Reproduction of sepsis and endocarditis by experimental infection of chickens with Streptococcus gallinaceus and Enterococcus hirae

Figures & data

Figure 1. Growth curves of S. gallinaceus (C13156) and E. hirae (C17410) expressed as optical density (OD 620 nm) and corresponding viable counts (log₁₀ CFU/ml). Inocula used for infection were adjusted to an OD 620 nm of 0.1 in PBS (approximately 10⁸ CFU/ml) taken from exponential growth of culture (approximately 9 h).

Table 1. Recovery of S. gallinaceus post mortem from birds experimentally infected via the brachial vein

		Bacterial isolation^a
Bird number	Days post-infection	Hock joint	Bone marrow	Liver	Spleen	Heart	Gross lesions^b
1	7	−	−	−	−	+	−
2	7	−	−	−	−	−	−
3	7	−	−	+	+	−	S
4	7	−	−	+	+	+	S
5	14	−	+	+	+	+	(S)
6	14	−	+	+	+	+	H, S, Ev
7	14	−	−	−	−	−	R
8	14	−	+	+	+	+	Ev
9	21	−	−	+	+	+	Ev
10	21	−	−	−	−	−	−
11	21	−	−	−	−	−	−
12	21	−	−	−	−	−	−
13	28	−	−	−	−	−	−
14	28	−	−	−	−	−	−
15	28	−	−	−	−	−	−
16	28	−	−	−	−	+	H, (S), Ev
17	28	−	−	−	−	−	−
		0/17	3/17	6/17	6/17	7/17
18^c	5	−	−	+	+	+	H, S, Ev
19	6	−	+	+	+	+	H, S, R, Ev
20	12	−	−	+	−	−	H, S, C, Pc, R
		0/3	1/3	3/3	2/3	2/3
Total (%)		0/20 (0%)	4/20 (20%)	9/20 (45%)	8/20 (40%)	9/20 (45%)
^aBacterial isolation: −, no isolation; +, isolation.
^bGross lesions: −, no lesion; C, circulatory disturbances, including general hyperaemia and subserosal discoloration of fat tissue; Ev, valvular (mitral) endocarditis; H, hepatomegaly; Pc, pericarditis; R, renomegaly; S, splenomegaly. Data in parentheses indicate less severe lesions.
^cBirds that were killed humanely due to development of clinical signs.

Figure 2. Gross postmortem lesions following S. gallinaceus jugular intravenous infection. 2a: hepatomegaly. 2b: splenomegaly with multiple, randomly distributed, irregular, well-demarcated areas of necrosis surrounded by a haemorrhagic zone (arrow). 2c: yellow–white vegetative valvular endocarditis (arrow).

Table 2. Recovery of S. gallinaceus post mortem from birds experimentally infected via the jugular vein

		Bacterial isolation^a
Bird number	Days post-infection	Hock joint	Bone marrow	Liver	Spleen	Heart	Gross lesions^b
1	7	−	+	+	+	+	S, Ev
2	7	−	+	+	+	+	S, Ev
3	7	−	+	−	+	+	−
4	7	−	+	+	+	+	S, Ev
5	7	−	−	−	−	−	S, Ev
6	14	−	−	−	−	(+)	Lc, Fp, Pc, S, Ev
7	14	−	−	−	−	(+)	Ev
8	14	−	−	−	−	(+)	H, Ev, MFNL
9	14	(+)	(+)	−	(+)	+	H, S, Lc, Ev, MFNS, FNM
10	14	−	−	−	−	−	S, Ev
11	21	−	−	−	+	+	H, S, Ev
12	21	−	+	+	+	+	H, S, MFNL/S, R, Ev, FNM
13	21	−	−	(+)	+	+	H, S, Em
14	21	−	−	−	−	−	(Ev)
15	21	−	+	−	−	(+)	(H, S, Ev)
16	23	−	−	−	−	−	S, (Ev)
17	23	−	−	−	−	−	(Lc), S, (Ev)
18	23	−	−	−	−	−	S, (Ev)
		1/18	7/18	5/18	8/18	12/18
19^c	7	−	−	(+)	(+)	+	H, S, C, Ev
20	7	−	−	−	−	+	Lc, C
21	7	−	−	−	+	+	C, H, S, MNFL
22	7	−	−	(+)	+	+	H, S, C, MFNL/S
23	12	−	−	(+)	(+)	+	C, MFNL, Fp, S, R, Ev
24	13	+	+	+	+	+	S, R, Ne, MFNL
		1/6	1/6	4/6	5/6	6/6
Total (%)		2/24 (8%)	8/24 (33%)	9/24 (38%)	13/24 (54%)	18/24 (75%)
^aBacterial isolation: −, no isolation; +, isolation; (+), sparse isolation.
^bGross lesions: −, no lesion; A, ascites; Br, bronchopneumonia; C, circulatory disturbances, including general hyperaemia and subserosal discoloration of fat tissue; Ev, valvular (mitral) endocarditis; Em, mural endocarditis; FNM, focal necrotizing myocarditis; Fp, fibrinous perihepatitis; H, hepatomegaly; Lc, liver cirrhosis; MFNL/S, multiple focal necrosis liver/spleen; Ne, necrotic enteritis; Pc, pericarditis; R, renomegaly; S, splenomegaly. Data in parentheses indicate less severe lesions.
^cBirds that were killed humanely due to development of clinical signs.

Table 3. Recovery of E. hirae and lesions seen post mortem in birds experimentally infected via the brachial vein

		Bacterial isolation^a
Bird number	Days post-infection	Hock joint	Bone marrow	Liver	Spleen	Heart	Gross lesions^b
1	7	−	−	+	+	+	−
2	7	−	−	+	+	+	−
3	7	−	−	+	+	+	S
4	7	−	−	−	−	−	H, S, A
5	7	−	−	+	+	−	Ev
6	14	−	−	−	−	−	−
7	14	−	−	+	+	+	Ev
8	14	−	−	+	−	+	Ev
9	21	−	−	−	−	−	A
10	21	−	−	−	−	−	H, S, C
11	21	−	−	−	−	−	A
12	21	−	−	−	−	−	R
13	28	−	−	−	−	−	−
14	28	−	−	−	−	−	−
15	28	−	−	−	−	−	−
16	28	−	−	−	−	−	−
17	28	−	−	−	−	−	−
18	28	−	−	+	−	+	−
		0/18	0/18	7/18	5/18	6/18
19^c	5	−	+	+	+	−	H, C, Pc, Br
20	5	−	+	+	+	+	C, Ev
		0/2	2/2	2/2	2/2	1/2
Total (%)		0/20 (0%)	2/20 (10%)	9/20 (45%)	7/20 (35%)	7/20 (35%)
^aBacterial isolation: −, no isolation; +, isolation; (+), sparse isolation.
^bGross lesions: −, no lesion; A, ascites; Br, bronchopneumonia; C, circulatory disturbances, including general hyperaemia and subserosal discoloration of fat tissue; Ev, valvular (mitral) endocarditis; H, hepatomegaly; Pc, pericarditis; R, renomegaly; S, splenomegaly. Data in parentheses indicate less severe lesions.
^cBirds that were killed humanely due to development of clinical signs.

Figure 3. Gross postmortem lesions following E. hirae jugular intravenous infection. 3a: hepatomegaly. 3b: splenomegaly with multiple, randomly distributed, irregular, well-demarcated areas of necrosis surrounded by a haemorrhagic zone (arrow). 3c: yellow–white lesion of vegetative valvular endocarditis (arrow denotes thickening of the mitral valve due to vegetative growth).

Table 4. Recovery of E. hirae and lesions seen post mortem in birds experimentally infected via the jugular vein

		Bacterial isolation^a
Bird number	Days post-infection	Hock joint	Bone marrow	Liver	Spleen	Heart	Gross lesions^b
1	7	+	+	+	+	+	H, S, MFNL/S, Fp, Ev
2	7	−	−	−	−	(+)	S
3	7	(+)	−	+	(+)	(+)	S
4	7	(+)	−	(+)	−	+	(S), (Ev)
5	7	(+)	+	+	+	+	H, S, MFNL, Ev
6	14	−	−	−	−	−	H, S,
7	14	(+)	−	−	−	−	Lc, Fp, A
8	14	−	−	+	+	+	S, (Ev)
9	14	−	−	−	−	−	S, Fp, (Ev)
10	14	−	+	+	+	+	H, S, MFNL/S, Pc, Ev
11	21	−	−	−	−	(+)	S, (Ev)
12	21	−	(+)	+	+	+	S, (Ev)
13	21	−	+	+	+	(+)	−
14	21	(+)	(+)	+	(+)	+	(S), (Ev)
15	21	(+)	−	(+)	+	(+)	(S), (Ev)
16	23	−	−	−	−	−	S
17	23	−	−	−	−	−	Pc, S, (Ev)
18	23	−	−	−	−	−	(S)
		7/18	6/18	10/18	9/18	12/18
19^c	7	+	+	+	+	+	S, C
20	12	+	+	+	+	+	S, C
21	13	(+)	−	+	+	+	MFNL, S, R, Ev
22	16	−	+	+	+	+	H, S, C
		3/4	3/4	4/4	4/4	4/4
Total (%)		10/22 (45%)	9/22 (41%)	14/22 (64%)	13/22 (59%)	16/22 (73%)
^aBacterial isolation: −, no isolation; +, isolation; (+), sparse isolation.
^bGross lesions: −, no lesion; A, ascites; Br, bronchopneumonia; C, circulatory disturbances, including general hyperaemia and subserosal discoloration of fat tissue; Ev, valvular (mitral) endocarditis; Em, mural endocarditis; FNM, focal necrotizing myocarditis; Fp, fibrinous perihepatitis; H, hepatomegaly; Lc, liver cirrhosis; MFNL/S, multiple focal necrosis liver/spleen; Ne, necrotic enteritis; Pc, pericarditis; R, renomegaly; S, splenomegaly. Data in parentheses indicate less severe lesions.
^cBirds that were killed humanely due to development of clinical signs.

Figure 4. Percent of positive cultures post mortem from specific tissues from birds at times post infection with (4a) S. gallinaceus via the brachial vein, (4b) E. hirae via the brachial vein, (4c) S. gallinaceus via the jugular vein, and (4d) E. hirae via the jugular vein.

Figure 5. Mitral valve endocarditis induced by S. gallinaceus 3 weeks post-infection. The bird had hepatomegaly and splenomegaly with necrosis, renomegaly and both valvular and mural endocarditis. There is formation of a vegetative lesion at the mitral valve (m) consisting of bacteria, fibrin and basophils (full arrow), surrounded by a corona consisting mainly of heterophils and erythrocytes in the periphery (open arrow).

Table 5. Comparison by Fisher Exact test of recovery of S. gallinaceus and E. hirae from different anatomical sites after experimental infection of 4-week-old birds the brachial vein and jugular vein

	Site of culture
Comparison group	Heart	Liver	Spleen	Bone marrow	Hock joint
Control versus
S. gallinaceus (jugular)	<0.001*	0.03*	<0.005*	0.07*	0.49
S. gallinaceus (brachial)	0.01*	0.07	0.07	0.53	NA
E. hirae (jugular)	<0.001*	<0.001*	<0.001*	0.002*	0.002*
E. hirae (brachial)	0.03*	0.01*	0.03*	NA	NA
S. gallinaceus (jugular) versus
S. gallinaceus (brachial)	0.063	0.75	0.14	0.29	0.49
E. hirae (jugular)	1.0	0.14	0.77	0.76	0.007*
S. gallinaceus (brachial) versus
E. hirae (brachial)	0.75	0.52	1.0	0.23	NA
E. hirae (jugular) versus
E. hirae (brachial)	0.029*	0.35	0.14	0.002*	<0.001*
Data are the P value for the probability that the two groups are similar. Statistical significance: *P<0.05. NA, not applicable due to zero values.