with the number of CCASP3-positive cells in the SIB poult. (P–R, SIB poult, CCASP3, compare with M–O). (P) CCASP3-positive cells in upper third of villi; (Q) CCASP3-positive cells along upper third of villus; (R) higher magnification showing detail of CCASP3-positive cells. Bars, low magnification = 50 μm, two higher magnifications both = 5 μm.

Fig. 5.

Numbers of immunopositive cells expressing PH3 (A) and CCASP3 (B) in SIB and TAU poults. Box plots show minimum, first quartile, median, third quartile, and maximum values. Significantly different values (p < 0.05) are enclosed in rectangles. (A) The number of PH3-positive cells was significantly higher in SIB poults from day 7 through 35, except for day 28 when the number of PH3-positive cells was also high in TAU poults. (B) Compared to TAU poults, SIB poults had greater numbers of CCASP3-positive cells from day 7 to 35, except for day 14. TAU poults had consistently low numbers of CCASP3-positive cells at all sampling times.