Unravelling fatty liver haemorrhagic syndrome: 2. Inflammation and pathophysiology

Figures & data

Table 1. Primer and probe sequences for qRT-PCR.

Target RNA	Probe/Primer^a	Sequence	Accession number
28S	P	5-(FAM)-AGGACCGCTACGGACCTCCACCA-(TAMRA)-3	X59733
	F	5-GGCGAAGCCAGAGGAAACT-3
	R	5-GACGACCGATTTGCACGTC-3
IL-1β	P	5-(FAM)-CCACACTGCAGCTGGAGGAAGCC-(TAMRA)-3	AJ245728
	F	5-GCTCTACATGTCGTGTGTGATGAG-3
	R	5-TGTCGATGTCCCGCATGA (FAM)-3
IL-6	P	5-(FAM)-AGGAGAAATGCCTGACGAAGCTCTCCA-(TAMRA)-3	AJ250838
	F	5-GCTCGCCGGCTTCGA-3
	R	5-GGTAGGTCTGAAAGGCGAACAG-3
IL-18	P	5-(FAM)-TCTTTACCAGCGTCCTACCTTGCGACA-(TAMRA)-3	AJ009800
	F	5-GCCCTCCTCCTGGTTTCAG-3
	R	5-TGGCACCGCAGCTCATT-3

^aP, probe; F, forward primer; R, reverse primer.

Table 2. Effect of oestrogen injections, and a single LPS injection on mortality, the incidence of FLHS, and liver haemorrhagic score of laying hens^a.

Treatments^b	Number of birds	FLHS^d (%)	Haemorrhagic score^d	Mortality (%)
Control^c	54	5.5^e	1–2	0
E2	18	66.7^g 33.3^f	4–5 2–3	11^f
LPS	18	5.5^e 11.1^e	2–3 1	0
E2&LPS	18	88.9^h 11.1^e	4–5 2–3	5.5^e

^aData are reported for the whole experimental period; number of birds sacrificed at each sampling point was three birds/treatment. At the end of the experimental period, all birds were sacrificed and underwent post mortem examination.

^bControl = untreated; E₂ = oestrogen-treated; LPS = LPS-treated; E₂ & LPS = E₂- & LPS-treated.

^cThere were no significant differences between untreated and oil-treated or saline-treated groups; therefore, data are pooled and presented together.

^dData on the occurrence of FLHS are divided to reflect haemorrhagic scores and % occurrence as diagnosed at necropsy.

^e,f,g,hValues within a column with different superscripts differ significantly (P < 0.05).

Table 3. Changes in blood cell counts (WBC and RBC), heterophil (H) and lymphocyte (L) percentages and H/L ratios in control and treated laying hens.^a,b

Treatment	Time	WBC^a (x10^9/L)	Heterophils (%)	Lymphocytes (%)	H/L ratio	RBC^b (x10^12/L)
CONT	3 h	18.0^c	29.6^c	61.9^c	0.48^c	2.5^c,d
E2		25.7^d	28.0^c	62.2 ^c	0.45^c	2.3^c
LPS		27.0^d	28.2^c	65.4 ^c	0.43^c	2.1^c
E2 & LPS		21.7^c,d	26.7^c	64.3 ^c	0.42^c	2.2^c
CONT	24 h	17.5^c	25.6^c	63.1 ^c	0.41^c	2.6^d
E2		28.2^d	29.0^c	64.1 ^c	0.45^c	2.2^c
LPS		35.8^d,e	53.6^e	37.8^d	1.42^e	2.3^c
E2 & LPS		43.8^d,e	55.6^e	36.2^d	1.54^e	2.2^c
CONT	1 wk	18.7^c	22.6^c	66.1^c	0.34^c	2.4^c,d
E2		42.7^d,e	35.9^d	54.4^c,d	0.66^d	2.3^c
LPS		31.9^d	48.8^d,e	38.0^d	1.28^e	2.0^c
E2 & LPS		34.8^d,e	51.6^e	42.9^d	1.20^e	2.5^c,d
CONT	2 wk	15.9^c	29.4^c	59.4^c	0.49^c	2.2^c
E2		52.3^e	36.1^d	55.5^c,d	0.65^d	2.6^d
LPS		18.6^c	22.0^c	66.5^c	0.33^c	2.4^c,d
E2 & LPS		35.5^d,e	30.3^d	58.2^c	0.52^d	2.2^c

^aE₂ = oestrogen-treated; LPS = LPS-treated; E₂ & LPS= E₂- and LPS-treated; CONT = data are pooled for all controls (not treated, corn-oil treated and PBS-treated birds); WBC = white blood cells; RBC = red blood cells;

^bValues are expressed as mean (n = 6). Means with different superscripts (c–e) within a column are significantly different (P < 0.05).

Figure 1. Plasma concentrations of fibrinogen, triglycerides (TG), and gamma glutamyl transferase (GGT) in treated and untreated hens. E₂ = oestrogen-treated; LPS = LPS-treated; E₂ & LPS= E₂- and LPS-treated; control = data are pooled for all controls (not treated, corn-oil treated and PBS-treated hens). Values are expressed as mean ± SEM (n = 6).

Figure 2. Histological sections (H&E stain) showing livers from control, LPS-treated, and E₂- & LPS-treated hens. Image A (×200) shows normal portal triad from control hens (V = portal venule; AR = hepatic artery; BD = bile duct), with some hepatocyte fat infiltration (thin arrows). Image B (×200) shows inflammatory cell infiltration (thick arrow) in the periportal area surrounding portal triad. Images C and D (×400) show periportal inflammation in livers from E₂-treated hens. Note moderate periportal inflammation (thick arrow) and fat infiltration (thin arrow). Images E and F (×400) show inflammation and haemorrhages in livers from LPS- & E₂-treated hens. Note severe parenchymal and sinusoidal (S) fat (thin arrow), leukocyte (thick arrow), RBC (double-headed arrow) infiltration and sinusoidal dilation (lined area), congestion and telangiectasia.

Figure 3. Cytokine mRNA expression profiles from real-time qRT-PCR analyses of hepatocytes of treated hens compared to baseline levels and controls at 3 h and 24 h post-treatments. E₂ = oestrogen-treated; LPS = LPS-treated; E₂ & LPS= E₂- and LPS-treated. All Ct values were corrected using the housekeeping gene 28S, and time point 0 was used as the calibrator. Values are expressed as mean ± SEM fold change relative to control (data are pooled for all controls: not treated, corn-oil treated and PBS-treated birds). Error bars show SEM from triplicate samples (n = 16) from two separate qRT-PCR experiments (P < 0.01, unpaired t-test).