Effects of cold stress and Salmonella Heidelberg infection on bacterial load and immunity of chickens

Figures & data

Table 1. Markers used for blood cell immunophenotyping.

Panel	Receptor	Fluorophore	Antibody
A	CD45	APC	Mouse Anti-Chicken CD45
	Macrophage/Monocyte Kul01	FITC	Mouse Anti-Chicken Monocyte/Macrophage
	MHCII	RPE	Mouse Anti-Chicken MHC class II clone 2G11
B	CD45	APC	Mouse Anti-Chicken CD45
	CD3	FITC	Mouse Anti-Chicken CD3 clone CT-3
	CD4	RPE	Mouse Anti-Chicken CD4 clone CT-4
	CD8 alfa	Biotin-streptavidin	Mouse Anti-Chicken CD8 clone CT-8
C	CD45	APC	Mouse Anti-Chicken CD45
	CD8 alfa	RPE	Mouse Anti-Chicken CD8 clone CT-8
	TCR1	Biotin-streptavidin	Mouse Anti-Chicken TCR γδ clone TCR-1

Note: FITC = Fluorescein ; RPE = R-phycoerithrin; APC = Allophycocyanin. Streptavidin conjugation was used to label biotinylated primary antibodies. All antibodies were produced by Southern Biotechcompany (Birmingham, AL, USA).

Figure 1. Effects of cold stress (19 ± 1°C, 6 h/day, from ED 1 to ED7) on corticosterone plasma levels (ng/ml) in SPF chickens studied at 7 (ED7) (a) and 21 (ED21) (b) days old that were infected (Group PCS) or not infected (Group CS) with Salmonella Heidelberg. Data are presented as the mean ± standard deviation (n = 8/group). *P < 0.05 compared to Control (Group C) and positive Salmonella Heidelberg control (Group PC) groups (two-way ANOVA followed by Tukey–Kramer test).

Table 2. Plasma concentrations of EPI and NOR in SPF chickens at ED7 and ED21 challenged or not with cold stress and/or Salmonella Heidelberg (SH).

Groups	Epinephrine ED7 (ng/ml) Mean ± SD	Norepinephrine ED7 (ng/ml) Mean ± SD	Epinephrine ED21 (ng/ml) Mean ± SD	Norepinephrine ED21 (ng/ml) mean ± SD
Not infected
C	2.82 ± 0.14	0.66 ± 0.56	1.47 ± 0.65	0.56 ± 0.40*
CS	2.65 ± 1.30	0.68 ± 0.58	2.97 ± 1.20	1.16 ± 0.24^#
Infected
PC	2.82 ± 0.63	2.53 ± 0.64	3.95 ± 1.18	1.05 ± 0.05*
PCS	1.52 ± 0.64	1.78 ± 0.68	3.53 ± 0.57	1.67 ± 0.11^§

Note: C = not infected and kept at thermoneutral zone; PC = SH-infected and kept at thermoneutral zone; CS = not infected and cold stressed at 19 ± 1°C, 6 h/day, from ED1 to ED7; PCS = SH infected and cold stressed at 19 ± 1°C 6 h/day/7 days from ED1 to ED7. Data are the means ± SD of eight birds per group.

*P < 0.05 compared with the respective control groups (Group C or Group PC).

^#P < 0.05 compared the C and PC groups.

^§P < 0.05 compared the PC and PCS groups.

Figure 2. Effects of cold stress (19 ± 1°C, 6 h /day, from ED 1 to ED7) on Salmonella Heidelberg caecal counts in SPF chickens at 7 (ED7) and 21 (ED21) days of age in both infected groups, with PC kept at a thermoneutral temperature during the entire trial versus the cold stressed (PCS) group. *P < 0.05 compared with the PC and PCS groups (ANOVA followed by Tukey–Kramer test). The mortality of the birds was 20% in the PC group and 40.6% in the PCS group. There was no mortality during the entire trial in the C and CS groups.

Table 3. Peripheral blood flow cytometry from SPF chickens at 7 days of age (ED7) challenged with cold stress and/or Salmonella Heidelberg (SH) groups or control groups.

	CD45^+
	CD3^+
Groups	Kul01/MHCII (% ± SD)	CD3^+ (% ± SD)	CD4^+ (% ± SD)	CD8^+ (% ± SD)	CD4^+/CD8^+ (% ± SD)	CD8^−TCRI^+ (% ± SD)	CD8^+TCRI^+ (% ± SD)
Not Infected
C	6.07 ± 1.4^#	2.15 ± 0.45	6.16 ± 4.37	55.24 ± 3.9^#	0.10 ± 0.07	12.54 ± 4.9^#	1.33 ± 0.6
CS	4.52 ± 0.72*	3.50 ± 0.49	8.67 ± 5.0	36.72 ± 10.6*	0.25 ± 1.1	7.74 ± 1.1*	0.81 ± 0.2
Infected
PC	0.20 ± 0.06	10.17 ± 2.6^§	20.3 ± 8.1	12.95 ± 4.1	1.91 ± 0.21	6.57 ± 2.4	0.45 ± 0.2
PCS	0.35 ± 0.07	7.48 ± 2.50*	23.97 ± 2.7	9.21 ± 4.2	2.11 ± 0.4	6.56 ± 3.0	0.77 ± 0.5

Note: C = not infected and kept at thermoneutral zone; PC = SH-infected and kept at thermoneutral zone; CS = not infected and cold stressed at 19 ± 1°C 6 h/day/7 days from ED1 to ED7; PCS = SH-infected and cold stressed at 19 ± 1°C, 6 h/day, from ED1 to ED7. Data are the means ± SD of eight birds per group.

*P < 0.05 compared with the respective control groups (Group C or Group PC).

^#P < 0.05 compared the C and PC groups.

^§P < 0.05 compared the PC and PCS groups.

Table 4. Peripheral blood flow cytometry from SPF chickens at 21 days of age (ED21) challenged with cold stress and/or Salmonella Heidelberg (SH) groups or control groups.

	CD45^+
	CD3^+
Groups	Kul01/MHCII^+ (% ± SD)	CD3^+ (% ± SD)	CD4^+ (% ± SD)	CD8^+ (% ± SD)	CD4^+/CD8^+ (% ± SD)	CD8^-TCRI^+ (% ± SD)	CD8^+TCRI^+ (% ± sd)
Not Infected
C	5.76 ± 1.65*	6.14 ± 0.81	17.07 ± 3.12*	5.94 ± 0.67	2.93 ± 0.8*^b	2.24 ± 0.34	8.18 ± 2.2^#
CS	7.22 ± 0.83^#	9.34 ± 1.2	27.75 ± 6.9^#	3.18 ± 0.61	8.85 ± 2.6^#	1.95 ± 0.49	6.04 ± 2.0*
Infected
PC	2.22 ± 0.92	18.36 ± 3.7^§	10.85 ± 2.2	11.16 ± 5.3^§	1.08 ± 0.63	1.25 ± 0.59	4.50 ± 0.48
PCS	3.13 ± 1.09	12.18 ± 6.5*	12.3 ± 6.4	6.33 ± 1.0*	1.85 ± 0.80	1.33 ± 0.33	3.82 ± 0.33

Note: C = not infected and kept at thermoneutral zone; PC = SH-infected and kept at thermoneutral zone; CS = not infected and cold stressed at 19 ± 1°C 6 h/day/7 days from ED1 to ED7; PCS = SH-infected and cold stressed at 19 ± 1°C, 6 h/day, 7 days ED1 to ED7. Data are the means ± SD of eight birds per group.

*P < 0.05 compared with the respective control groups (Group C or Group PC).

^#P < 0.05 compared the C and PC groups.

^§P < 0.05 compared the PC and PCS groups.