Subclinical circulation of avian hepatitis E virus within a multiple-age rearing and broiler breeder farm indicates persistence and vertical transmission of the virus

Figures & data

Table 1. Time points of sampling of broiler breeders and day-old chicks.

	Weeks of age
	6	10	25	45	50	52	62
Serology	20^a/n.a.	20/n.a.	16/12^b	10/10			20^c/10
RT-PCR (FTA^® cards)	20/n.a.	20/n.a.	16/12	10/10
RT-PCR (tissues)						5/n.a.	20^c/10
RT-PCR (pooled faeces)					2/n.a.
Histopathology					5/n.a.	5/n.a.	20^c/n.a.

n.a., not applicable. ^aNumber of tested broiler breeders. ^bNumber of tested day-old chicks. ^c10 hens and ten cockerels.

Figure 1. Egg production and mortality. Starting from week 38 there was a slight decrease in egg production but the egg production was still considered normal and the mortality did not increase.

Table 2. Results for individual birds obtained by conventional RT-PCR, real-time RT-PCR and ELISA.

Weeks of age
6	10	25	45	52	62	62 (male)	25 (DOC)	45 (DOC)	62 (DOC)
−/−/94^a	+(C)^b/−/13	−/−/100	+(H,C)/1.8 × 10^9^c/283	−/−/n.a.	+(H,C)/1.0 × 10^7/123	−/−/397^a	+(H)/1.1 × 10^3/1	−/−/1	−/−/38
−/−/1	−/−/1	−/1.5 × 10^3/346	+(H,C)/1.3 × 10^9/1600^d	+(H,C)/6.8 × 10^6/n.a.	−/−/815^d	−/−/180	+(H)/4.1 × 10^2/1	−/−/1	−/−/1
+(C)/−/68	−/−/1	−/3.5 × 10^1/147	+(H)/−/935^d	+(H,C)/6.4 × 10^7/n.a.	−/−/316	−/−/365	−/1.4 × 10^2/1	−/−/67	−/−/1
−/−/36	−/−/1	−/−/106	+(H,C)/2.8 × 10^8/63	−/5.0 × 10^2/n.a.	−/−/235	−/−/304	−/−/41	−/−/1	−/−/1
+(C)/−/304	−/−/1	−/−/16	+(H,C)/5.1 × 10^8/376	+(H,C)/1.0 × 10^7/n.a.	−/−/415^d	−/−/268	−/−/81	−/−/719^d	−/−/38
−/−/277	−/−/1	−/−/78	−/−/33		−/−/2420^d	−/−/180	−/−/1	−/−/4	−/−/10
−/−/94	−/−/1	−/−/1	+(H,C)/2.0 × 10^8/137		−/−/1908^d	−/−/110	−/1.7 × 10^1/13	−/−/1	−/−/127
+(C)/−/158	−/−/1	−/−/1	+(H,C)/1.2 × 10^9/2453^d		+(H,C)/8.6 × 10^7/352	−/−/123	−/−/1	−/−/1	−/−/7
−/−/213	−/−/85	−/−/203	+(H,C)/6.6 × 10^7/1432^d		−/−/209	−/−/196	−/−/213	−/−/1408^d	−/−/231
−/−/68	−/−/1	−/−/29	+(H,C)/5.7 × 10^8/428^d		−/−/487^d	−/−/304	+(H,C)/−/1	−/−/1	−/−/10
−/−/87	−/−/1	−/1.8 × 10^2/81					+(H)/2.5 × 10^3/1
−/−/256	−/−/170	−/−/190					−/−/1
−/−/57	−/−/72	−/−/91
−/−/1	+(C)/−/1	−/−/21
−/−/8	−/−/19	−/−/54
−/−/1	−/−/522^d	−/6.7 × 10^4/47
−/−/13	−/−/117
+(C)/−/81	−/−/333
−/−/1	−/−/1
+(C)/−/11	+(C)/−/11

^a For each bird, the results of conventional RT-PCR/real-time RT-PCR/ELISA are given. ^bIn the case of a positive RT-PCR result, the primer is indicated in parentheses. ^cIn the case of a positive real-time RT-PCR result, the viral load is given in copies per reaction. ^dTitre above the cut-off value. DOC, day-old chicks; +, positive; −, negative; H, helicase primers; C, capsid primers; n.a., not applicable.

Figure 2. Viral load of samples from broiler breeders (BB) and day-old chicks (DOC); only positive samples are shown. FTA^® cards and tissue samples obtained from broiler breeders and day-old chicks were investigated by quantitative RT-PCR and the viral loads (copies of avian HEV RNA per reaction) were determined.

Figure 3. Avian HEV antibody titres of broiler breeders (BB) and day-old chicks (DOC) measured with the BLS ELISA antibody detection kit (BioChek).

Figure 4. Histopathological lesions in the spleen (4a, 4b) and liver (4c, 4d) of a 52-week-old broiler breeder hen. Spleen showing lymphoid hyperplasia and presence of numerous lymphoid nodules. Liver showing periportal infiltrations of mononuclear inflammatory cells. 4a, 4c: bar =100 µm. 4b, 4d: bar =20 µm. Haematoxylin and eosin staining.

Figure 5. Phylogenetic trees constructed by the neighbour-joining method as implemented in the Lasergene software (DNASTAR Inc.) based on the sequences of (5a) the helicase gene of avian HEV (130-base-pair fragment) and (5b) the capsid gene of avian HEV (124-base-pair fragment), respectively. The nucleotide substitution per 100 residues is given; bootstrap values are indicated for the major nodes. Sequences retrieved within this study are written in bold. The type of bird (BB, broiler breeder or DOC, day-old chick) and the week of age are indicated. *Samples from this farm (also from previous investigations).