Reproducibility of swollen sinuses in broilers by experimental infection with avian metapneumovirus subtypes A and B of turkey origin and their comparative pathogenesis

Figures & data

Figure 1.  Percentage of broilers per group showing clinical signs and clinical score indices from (1a) Experiment 1 and (1b) Experiment 2. Bar graph shows the percentage of birds/group showing clinical signs, line graph shows clinical score indices of the groups. n = 5/group and experimental day. Clinical score index = the sum of scores observed/total number of chickens per group at each day point. Chickens that received virus-free TOC medium were free of clinical signs throughout both experiments.

Figure 2.  Clinical signs of aMPV-infected broilers at 9 d.p.i. 2a: Control broiler. 2b: aMPV subtype-B-inoculated broiler showing swelling of periorbital sinuses and the area around the eye (black arrow) and clear nasal exudate (white arrow). These clinical signs were also observed in aMPV subtype-A-inoculated birds (data not shown).

Figure 3.  Histopathological lesions in the nasal turbinate at 6 d.p.i. (H & E stain, 200x magnification). 3a: Intact ciliated epithelium of a control broiler (arrows). 3b: Nasal turbinate of an aMPV subtype-B-inoculated broiler showing infiltration of lymphoid cells in the epithelium (black arrow) and hypertrophy of the mucous gland (white arrow) (score 3). Nasal turbinates of aMPV subtype-A-inoculated broilers show similar lesions.

Table 1.  Mean histopathological lesion scores in aMPV-inoculated and control broilers

	Mean histopathological lesion score±standard error of the mean
	Nasal turbinate			Trachea
Days post inoculation	Control^a	aMPV subtype A^b	aMPV subtype B^c	Control	aMPV subtype A	aMPV subtype B
Experiment 1
3	0.0±0.00^B	1.2±0.37^A	1.8±0.37^A	0.0±0.00	0.4±0.24	0.8±0.37
6	0.0±0.00^B	2.4±0.24^A	2.4±0.24^A	0.0±0.00^B	2.0±0.45^A	2.4±0.24^A
11	0.0±0.00^B	2.0±0.32^A	2.6±0.24^A	0.0±0.00^B	2.2±0.37^A	2.4±0.32^A
14	0.0±0.00	0.8±0.37	0.6±0.24	0.0±0.00	0.6±0.24	0.8±0.37
17	0.0±0.00	0.4±0.24	0.4±0.24	0.0±0.00	0.2±0.20	0.6±0.24
Experiment 2
3	0.0±0.00^B	1.4±0.40^A	1.6±0.24^A	0.00±0.00	1.2±0.37	1.0±0.55
6	0.0±0.00^B	2.6±0.24^A	2.6±0.24^A	0.00±0.00^B	2.2±0.37^A	2.8±0.20^A
10	0.0±0.00^B	1.6±0.40^A	2.4±0.40^A	0.00±0.00^B	1.6±0.51^A	2.2±0.37^A
14	0.0±0.00^B	1.4±0.24^A	1.2±0.48^A	0.00±0.00^B	1.2±0.49^A	1.2±0.20^A
21	0.0±0.00	0.4±0.24	0.4±0.24	0.00±0.00	0.2±0.20	0.2±0.20
^aControl chickens, inoculated with virus-free TOC media. ^bInoculated with 10^4 CD50 aMPV subtype A. ^cInoculated with 10^4 CD50 aMPV subtype B. ^A,BMeans with different uppercase superscript letters within the same row and organ are significantly different (P < 0.05, Duncan's multiple range test, n = 5).

Figure 4.  Histopathological lesions of the trachea at 6 d.p.i. (H & E stain, 200x magnification). 4a: Intact ciliated epithelium of a control broiler (arrow). 4b: Trachea of an aMPV subtype-A-inoculated broiler with thickening due to oedema and infiltration of inflammatory cells in the epithelium (black arrow) and focal exfoliation (white arrow) (score 2). Tracheae of aMPV subtype-A-inoculated broilers show similar lesions.

Figure 5.  Histopathological lesions of the infraorbital sinus epithelium at 6 d.p.i. (H & E stain, 100x magnification). 5a: Control group. 5b: Infraorbital sinus epithelium of an aMPV subtype-B-inoculated broiler showing infiltration of lymphoid cells (black arrow) and destruction of the epithelium (white arrow). Infraorbital sinus epithelium of an aMPV subtype-A-inoculated broiler shows similar lesions.

Figure 6.  Histopathological lesions of the secondary bronchi of the lung, at 6 d.p.i. (H & E stain, 100x magnification). 6a: Control group. 6b: Secondary bronchi of an aMPV subtype-B-inoculated broilers showing formation of lymphoid follicles in the submucosa (arrows). Secondary bronchi of an aMPV subtype-A-inoculated broiler show a similar lesion.

Figure 7.  Histopathological lesions of the Harderian gland at 6 d.p.i. (H & E stain, 200x magnification). 7a: Control group. 7b: Harderian gland of an aMPV subtype-B-inoculated broiler showing infiltration of lymphocytes and lymphoid follicles around the secondary collecting duct (arrows). The Harderian gland of an aMPV subtype-A-inoculated broiler shows a similar lesion.

Figure 8.  Histopathological lesions of the lachrymal gland at 6 d.p.i. (H & E stain, 100x magnification). 8a: Control group. 8b: Lachrymal gland of an aMPV subtype-B-inoculated broiler showing infiltration of lymphoid cells in the interstitial tissues and interlobular tissues (arrows). Lachrymal glands of an aMPV subtype-A-inoculated broiler show similar lesions.

Table 2.  Detection of the aMPV genome in broiler tissues by RT-nested PCR

	Detection of aMPV in Experiment 1/Experiment 2
	3 d.p.i.		6 d.p.i.		11/10 d.p.i.
Tissue sample	aMPV subtype A^a	aMPV subtype B^b	aMPV subtype A	aMPV subtype B	aMPV subtype A	aMPV subtype B
Nasal turbinate	+/ +	+/ +	+/ +	+/ +	−/ −	−/ −
Harderian gland	+/ +	+/ +	+/ −	+/ +	−/ −	−/ +
Trachea	−/ +	+/ +	+/ −	+/ +	−/ −	+/ −
Lung	−/ −	−/ +	−/ −	+/ +	−/ −	−/ −
Spleen	−/ −	−/ −	−/ −	+/ +	−/ −	−/ −
Caecal tonsil	nd/ −	nd/ −	nd/ −	nd/ +	nd/ −	nd/ −
Bursa cloacalis	−/ −	−/ +	−/ −	+/ −	−/ −	−/ −
Cloacal swabs	nd/ −	nd/ −	nd/ −	nd/ +	nd/ −	nd/ −
Chickens that had received virus-free TOC media were free of aMPV throughout the experimental period.+, aMPV-positive; –, aMPV-negative; nd, not done. ^aChickens inoculated oculonasally with 10^4 CD50 aMPV subtype A. ^bChickens inoculated oculonasally with 10^4 CD50 aMPV subtype B.

Table 3.  aMPV-antibody detection in broilers inoculated with aMPV subtype A or subtype B

	Number of birds positive for aMPV antibodies/number tested
	Experiment 1		Experiment 2
Days post inoculation	aMPV subtype A^a	aMPV subtype B^b	aMPV subtype A	aMPV subtype B
0	0/8	0/8	0/8	0/8
3	0/8	0/8	0/8	0/8
6	0/8	0/8	0/8	1/8
10 to 11	4/8	7/8	7/8	8/8
14	8/8	8/8	6/8	8/8
17	7/8	8/8	7/8	8/8
20 to 21	6/8	8/8	8/8	8/8
24	8/8	8/8	7/7	7/7
Control chickens inoculated with virus-free medium were negative for aMPV antibodies at all indicated time points. ^aInoculated with 10^4 CD50 aMPV subtype A. ^bInoculated with 10^4 CD50 aMPV subtype B.