Induction of eggshell apex abnormalities by Mycoplasma synoviae: field and experimental studies

Figures & data

Table 1.  Serology and mycoplasma culture of oviducts of hens from farms producing eggs with EAA and farms producing unaffected eggs

	Farm number	Number of birds investigated	Serum antibodies against M. synoviae ^a	Oviduct positive by mycoplasma culture^b
Producing eggs with EAA	1	3	1	1
	2	6	6	1
	3	3	1	2
Producing unaffected eggs	4	5	5	0
	5	5	3	0
	6	5	5	0
^aAgglutination at dilution ≥ 1:8. ^bMycoplasma cultures identified as M. synoviae by PCR.

Table 2.  Egg quality measurements, mycoplasma serology and mycoplasma culture of oviducts in Field study 2 (treatment trial)

		Farm producing eggs with EAA			Control farm
	Week	Untreated hens producing eggs with EAA	Treated hens producing eggs with EAA^a	Hens producing unaffected eggs	Hens producing unaffected eggs
M. synoviae RPA test^b,c	0	20/20^A	20/20^A	–	–
	3	–	–	–	–
	4	10/10^A	8/10^A	–	–
	7	10/10^A	10/10^A	9/10^A	9/9^A
Mycoplasma culture oviduct^b,e	4	8/10^A	7/10^A	–	–
	7	9/10^A	9/10^A	2/10^B	0/9^B
Eggshell strength (N) (n = 10 to 20)^d	3	15.9 (2.2)^A	–	–	34.1 (1.7)^B
	4	14.4 (3.2)^A	30.4 (3.0)^B	–	32.4 (1.3)^B
	7	16.2 (1.8)^A	13.9 (1.8)^A	–	31.1 (1.8)^B
Haugh units (n = 12 to 20)^d	3	90.1 (1.5)^A	^-	–	87.2 (1.5)^A
	4	85.1 (2.3)^A	82.5 (1.2)^A	–	80.1 (1.2)^A
	7	74.2 (5.3)^A	81.2 (2.0)^A		80.1 (1.6)^A
Egg white thickness (mm) (n = 10 to 20)^d	3	8.4 (0.3)^A	–	–	7.8 (0.3)^A
	4	7.4 (0.4)^A	6.9 (0.2)^A	–	6.6 (0.2)^A
	7	6.4 (0.4)^A	6.9 (0.3)^A	–	6.8 (0.3)^A
Means within the same row with the same uppercase superscript letter are not significantly different (P > 0.05). ^aTreated birds were given a single dose of long-acting oxytetracycline at 3 weeks. ^bTwo-sample proportion test. ^cAgglutination at dilution ≥1:8 were considered positive. ^dMean (± standard error of the mean) and statistical analysis with Kruskal–Wallis one-way analysis of variance. ^eBetween two and five mycoplasma positive cultures were tested by PCR and identified as M. synoviae.

Figure 1.  Total egg production and production of eggs with EAA in Field study 2. Boxed numbers (21 to 40) indicate the chickens treated with long-acting oxytetracycline at day 25. Note the temporary production of normal eggs for 12 days (day 28 until day 40) after administration of antibiotic.

Figure 2.  2a: Eggshell apex abnormality characterized by an altered shell surface, shell thinning, increased translucency, and the occurrence of cracks and breaks. Eggshell pathology is confined to a region approximately 2 cm from the apex. The picture shows eggs originating from brown and white layer chickens. 2b: The clear demarcation zone separating the affected eggshell from the remainder of the shell is more apparent at candling. The abnormal eggshell has increased translucency.

Table 3.  Serology, mycoplasma culture of the oviduct, growth, eggshell strength, egg production and production of eggs with EAA in the experimental infection study

	Week	Control	M. synoviae (i.v.)	M. synoviae (i.v.) + IBV	M. synoviae (i.t)	M. synoviae (i.t.) + IBV
n		12	18	17	18	17
Mean IBV HI titre^a	0	–	–	4.4 (0.2)^A	–	4.4 (0.2)^A
	4	4.5 (0.2)^A	3.6 (0.3)^A	7.1 (0.3)^B	4.9 (0.3)^A	7.1 (0.3)^B
	8	4.5 (0.3)^A	4.8 (0.2)^A	6.9 (0.3)^B	3.9 (0.1)^A	7.8 (0.4)^B
	11	4.4 (0.1)^A	4.1 (0.1)^A	6.8 (0.2)^B	4.2 (0.3)^A	7.4 (0.4)^B
M. synoviae RPA test positive^b,c	0	0/12^A	0/18^A	0/17^A	0/18^A	0/17^A
	4	0/12^A	15/18^B	12/17^B	14/18^B	12/17^B
	8	0/12^A	17/17^B,f	17/17^B	17/17^B,f	13/17^B
	11	0/12^A	13/17^B	13/16^B,f	15/17^B	11/17^B
Oviduct Mycoplasma culture positive^b,d	11	0/12^CD	0/17^C	1/16^AC	6/17^ABD	7/17^B
Weight (g)^a	0	1230 (19)^A	1248 (17)^A	1261 (14)^A	1261 (18)^A	1258 (18)^A
Increase in body weight (g)^a	0 to 11	474 (20)^A	384 (21)^AB	368 (18)^B	486 (28)^A	372 (20)^B
Mean daily egg production per chicken^a	1 to 11	0.75 (0.02)^A	0.61 (0.03)^B	0.62 (0.03)^B	0.49 (0.02)^C	0.54 (0.03)^B,C
Eggs with EAA	4 to 11	0	0	15	49	96
Mean daily EAA eggs per chicken^a	4 to 11	0.0 (0.00)	0.0 (0.00)	0.02 (0.00)^A	0.06 (0.01)^B	0.1 (0.01)^C
Eggshell strength (N)
Unaffected eggs (n=60 per group)^e	4 to 5	37.7 (1.4)^A	–	38.2 (1.5)^A	–	–
EAA eggs of 3 groups (n=60)^e	4 to 5	–	–	16.8 (0.9)^B	–	–
Unaffected eggs (n=30 per group)^e	9 to 11	31.7 (1.8)^A	35.1 (1.8)^A	35.2 (2.0)^A	35.6 (1.6)^A	37.2 (1.6)^A
EAA eggs of 3 groups (n=30)^e	9 to 11	–	–	12.7 (1.0)^B	–	–
Means with the same uppercase superscript letter within the same row are not significantly different (P > 0.05). ^aMean (± standard error of the mean) and statistical analysis with Kruskal–Wallis one-way analysis of variance. ^bTwo-sample proportion test. ^cAgglutination at dilution ≥1:8 was considered positive. ^dBetween one and two mycoplasma positive cultures were tested by PCR and identified as M. synoviae. ^eMean (± standard error of the mean) and statistical analysis with Kruskal–Wallis one-way analysis of variance; means within the same row and between rows (no eggs with EAA and eggs with EAA) with the same superscript are not significantly different (P > 0.05). ^fOne bird died.

Figure 3.  Production of eggs with EAA after experimental infection. Eggs with EAA were not produced in the control group and the group inoculated only with M. synoviae intravenously.

Figure 4.  Dendrogram constructed from the unweighted paired group method with arithmetic averages analysis of HindIII/HhaI AFLP patterns. The strains and isolates included are: from Field study 1, SP2005-00257 A (Farm 1) and SP2005-00255 B (Farm 3, also inoculum used for experimental infection); from Field study 2, SP2007-00333-3 (untreated bird), 00333-4 (untreated bird), 00333-14 (treated bird), 00333-15 (treated bird) and SP20007-00426-1 (bird producing unaffected eggs); from the experimental infection study, SP2007-00597-15 and SP2007 00601-24; and two additional field isolates, SP2002-00804 and SP2007-01054. Three different clusters of M. synoviae strains could be identified. M. synoviae isolates from oviducts of hens producing eggs with EAA were not clonal. The inoculum strain had 88% similarity with the oviduct reisolates from the animal experiment. Isolate SP2007-00426-1 from Field study 2 had 85% similarity with the other oviduct isolates from this study.

Figure 5.  5a: SEM image of an unaffected eggshell showing the inner membranes (A), the mammillary knob layer (B) and part of the palisade layer (C). Bar = 50 µm. 5b: SEM image of an abnormal apical eggshell. The mammillary knob layer is absent and only part of the palisade layer is present: inner membranes (A), palisade layer (B), vertical layer (arrow) and cuticule (arrowhead). Note also the increased density of the inner membranes. Bar = 100 µm.