Fowl aviadenovirus serotype 1 confirmed as the aetiological agent of gizzard erosions in replacement pullets and layer flocks in Great Britain by laboratory and in vivo studies

Figures & data

Figure 1. Gross lesions in gizzards from 41-day-old pullets (pullet flock P-A). Erosions in the koilin layer (arrows).

Table 1. Data on the pullet and layer flocks investigated together with results from histopathological and virological investigations of gizzards.

Case^a	Date	Age, husbandry^b	Histopathology	ISH (probe “FAdV-A”)^c	ISH (probe “FAdV-E”)^d	Real-time PCR (FAdV 52 K)^e	Cell culture and PCR (HexA/HexB)^f	PCR (FAdV-A Fib-1 and Fib-2)^g
P1	November 2015	41 days, NR	Ventriculitis and degeneration of glandular epithelium; basophilic intranuclear inclusion bodies	Positive	nd^h	Positive FAdV-A^i	nd	nd
P2	January 2016	41 days, NR	Ventriculitis and degeneration of glandular epithelium; basophilic intranuclear inclusion bodies	Positive	nd	Positive FAdV-A^i	nd	nd
P3	February 2016	35 days, NR	Ventriculitis and degeneration of glandular epithelium; basophilic intranuclear inclusion bodies	Positive	Negative	Positive FAdV-A^i FAdV-E	nd	nd
P4	March 2016 “P-A”	41 days, NR	Degeneration and erosion of gizzard epithelial cells, infiltration of inflammatory cells; basophilic intranuclear inclusion bodies	Positive	Negative	Positive FAdV-A^i FAdV-E	Positive FAdV-A, FAdV-1; FAdV-E, FAdV-8a; FAdV-C, FAdV-4	Positive
L1	August 2009	24 weeks, NR	Presence of intranuclear inclusion bodies within glandular epithelial cells	Positive	nd	Negative	nd	nd
L2	September 2009	28 weeks, NR	Subacute ventriculitis with glandular degeneration, necrosis; basophilic intranuclear inclusion bodies	Positive	nd	Positive FAdV-A^i	nd	nd
L3	July 2011	21 weeks, CG	Chronic non-suppurative gizzard erosion and ulceration; suspected inclusion bodies	Positive	nd	Negative	nd	nd
L4	October 2011	25 weeks, FR	Subacute to chronic focal ventriculitis with glandular necrosis and ulceration; large intranuclear inclusion bodies	Positive	nd	Positive FAdV-A^i	nd	nd
L5	April 2012	29 weeks, NR	Focal ulceration and ventriculitis, with glandular degeneration and intranuclear inclusion bodies	Positive	nd	Negative	nd	nd
L6	April 2014 “L-A”	25 weeks, FR	Extensive acute to subacute non-suppurative ventriculitis; large basophilic intranuclear inclusions	Positive	nd	Positive FAdV-A^i	Positive FAdV-A, FAdV-1^j	Positive
L7	June 2014	35 weeks, FR	Fibrinonecrotising ventriculitis with inclusion bodies	Positive	nd	Positive FAdV-A^i	nd	nd
L8	November 2014	24 weeks, FR	Multifocal extensive haemorrhagic ventriculitis with loss of glandular epithelium; inclusion bodies detected in gizzards	Positive	nd	Positive FAdV-A^i	nd	nd
L9	January 2015	26 weeks, FR	Focally extensive subacute non-suppurative ventriculitis with ulceration and regeneration; no inclusions observed	Positive	nd	Positive FAdV-A^i	nd	nd
L10	May 2015	21 weeks, CG	Extensive subacute ulcerative and non-suppurative ventriculitis with degeneration, loss of epithelium; basophilic intranuclear inclusion bodies	Positive	nd	Positive FAdV-A^i	nd	nd
L11	July 2015 “L-B”	21 weeks, FR	Extensive subacute ulcerative and non-suppurative ventriculitis, loss of epithelium; basophilic intranuclear inclusion bodies	Positive	nd	Positive FAdV-A^i	Positive FAdV-A, FAdV-1^j	Positive

^aP, pullet flock; L, layer flock.

^bFR, free range; CG, enriched cages; NR, not reported.

^cIn situ hybridization for the detection of FAdV-A DNA.

^dIn situ hybridization for the detection of FAdV-E DNA.

^eReal-time PCR (FAdV 52 K) for the detection FAdV DNA, sequencing differentiates between species FAdV-A to FAdV-E.

^fPCR (HexA/HexB) = conventional PCR for the detection of FAdV, sequencing differentiates between serotype FAdV-1 to FAdV-11.

^gPCR (FAdV-A Fib-1) = conventional PCR for the detection of FAdV-A long fibre; PCR (FAdV-A Fib-2) = conventional PCR for the detection of FAdV-A short fibre.

^hnd, not done.

^i,jsamples with 100% nucleotide sequence identity in the investigated gene regions.

Figure 2. Weekly mortality (%) in (a) the affected pullet flock (P-A) and (b) the affected layer flocks (L-A and L-B - black and grey bars, respectively).

Figure 3. Histopathology of gizzards from 35-day-old pullets (pullet flock P3). (a) Erosion of the epithelium and infiltration of inflammatory cells in the mucosal membrane. Basophilic intranuclear inclusion bodies (arrows) can be observed in gizzard epithelial cells. H&E. Bar = 100 µm. (b) Inclusion bodies with FAdV-A DNA. ISH. Bar = 20 µm.

Figure 4. Box plot presentation of VNT results from pullet and layer cases. Titres (log₂) of FAdV-1-, FAdV-4- and/or FAdV-8a-specific antibodies from pullet flocks with (P-A) and without gizzard erosions (P-B, “control”) and from a layer flock (L-B) collected at the time disease was diagnosed (P-A1, P-B1 & L-B1) and approximately six (P-A2, P-B2)/three (L-B2) weeks later are shown. Titres ≤ 3 log₂ are considered negative. Different letters (a–d) indicate statistically significant differences (P < 0.05).

Figure 5. Box plot presentation of VNT results from in vivo study. Titres (log₂) of FAdV-1- and FAdV-8a-specific antibodies from SPF pullets orally infected with FAdV-1 or FAdV-8a. Titres ≤ 3 log₂ are considered negative.

Table 2. Pathological changes in the gizzard during the in vivo study. Twenty-one-day-old SPF pullets orally infected with FAdV-1 or FAdV-8a examined at 7, 10 and 14 DPI.

		Gross pathological changes		Histology results
Group	DPI^a	Koilin layer	Gizzard mucosa	Epithelial degeneration and inflammation	ISH (probe “FAdV-A”)	ISH (probe “FAdV-E”)
FAdV-1	7	4/4 (1.25)^b	4/4 (1.25)	4/4	3/4	0/4
	10	3/4 (0.75)	1/4 (0.25)	3/4	0/4	0/4
	14	1/4 (0.25)	1/4 (0.25)	3/4	0/4	0/4
FAdV-8a	7	0/4	0/4	0/4	0/4	0/4
	10	0/4	0/4	0/4	0/4	0/4
	14	0/4	0/4	0/4	0/4	0/4

^aDPI, day post infection.

^bNumber of birds positive/number examined (mean lesion scores in parenthesis; no lesion = 0, mild lesions = 1, moderate lesions = 2, severe lesions = 3).

Table 3. Detection of viral DNA from organs and cloacal swabs in the in vivo study. Samples from 21-day-old SPF pullets orally infected with FAdV-1 or FAdV-8a were investigated at 7, 10 and 14 DPI by real-time PCR.

Group	DPI^a	Cloacal swabs	Gizzard	Liver
FAdV-1	0	0/4^b	NE^c	NE
	7	4/4	4/4	1/4
	10	2/4	4/4	0/4
	14	0/4	4/4	1/4
FAdV-8a	0	0/4	NE	NE
	7	4/4	2/4	0/4
	10	1/4	2/4	0/4
	14	0/4	0/4	1/4

^aDPI, day post infection.

^bNumber of positive samples/number tested.

^cNE, not examined.