Exploring surface water as a transmission medium of avian influenza viruses – systematic infection studies in mallards

Figures & data

Figure 1. General settings of infection experiments in mallards using two infected “seeder” ducks (light colour), eight contact mallards (dark colour) and two different sources of water (a) bell drinker versus and (b) pool. Seeder ducks of (a) received LP H4N6 while those of (b) were inoculated with HP H5N8. Setting (c) provides artificially contaminated pool water to four ducks as a source of infection and (d) uses a non-infected “seeder” duck with virus-contaminated plumage as contact to eight contact mallards. Created with BioRender.com. An observation time line is indicated at the top; I – Inoculation of two seeder ducks (a, b) B – blood sample, S – Swab sample (oropharyngeal, cloacal, plumage), F – Feather sample (secondary flight feather); red circled dots indicate presence of virus in water (c) or adhering to plumage (d).

Figure 2. Seroconversion at the beginning and at the end of the observation period (11 or 13 dpi, respectively) against AIV nucleoprotein as measured by blocking ELISA in inoculated (black squares) and naïve (open circles) mallards exposed to LPAIV H4N6 (LP) or HPAIV H5N8 (HP) via inoculated “seeder” ducks (a) or via pool water (b) artificially loaded with HPAIV H5N8 at 100, 1000 or 10,000 TCID₅₀ L⁻¹. The dotted line represents the ELISA threshold. % inhibition indicates the blocking efficacy of mallard sera.

Figure 4. Dynamics of influenza A virus infection of four naïve mallards each exposed to artificially pool water containing 10², 10³ or 10⁴ TCID₅₀ L⁻¹ of HPAIV H5N8. (1) Qualitative results; percentage of mallards excreting viral RNA orally, cloacally, and of RNA adhering to the breast plumage (percentage RT-qPCRs-positive mallards per group) are shown. (2) Quantitative results of viral RNA excretion compared between exposure groups 10² (open circle), 10³ (filled circle), and 10⁴ (open triangle) in (a) oral, (b) cloacal and (c) plumage swabs.

Table 1. Detection of viral infectivity in selected RT-qPCR-positive plumage swab samples obtained from mallards in experiments 1 and 2.

Animal/experiment	Group	Day (p.i.)	Cq value^a	Virus replication
57/1	LP-Pool	7	29.64	+++^b
57/1	LP-Pool	8	32.40	++-
74/1	LP bell drinker	3	26.12	- - -
16/1	HP bell drinker	9	29.11	- - -
17/1	HP bell drinker	11	31.24	- - -
23*/1	HP bell drinker	11	31.30	- - -
496/2	HP 10,000	4	35.94	++-
496/2	HP 10,000	6	33.62	++-
382/2	HP 10,000	6	33.91	- - -
363/2	HP 10,000	13	33.48	- - -

*indicates an inoculated animal.

^a Cq value of the original sample as measured by generic RT-qPCR targeting the NP gene.

^b  + indicates virus-positive embryonated chicken egg (three eggs per sample used).

Table 2. Detection of viral RNA by generic RT-qPCR in water samples of infection experiments 1 (a) and 2 (b) and (c) detection of viral infectivity in selected RT-qPCR-positive water samples obtained from experiments 1 and 2.

(a)
Days p.i.
	0	1	2	3	4	5	6	7	8	9	11
LP bell drinker	neg	neg	22.10	20.15	18.55	18.16	19.93	23.25	25.11	25.17	26.25
LP pool	neg	neg	34.44	neg	neg	27.59	20	24	19.48	23.72	25.36
HP bell drinker	neg	neg	29.18	34.25	neg#	29.37	26.65	27.11	neg	neg	n.e.
HP pool	neg	25.54	29.08	25.04	25.28	23.43	24.77	26.17	30.85	32.36	36.01
(b)
Days p.i.
	0	1	2	3	4	5	6	7	8	9	11	12	13
HP 100	neg	neg	neg	36.06	neg	neg	37.75	28.17	27.41	34.44	neg	neg	neg
HP 1,000	neg	neg	neg	30.87	28.09	30.90#	34.72#	34.03#	34.13#	30.599	neg	neg	neg
HP 10,000	neg	30.84	30.98	31.03	28.17	25.78	27.06	29.14	30.09	29.23	32.98	31.71	38.15
(c)
Group/ experiment	Day p.i.	Cq value^a	Virus replication^b
LP bell drinker/1	5	18.16	++
LP pool/1	6	20	++
HP bell drinker/1	6	26.65	- -
HP pool/1	6	24.77	+-
HP 100/2	7	28.17	- -
HP 10,000/2	5	25.78	++

^a Cq value of the original sample as measured by generic RT-qPCR targeting the NP gene.

^b  + indicates virus-positive embryonated chicken egg (two eggs per sample used).

Neg – Cq value ≥40; n.e. – not examined; # RNA extraction by Macherey-Nagel NucleoMag® VET-Kit. All other samples were extracted by the Zymo Environ Water RNA Kit.

Figure 5. Schematic view of entry (1), dispersal (2–5) and exit (6) of avian influenza viruses within a waterbird habitat by infected aquatic wild birds. Red dots/clouds symbolize infectious virus.

Data availability statement

All data pertinent to this study are presented in tables and figures in the main text or in the supplementary materials.