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Co-infection of mallards with low-virulence Newcastle disease virus and low-pathogenic avian influenza virus a

b

b

c

c

d

M. França , E. W. Howerth , D. Carter , A. Byas , R. Poulson , C. L. Afonso & D. E. c

Stallknecht a

Poultry Diagnostic and Research Center, Department of Population Health, College of Veterinary Medicine, The University of Georgia, Athens, Georgia, USA b

Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia, USA c

Southeastern Cooperative Wildlife Disease Study, Department of Population Health, College of Veterinary Medicine, The University of Georgia, Athens, Georgia, USA d

Southeast Poultry Research Laboratory, USDA ARS, Athens, Georgia, USA Published online: 27 Jan 2014.

To cite this article: M. França, E. W. Howerth, D. Carter, A. Byas, R. Poulson, C. L. Afonso & D. E. Stallknecht (2014) Co-infection of mallards with low-virulence Newcastle disease virus and low-pathogenic avian influenza virus, Avian Pathology, 43:1, 96-104, DOI: 10.1080/03079457.2013.876530 To link to this article: http://dx.doi.org/10.1080/03079457.2013.876530

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Avian Pathology, 2014 Vol. 43, No. 1, 96–104, http://dx.doi.org/10.1080/03079457.2013.876530

ORIGINAL ARTICLE

Co-infection of mallards with low-virulence Newcastle disease virus and low-pathogenic avian influenza virus M. França1*, E. W. Howerth2, D. Carter2, A. Byas3, R. Poulson3, C. L. Afonso4, and D. E. Stallknecht3 1

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Poultry Diagnostic and Research Center, Department of Population Health, College of Veterinary Medicine, The University of Georgia, Athens, Georgia, USA, 2Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia, USA, 3Southeastern Cooperative Wildlife Disease Study, Department of Population Health, College of Veterinary Medicine, The University of Georgia, Athens, Georgia, USA, and 4Southeast Poultry Research Laboratory, USDA ARS, Athens, Georgia, USA

Waterfowl are considered the natural reservoir of low-virulence Newcastle disease viruses (loNDVs) and lowpathogenic avian influenza viruses (LPAIVs). The objective of this study was to investigate the effect of coinfections with loNDV and LPAIV on the infectivity and excretion of these viruses in mallards. One-month-old mallards were inoculated intranasally with 106 median embryo infectious doses of a wild-bird-origin loNDV and A/Mallard/MN/199106/99 (H3N8) LPAIV on the same day or received the LPAIV 2 or 5 days after loNDV inoculation. All mallards became infected with both viruses based on detection of seroconversion and viral shedding. Co-infection resulted in a higher number of cloacal swabs detected positive for LPAIV and a lower number of cloacal swabs detected positive for loNDV in some groups, although differences between groups were not statistically significant. Co-infection did not affect replication of LPAIV in epithelial cells of the lower intestine and bursa of Fabricius. In summary, the results of this study indicate that co-infection with LPAIV and loNDV does not affect the ability of mallards to be infected with either virus although it may have minimal effects on patterns (source and timing) of viral shedding.

Introduction Wild, free-ranging waterfowl are reservoirs for avian paramyxoviruses (APMVs) 1 to 4 and AMPVs 6 to 9, and wild birds are reservoirs for all 16 haemagglutinin and nine neuraminidase subtypes of avian-origin influenza A viruses (AIVs) (Alexander, 2000; Stallknecht & Brown, 2008). Newcastle disease virus (NDV) is the most prevalent APMV detected in ducks (Alexander, 2000), and lowvirulence NDV (loNDV) of all class I genotypes and loNDV from genotypes I and II of class II are commonly detected from wild birds (Kim et al., 2007). Both loNDV and low-pathogenic avian influenza virus (LPAIV) may be transmitted from waterfowl to domestic poultry and have the potential to evolve into highly pathogenic viruses (Collins et al., 1993; Takakuwa et al., 1998; Swayne & Pantin-Jackwood, 2008). Mixed infections with NDV and LPAIV have been reported in waterfowl, and numerous NDV isolations have been reported as a byproduct of AIV surveillance in the USA (Rosenberger et al., 1974; Slemons & Easterday, 1976; Smitka & Maassab, 1981; Deibel et al., 1985; Hinshaw et al., 1985; Stallknecht et al., 1991; Hanson et al., 2005; Dormitorio et al., 2009; Coffee et al., 2010; Goekjian et al., 2011; El Zowalaty et al., 2011). Similar to LPAIVs, detection of APMVs in wild ducks varies

depending on bird species, age and season (Stallknecht et al., 1991). Although loNDV and LPAIV co-exist in waterfowl, questions remain regarding potential interactions between these viruses in wild-bird populations as well as in individual co-infected birds. The objective of this study was to evaluate the effect of co-infections with loNDV and LPAIV on the infectivity, viral shedding and pathogenesis of these viruses in mallards.

Materials and Methods Viruses. Both the LPAIV (A/Mallard/MN/199106/99 [H3N8]) and NDV used in this study originated from wild mallards. The NDV is Mallard/US (MN)/AI06-978/2006 and was previously determined to be a loNDV class I and genotype 7 virus, similar to other US isolates such as Mallard/US(MD)/ 03-808/2003 (Mia Kim et al., 2008). Viral stocks were prepared and titrated in 9-day-old to 10-day-old specific pathogen free embryonated chicken eggs (Woolcock, 2008); the median embryo infectious dose (EID50) was calculated using previously reported methods (Reed & Muench, 1938). The viral stocks were diluted in sterile brain–heart infusion medium containing antimicrobials to yield a final titre of 106 EID50/0.1 ml.

Birds. One-day-old male and female mallards (Anas platyrhynchos) were purchased from a commercial hatchery (Murray McMurray Hatchery, Webster City, Iowa, USA) and raised under confined conditions until the birds were 1 month old. The 1-month-old mallards were transferred to a

*To whom correspondence should be addressed. Tel: +1 470 222 0577. E-mail: [email protected] (Received 5 November 2013; accepted 8 December 2013) © 2014 Houghton Trust Ltd

Experimental NDV and LPAIV co-infection in mallards

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BSL-Ag2+ facility with isolator units ventilated under negative pressure with high-efficiency particulate air filters in the incoming air supply and the exhaust. The birds were acclimatized for 3 days prior to inoculation. Feed and water were provided ad libitum. Blood samples as well as oropharyngeal (OP) and cloacal (CL) swabs were collected from all birds prior to inoculation. Bird care was provided according to an animal use protocol approved by the Institutional Animal Care and Use Committee at the University of Georgia.

Experimental design. Sixty-three birds were randomly divided into three co-infection groups, two single infection groups and one sham-inoculated control group. Each single infection group and co-infection group had 11 birds while the sham-inoculated control group had eight birds. Single infection groups were inoculated with 106 EID50/0.1 ml loNDV (loNDV-single) or A/Mallard/MN/199106/99 (H3N8) (LPAIV-single) via choanal cleft, and the sham-inoculated control group received 0.1 ml brain– heart infusion solution via the same route. In these groups, swabs (OP and CL) were collected at 1, 2, 3, 4, 5, 7, 9, 11 and 14 days post inoculation (d.p.i.) and blood samples were collected at 7 d.p.i. from the jugular vein. Two birds in the single infection groups were euthanized and necropsied daily from days 1 to 3 after loNDV or LPAIV inoculation. The shaminoculated control group had one bird euthanized and necropsied daily from 1 to 3 d.p.i. The remaining five birds in each group were euthanized at 14 d.p.i. and blood samples were collected from the femoral vein. The birds in the co-infection groups were inoculated with 106 EID50/0.1 ml loNDV via choanal cleft on day 0 and sequentially inoculated with 106 EID50/0.1 ml A/Mallard/MN/199106/99 (H3N8) on the same day (loNDVxLPAIV-D0), on day 2 (loNDVxLPAIV-D2) or on day 5 (loNDVxLPAIV-D5) after loNDV inoculation. Birds in the co-infection groups had OP and CL swabs collected daily after loNDV inoculation and on days 1, 2, 3, 4, 5, 7, 9, 11 and 14 after LPAIV inoculation. Two birds were euthanized and necropsied daily from days 1 to 3 after LPAIV inoculation. Blood samples were collected from the jugular vein at day 7 post LPAIV inoculation. The remaining five birds in each group were euthanized at day 14 post LPAIV inoculation and blood samples were collected from the femoral vein. All birds were monitored twice a day for overt clinical signs. The birds were euthanized in a carbon dioxide chamber and necropsied birds had samples of the upper and lower respiratory tract (turbinates, sinuses, trachea, lung), small and large intestines (duodenum, jejunum, ileum, caeca and colon), spleen and bursa of Fabricius collected in 10% buffered formalin for histopathology and immunohistochemistry (IHC). All swabs were collected in sterile brain–heart infusion medium containing antimicrobial drugs and stored at –70°C prior to testing. The blood samples were centrifuged and the sera were stored at –20°C prior to testing. Back titres of the viral inocula determined by titration in specific pathogen free embryonated chicken eggs after inoculation were 106.2 EID50/0.1 ml for Mallard/US(MN)/AI06-978/2006 loNDV and 105.9 to 106.3 EID50/0.1 ml for A/Mallard/MN/199106/99 (H3N8) LPAIV.

RNA extraction and real-time reverse transcriptase polymerase chain reaction. RNA was extracted from OP and CL swabs using the MagMAX™-96 AI/ND Viral RNA Isolation Kit (Ambion, Austin, TX, USA) and the Thermo Electron KingFisher magnetic particle processor (Thermo Electron Corporation, Waltham, Massachusetts, USA) as described previously (Das et al., 2009). The extracted RNA was tested for AIV by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) to target the influenza matrix gene according to previously described methods (Spackman et al., 2002). Positive and negative control samples were run with test samples during RNA extraction and rRT-PCR. The results of AIV rRT-PCR were considered positive in swab samples with a cycle threshold (Ct) value equal to or less than 40.00 (Spackman & Suarez, 2008). RNA extraction and rRT-PCR were performed on serial dilutions of the titrated virus stocks and standard curves were generated to convert the experimental Ct values to viral titres in EID50/ml equivalents in order to provide a quantitative approximation of infectious doses of virus in the samples. However, it was recently described that this approximation may not provide accurate results as infectious titres may be overestimated in CL swabs and in samples with lower viral concentrations (Brown et al., 2012). Some extracted RNA samples were also tested for NDV by rRT-PCR targeting the L-gene as described previously (Mia Kim et al., 2008). NDV

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rRT-PCR was performed for all OP and CL swab samples collected from the loNDV-single group. In the co-infection groups, NDV rRT-PCR was performed on OP swab samples collected prior to loNDV inoculation and on days 1, 2, 3 and 5 post loNDV inoculation. CL swabs from co-infection groups collected prior to loNDV inoculation and on days 1, 2, 3, 5, 7 and 9 post loNDV inoculation were also tested by NDV rRT-PCR. Ct values of NDV below 35.00 were considered positive (Mia Kim et al., 2008).

Serology. All serum samples were tested for NDV and avian influenza antibodies by haemagglutination inhibition (HI) test according to previously described methods (Pedersen, 2008; Thayer & Beard, 2008). Samples with HI titre > 8 were considered positive and geometric mean titres (GMTs) were calculated as described previously (Brugh, 1978).

Histopathology. All tissues were routinely processed for histology. The nasal turbinates were decalcified in Kristensen’s solution prior to processing (Kristensen, 1948). Paraffin-embedded tissue sections were stained with haematoxylin and eosin and examined in a bright-field microscope.

Immunohistochemistry. IHC for AIV was performed similarly to previously described methods (Driskell et al., 2010). For IHC, a mouse monoclonal antibody that recognizes the influenza virus nucleoprotein (NP) (Biodesign International, Sako, Maine, USA) was used at 1:1000 dilution. The antigen was detected with a horseradish peroxidase-labelled polymer and 3,3′-diaminobenzidine (Vector Laboratories, Burlingame, CA, USA) was used as a chromogen. For NDV IHC, paraffin-embedded tissue sections were deparaffinized in xylene and hydrated in decreasing alcohol solutions. Antigen retrieval was performed in citrate buffer at pH 6.0 for 45 min using a steamer. Sections were blocked with Universal Blocking solution (Biogenex, San Ramon, CA, USA) for 5 min prior to immunostaining. Tissue sections were incubated overnight at 4°C with a 1:8000 dilution of a rabbit primary polyclonal antibody raised against the nucleoprotein. After washing steps, blocking of the peroxidase activity was performed with a 3% H2O2 solution and the tissues were incubated with MACH3 rabbit probe and polymer (Biocare Medical, Concord, CA, USA), following the manufacturer’s recommendations. After washing steps, the tissues were incubated with 3,3′-diaminobenzidine (Vector Laboratories, Burlingame, CA, USA) for 10 min at room temperature and counterstained with haematoxylin.

Statistical analysis. Repeated-measures analysis of variance (ANOVA) was used to compare Ct values of LPAIV shedding over time and between groups. Degrees of freedom for F tests of within-subject factors were corrected for deviance from sphericity assumption using the Greenhouse– Geisser correction. Post-hoc testing was performed using the Bonferroni procedure to limit the type I error probability to 5% over all comparisons. Samples with a negative LPAIV rRT-PCR result were arbitrarily assigned a Ct value of 45 for statistical analyses. The number of OP and CL swabs detected positive for LPAIV and loNDV and the mean number of viral shedding days per group were analysed using Kruskal–Wallis nonparametric one-way ANOVA, followed by Dunn’s multiple comparisons test. All testing assumed a two-sided alternative hypothesis and P < 0.05 was considered statistically significant. Analyses were performed using commercially available statistical software (GraphPad Prism 6; GraphPad Software, Inc., La Jolla, CA, USA).

Results Clinical signs. No overt clinical signs were observed in any of the virus-inoculated and sham-inoculated control birds during the entire length of the experiment. Infection and viral shedding. All birds were negative for loNDV and LPAIV by serology and rRT-PCR prior to inoculation. All birds in all co-infection groups became infected with H3N8 LPAIV as determined by detection of the LPAIV matrix gene in OP and CL swabs; however,

98

M. França et al. Table 1. Viral shedding a of LPAIV detected in OP and CL swabs after single infection or co-infection with loNDV and H3N8 LPAIV in mallards.

Day post LPAIV inoculation Swab OP

CL

Group

1

2

3

4

5

7

9

11

14

Total b

Meanc

LPAIV-single loNDVxLPAIV-D0 loNDVxLPAIV-D2 loNDVxLPAIV-D5 LPAIV-single loNDVxLPAIV-D0 loNDVxLPAIV-D2 loNDVxLPAIV-D5

4/5d 1/5 1/5 2/5 1/5 5/5 2/5 1/5

2/5 3/5 5/5 4/5 3/5 4/5 5/5 5/5

5/5 2/5 2/5 5/5 5/5 5/5 5/5 5/5

5/5 5/5 5/5 4/5 5/5 5/5 5/5 5/5

2/5 2/5 4/5 4/5 5/5 5/5 4/5 5/5

1/5 0/5 0/5 0/5 3/5 5/5 4/5 2/5

0/5 0/5 0/5 0/5 2/5 4/5 2/5 2/5

0/5 0/5 0/5 0/5 1/5 0/5 1/5 2/5

0/5 0/5 0/5 0/5 0/5 2/5 2/5 0/5

19 13 17 19 25 35 30 27

3.8 2.6 3.4 3.8 5.0 7.0 6.0 5.4

a

Determined by rRT-PCR. Total number of positive swabs. c Mean number of viral shedding days. d Number of positive birds/total number of birds (n = 5 indicates the number of birds that were sampled during the entire study period, excluding the birds that were euthanized in the first 3 days post single infection or co-infection).

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b

there were some variations between groups in the detection of viral shedding post inoculation (Table 1). The total number of swabs detected positive and the duration of shedding also varied between groups. For OP shedding, birds in the loNDVxLPAIV-D0 and loNDVxLPAIV-D2 groups had fewer OP swabs detected positive by rRT-PCR and a lower mean number of days of LPAIV OP shedding than the LPAIV-single and loNDVxLPAIV-D5 co-infection group, with a greater reduction in the loNDVxLPAIV-D0 group (Table 1). The total number of CL swabs detected positive for LPAIV was higher and the duration of CL viral shedding was longer in the loNDVxLPAIV-D0 and loNDVxLPAIV-D2 co-infection groups than in the LPAIVsingle and loNDVxLPAIV-D5 groups (Table 1). The differences in number of swabs detected positive between groups were not statistically significant for both OP (P = 0.9246) and CL (P = 0.6454) swabs. Similarly, differences in the mean number of LPAIV shedding days between groups were not statistically significant based on the results obtained from OP (P = 0.3366) and CL (P = 0.1475) swabs.

The time to peak LPAIV shedding in OP swabs varied between groups as determined by Ct values (Figure 1). The LPAIV-single group had two similar peaks of LPAIV OP shedding at 1 and 4 d.p.i. The peak of LPAIV OP shedding was delayed by 1 day in the loNDVxLPAIV-D2 coinfection group, by 2 days in the loNDVxLPAIV D5 group and by 3 days in the loNDVxLPAIV-D0 group, compared with the first peak of OP shedding in the LPAIV-single group (Figure 1). There were no statistically significant differences in the total amount of OP (P = 0.7313) shedding between groups. Statistically significant differences in OP shedding were observed between some groups in some days post LPAIV inoculation. The mean Ct value of OP shedding for the loNDVxLPAIV-D2 group was significantly lower (higher viral shedding) than those of the loNDVxLPAIV-D0 and LPAIV-single groups at day 2 post LPAIV inoculation (P = 0.0451). The mean Ct value of OP shedding for the loNDVxLPAIV-D5 group was also significantly lower than those of the loNDVxLPAIV-D0 and loNDVxLPAIV-D2 groups at day 3 post LPAIV inoculation (P = 0.0102). There

LPAIV OP shedding 30 4.7 EID50 /ml

LPAIV-single loNDVxLPAIV-D0 loNDVxLPAIV-D2

35 3.4 EID50 /ml

loNDVxLPAIV-D5

40 2.2 EID50 /ml

45 0.9 EID50 /ml 0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

days after LPAIV inoculation

Figure 1. Mean Ct values of LPAIV detected in OP swabs per day post H3N8 LPAIV inoculation in single infection and co-infection groups. Dotted line, diagnostic cut-off value (Ct value < 40.00). Viral titres reported in log10 EID50/ml equivalents are also shown.

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Experimental NDV and LPAIV co-infection in mallards

were no significant differences in OP shedding between groups at day 1 (P = 0.1016), day 4 (P = 0.1014), day 5 (P = 0.2534), day 7 (P = 0.2673), day 9 (P = 0.6247), day 11 (P = 1.00) or day 14 (P = 1.00) post LPAIV inoculation. The time to peak of LPAIV shedding detected in CL swabs also varied between groups (Figure 2). The peak of LPAIV CL shedding was reached at 3 d.p.i. in the LPAIV-single group and was delayed by 2 days in the loNDVxLPAIV-D0 co-infection group and by 1 day in the loNDVxLPAIV-D5 group (Figure 2). In the loNDVxLPAIV-D2 group, the peak of LPAIV CL shedding was reached 1 day earlier than in the LPAIV-single group (Figure 2). There were no significant differences in the total amount of CL (P = 0.0890) shedding between groups. The overall ANOVA showed a statistically significant difference in CL shedding between groups at day 2 post LPAIV inoculation (P = 0.0273), but there were no significant pair-wise comparisons between groups when the Bonferroni procedure was used to limit the type I error probability to 5%. There were no significant differences in CL shedding between groups at day 1 (P = 0.0576), day 3 (P = 0.0576), day 4 (P = 0.8096), day 5 (P = 0.1950), day 7 (P = 0.0732), day 9 (P = 0.2687), day 11 (P = 0.5043) or day 14 (P = 0.1899) post LPAIV inoculation. NDV OP viral shedding was intermittent and only detected between days 1 and 3 post loNDV inoculation in the loNDVsingle group and in the co-infection groups loNDVxLPAIVD2 and loNDVxLPAIV-D5. The loNDVxLPAIV-D0 group only had one bird detected positive for NDV in the OP swab at 1 d.p.i. (Table 2). Differences in the mean number of loNDV shedding days via the oropharynx (P = 0.0875) and the number of OP swabs detected positive for loNDV (P = 0.4311) between groups were not statistically significant. NDV CL shedding was detected from 3 to 9 d.p.i. in the loNDV-single group with detection of a 100% CL shedding rate at 5 d.p.i. (Table 2). There was a marked reduction in the number of CL swabs detected positive for NDV and in the mean number of days of NDV CL shedding in the coinfection groups (Table 2). There were statistically significant differences in the mean number of CL shedding days between the loNDV-single and the co-infection groups

99

loNDVxLPAIV-D0 and loNDVxLPAIV-D5 (P = 0.0046). However, the number of CL swabs detected positive for loNDV between groups was not statistically different (P = 0.0631). The Ct values of loNDV shedding obtained from OP and CL swabs in the single infection and co-infection groups are shown in Table 2. All sham-inoculated control birds were negative for NDV and LPAIV in OP and CL swabs by rRT-PCR throughout the entire length of the experiment. Birds in the LPAIV-single group were negative for NDV by rRT-PCR in the examined swab samples and birds in the loNDV-single group were negative for LPAIV during the entire study period. Serology. All sham-inoculated control birds remained seronegative for NDV and H3N8 LPAIV. All birds in the single infection groups were serologically positive for the inoculated viruses by HI test at 7 and 14 d.p.i. All birds in the co-infection groups were detected positive for NDV and LPAIV (Table 3) by HI test on days 7 and 14 following LPAIV inoculation. Birds in the loNDVxLPAIV-D0 group had the lowest HI GMT for LPAIV on both days 7 and 14 post LPAIV inoculation. Birds in the loNDVxLPAIV-D0 also had lower GMT for NDV than the loNDV-single group at 7 and 14 d.p.i. At 7 d.p.i., the loNDV-single group had a NDV GMT 84, while the loNDVxLPAIV-D0 had a GMT 27. At 14 d.p.i., the loNDV-single group had a NDV GMT 64, while the loNDVxLPAIV-D0 had a GMT 12. The GMT for NDV of the loNDVxLPAIV-D2 group at 7 and 14 days post LPAIV inoculation was 64 and 73, respectively. The GMT for NDV of the loNDVxLPAIV-D5 group at 7 and 14 days post LPAIV inoculation was 48 and 42, respectively. Comparisons of NDV GMT between loNDVxLPAIV-D2 and loNDVxLPAIV-D5 and other groups were not done because blood samples in these groups were collected on different days following loNDV inoculation. Pathology. Table 4 summarizes the microscopic lesions in birds after single infection or co-infection with loNDV and H3N8 LPAIV. Some birds in the loNDV-single and

LPAIV CL shedding LPAIV-single

20 7.2 EID50 /ml

loNDVxLPAIV-D0 loNDVxLPAIV-D2

25 5.9 EID50 /ml

loNDVxLPAIV-D5

30 4.7 EID50 /ml

35 3.4 EID50 /ml

40 2.2 EID50 /ml

45 0.9 EID50 /ml 0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

days after LPAIV inoculation Figure 2. Mean Ct values of LPAIV detected in CL swabs per day post H3N8 LPAIV inoculation in single infection and co-infection groups. Dotted line, diagnostic cut-off value (Ct value < 40.00). Viral titres reported in log10 EID50/ml equivalents are also shown.

100

M. França et al. Table 2.

Viral sheddinga of NDV detected in OP and CL swabs in single infection and co-infection groups.

Day post NDV inoculation Swab OP

loNDV-single loNDVxLPAIV-D0 loNDVxLPAIV-D2 loNDVxLPAIV-D5

CL

loNDV-single loNDVxLPAIV-D0 loNDVxLPAIV-D2

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1

2

3

5

7

9

11

14

Total b

Meanc

5/5d 31.5±0.5e 1/5 29.8 3/5 29.9±0.9 4/5 29.7±0.8 0/5 0 0/5 0 0/5 0 0/5

0/5 0 0/5 0 2/5 32.8±0.2 2/5 33.9±0.4 0/5 0 0/5 0 0/5 0 0/5

1/5 34.6 0/5 0 1/5 32.2 1/5 33.6 2/5 29.3±3.7 0/5 0 2/5 29.2±4.3 0/5

0/5 0 0/5 0 0/5 0 0/5 0 5/5 30.6±0.7 1/5 34.6 2/5 33.6±1 0/5

0/5 0 ND ND ND ND ND ND 2/5 31.9±0.9 0/5 0 0/5 0 0/5

0/5 0 ND ND ND ND ND ND 1/5 33.5 0/5 0 0/5 0 0/5

0/5 0 ND ND ND ND ND ND 0/5 0 ND ND ND ND ND

0/5 0 ND ND ND ND ND ND 0/5 0 ND ND ND ND ND

6

1.2

1

0.2

6

1.4

7

1.4

10

2.0

1

0.2

4

0.8

0

0.0

Group

loNDVxLPAIV-D5 a

Determined by rRT-PCR. ND, not done. Total number of positive swabs. c Mean number of viral shedding days. d Number of positive birds/total number of birds (n = 5 indicates the number of birds that were sampled during the entire study period, excluding the birds that were euthanized in the first 3 days post single infection or co-infection). e Mean or mean ± standard error of the mean of Ct values obtained from positive swabs. b

loNDVxLPAIV-D5 groups had an enlarged spleen with lymphoid hyperplasia on days 1 to 3 post loNDV and LPAIV inoculation, respectively. Some birds in the loNDVsingle and loNDVxLPAIV-D0 groups had moderately distended and fluid-filled caeca and lymphoplasmacytic colitis at 3 d.p.i. Some birds infected with LPAIV had moderate lymphocytic and heterophilic rhinitis and sinusitis on days 1 and 2 post LPAIV inoculation. Additional microscopic findings were similar between infected and sham-inoculated control birds.

Immunohistochemistry. Table 5 summarizes the distribution of AIV NP antigen in tissues of mallards after LPAIV infection in single infection and co-infection groups. Mallards from all groups had AIV NP antigen detected in epithelial cells of the lower intestinal tract (Figure 3) and bursa of Fabricius with some variations between individual birds and d.p.i. The LPAIV-single and loNDVxLPAIV-D0 groups were the only groups that had some birds with AIV Table 3. Serological status for LPAIV, as determined by HI test, of mallards at 7 and 14 days post H3N8 LPAIV inoculation in single infection and co-infection groups.

HI resultsa

Group Day 7 LPAIV-single loNDVxLPAIV-D0 loNDVxLPAIV-D2 loNDVxLPAIV-D5 a

b

5/5 5/5 5/5 5/5

(294) (147) (194) (256)

Day 14 5/5 (111) 5/5 (84) 5/5 (128) 5/5 (168)

Number of positive birds/total (GMT). Number of positive birds/total number of birds (n = 5 indicates the number of birds that were sampled during the entire study period, excluding the birds that were euthanized in the first 3 days post single infection or co-infection). b

NP antigen detected in the lower intestine and bursa of Fabricius at day 1 post LPAIV inoculation. Some coinfected birds had moderate to abundant AIV NP antigen in these tissues between days 2 and 3 after LPAIV inoculation (Table 5). None of the birds had AIV NP antigen detected in the respiratory tract, spleen, duodenum or jejunum by IHC. The distribution of NDV NP antigen in tissues of mallards after single infection and co-infection are summarized in Table 6. IHC for NDV revealed varying staining for NDV NP antigen in epithelial cells of the trachea and lamina propria macrophages of the trachea and intestines (Figure 4) in the loNDV-single and loNDVxLPAIV-D0 groups. One bird from the loNDVxLPAIV-D0 group also had moderate staining for NDV NP antigen in bronchial epithelial cells and lamina propria macrophages at 2 d.p.i. The loNDVxLPAIV-D2 and loNDVxLPAIV-D5 groups did not have NDV antigen detected in the respiratory tract. The loNDVxLPAIV-D2 group was also negative for NDV antigen in the intestines, while the loNDVxLPAIV-D5 group had varying amounts of NDV antigen detected in lamina propria macrophages of the intestine on days 1 and 2 post LPAIV inoculation (days 6 and 7 post loNDV inoculation) and mild staining in epithelial cells of the villous tips at day 1 post LPAIV inoculation (Figure 5) (day 6 post loNDV inoculation). None of the examined birds had NDV antigen detected in the bursa of Fabricius and spleen. Birds in the sham-inoculated control group were negative for LPAIV and loNDV by IHC.

Discussion Mallards in all co-infection groups became infected with loNDV and LPAIV as evidenced by seroconversion and/or viral shedding detected by rRT-PCR. A higher number of CL swabs detected positive for LPAIV was observed when mallards were co-infected with both viruses on the same day (loNDVxLPAIV-D0 group). Conversely, this group had

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Table 4. Microscopic findings in mallards after single infection or co-infection with loNDV and H3N8 LPAIV.a

Days post inoculationb 1

2

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3

Rhinitis / sinusitis

Group LPAIV-single loNDVxLPAIV-D0 loNDVxLPAIV-D2 loNDVxLPAIV-D5 loNDV-single Control LPAIV-single loNDVxLPAIV-D0 loNDVxLPAIV-D2 loNDVxLPAIV-D5 loNDV-single Control LPAIV-single loNDVxLPAIV-D0 loNDVxLPAIV-D2 loNDVxLPAIV-D5 loNDV-single Control

Tracheitis

Bronchitis

Enteritis

Ileitis / typhlitis

+ – ± ±/+ + ± ±/+ ± + ± –/± ± + + ±/+ –/± + ±

– ±/+ ± –/+ ± ± – –/+ –/± –/± ±/+ ± ± –/± – – ± ±

– – – – – – – –/+ –/± – – – – – – – – –

–/+ ± + –/± –/+ + ±/+ + –/+ ± –/± – –/+ ±/+ ±/+ – + ±

c

±/+ +/+ + ± ±/+ + + + +/+ + ±/+ + ±/+ + ± ±/+ ± + + + –/± ±/+ ±

Colitis

Lymphoid depletion in bursa

Lymphoid hyperplasia in spleen

–/+ – + –/± – ± + + + ±/+ –/± ± –/± +/+ + –/+ –/± +/+ + ±

–/+ –/+ + – – – – – –/± –/± – –/+ ± –/± ± – – ±/+ +

++ ++ ++ +/+ ++ +/+ + + + +/+ + +/+ + ++ +/+ + + + +/+ + +/+ + +/+ + + + /+ + + ++

–, no lesions; ±, minimal; +, mild; ++, moderate; + + +, marked. Days post inoculation for the loNDV-single group is the number of days following loNDV inoculation. For all other groups this refers to days following LPAIV inoculation. c Variation between individual birds separated by slash (/). a

b

sample size and relatively small differences between groups rather than the absence of an actual effect. A mild effect of co-infection may also explain the variations in the time to peak of LPAIV OP and CL shedding between groups. The gross and microscopic findings in the spleen and caeca of co-infected birds were probably caused by loNDV infection since infection with LPAIV has not been associated with these findings in experimentally and naturally infected mallards (Daoust et al., 2011; Franca et al., 2012). Moderate rhinitis in some birds from co-infection groups was possibly caused by LPAIV infection as one bird in the LPAIV-single infection group had similar findings at 2 d.p.i. Although birds infected with LPAIV had rhinitis and OP viral shedding, IHC did not reveal AIV NP antigen in the nasal passages and trachea, possibly due to low viral titres in these tissues. Antigen was only detected in the lower

fewer OP swabs detected positive for LPAIV and loNDV and lower HI GMT for these viruses. Co-infection was also associated with a marked reduction of loNDV CL shedding. The peak of LPAIV CL shedding occurred at the same time of loNDV CL shedding detected in the loNDVsingle group, and it is possible that viral interference might have caused the decrease in the number of CL swabs detected positive for loNDV in the loNDVxLPAIV-D0 and loNDVxLPAIV-D2 groups. The total decrease in loNDV CL shedding in the group that received LPAIV 5 days post loNDV inoculation (loNDVxLPAIV-D5 group) may also possibly be explained by interference between these viruses since the peak of loNDV CL shedding was observed at 5 d.p.i. in the loNDV-single group. Differences in the number of swabs detected positive between groups were not statistically significant, and this is probably due to small

Table 5. Distribution of AIV NP antigen in mallards after LPAIV infection in single infection and co-infection groups.a

Days post LPAIV inoculation 1

2

3

Group

Turbinates /sinuses

Trachea

Lung

Duodenum / jejunum

LPAIV-single loNDVxLPAIV-D0 loNDVxLPAIV-D2 loNDVxLPAIV-D5 LPAIV-single loNDVxLPAIV-D0 loNDVxLPAIV-D2 loNDVxLPAIV-D5 LPAIV-single loNDVxLPAIV-D0 loNDVxLPAIV-D2 loNDVxLPAIV-D5

– – – – – – – – – – – –

– – – – – – – – – – – –

– – – – – – – – – – – –

– – – – – – – – – – – –

Ileum / caeca ± ±/+ – – ±/+ ±/+ ±/+ + –/+ + – ±/+ + ±/+ ±

Colon

Bursa

b

–/± –/± – – – – ±/+ ++ ++ –/± ±/+ + ± –

+/+ + ±/+ + – – ±/+ ±/+ ++ ±/+ ++ +/+ ++ ±/+ +++ –/±

–, no staining; ±, rare staining; +, staining of small number of cells; ++, staining of moderate numbers of cells; + + +, staining of numerous cells. Variation between individual birds separated by slash (/).

a

b

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M. França et al.

loNDVxLPAIV-D5 co-infection groups did not have NDV NP antigen detected in the respiratory tract probably because evaluation in these groups started from day 3 (loNDVxLPAIV-D2) and day 6 (loNDVxLPAIV-D5) post loNDV inoculation, when NDV OP shedding is decreased or ceased, respectively. One bird in the loNDVxLPAIV-D5 group had NDV NP antigen in some epithelial cells of the large intestine at day 6 post loNDV inoculation (day 1 post LPAIV inoculation), which indicates that loNDV also has tropism for epithelial cells of the large intestine in mallards at later time points after infection. It is also possible that loNDV replicated in low titres in the intestine not detected by IHC in other groups infected with this virus. Further studies evaluating intestinal distribution of NDV at different time points after single loNDV infection should be performed to clarify this. Our results indicate that prior or concurrent infection with loNDV may not be detrimental to LPAIV infection and shedding in naturally infected mallards, but may have an effect on loNDV transmission in these birds. However, infection dynamics and transmission mechanisms of NDV are not well described in wild ducks, and additional studies are needed to better understand the ecology of these viruses. The viral shedding and immunohistochemistry results in this study suggest that LPAIV may have reduced or inhibited loNDV replication in the intestines. As LPAIV are shed in high titres in faeces and are transmitted mainly by a faecal–oral route, the preferred tropism of loNDV for the respiratory tract would probably not limit LPAIV transmission in populations of wild ducks. However, this may not be the case with H5N1 highly pathogenic AIVs as these viruses mainly replicate in the respiratory tract of ducks (Swayne & Pantin-Jackwood, 2008). Our study demonstrated a reduction of LPAIV OP shedding when mallards were co-infected on the same day. Competition for replication sites may explain these effects. Both NDV and AIV bind to sialic acid-linked glycoconjugates on host cells (Markwell, 1991; Couceiro et al., 1993) and may also compete for host cell machinery during viral replication. Modification of cellular receptors after the primary viral infection is another possible mechanism. One study reported that chicken embryo cells expressing the haemagglutinin–neuraminidase protein of NDV were resistant to infection with NDV and an H1N1 influenza virus, possibly

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Figure 3. Photomicrograph of the colon showing abundant AIV NP antigen in enterocytes in a mallard from the loNDVxLPAIV-D2 group at day 3 post LPAIV inoculation. Immunoperoxidase labelling, haematoxylin counterstain. Bar = 20 µm.

intestinal tract and bursa of Fabricius without significant differences between groups. This supports a previous finding by our group that the main sites of LPAIV replication are the enterocytes of the lower intestinal tract and epithelial cells of the bursa of Fabricius in mallards inoculated with these viruses via choanal cleft (Franca et al., 2012), which is a natural route of exposure. Earlier detection of AIV NP antigen in these tissues was observed in birds from the LPAIV-single and loNDVxLPAIV-D0 groups, which was associated with lower Ct values (higher viral shedding) detected in CL swabs in these individual birds. While the main sites of LPAIV replication are epithelial cells of the lower intestine and bursa of Fabricius in mallards, NDV antigen was mainly detected in the upper respiratory tract and intestinal macrophages in the first 3 days after infection. The presence of NDV NP antigen in the respiratory tract of birds in the loNDV-single group correlated with the detection of OP shedding. These findings support a previous report that APMV-1 preferentially replicates in the trachea of experimentally infected mallards (Kim et al., 2012). The loNDVxLPAIV-D2 and

Table 6. Distribution of NDV NP antigen in mallards after single infection or co-infection with loNDV and H3N8 LPAIV.a

Days post inoculationb 1

2

3

Group loNDV-single loNDVxLPAIV-D0 loNDVxLPAIV-D2 loNDVxLPAIV-D5 loNDV-single loNDVxLPAIV-D0 loNDVxLPAIV-D2 loNDVxLPAIV-D5 loNDV-single loNDVxLPAIV-D0 loNDVxLPAIV-D2 loNDVxLPAIV-D5

Turbinates /sinuses − − − − −/+ −/+ − − − − − −

Trachea c

+/+ + +/+ + − − + + − − −/+ + − − −

Lung

Duodenum / jejunum

Ileum /caeca

Colon

Bursa

− − − − − −/+ + − − − − − −

−/+ + − − −/+ + +/+ + −/+ − −/± + − − −

− − − −/+ +/+ + −/+ − −/+ −/+ − − −

− − − −/+ ±/+ ++ −/+ − − − − − −

− − − − − − − − − − − −

–, no staining; ±, rare staining; +, staining of small number of cells; ++, staining of moderate numbers of cells; + + +, staining of numerous cells. Days post inoculation for the loNDV-single group is number of days following loNDV inoculation. For all other groups this refers to days following LPAIV inoculation. c Variation between individual birds separated by slash (/). a

b

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In summary, mallards inoculated with loNDV and LPAIV became infected with and seroconverted to both viruses. Co-infection caused more productive LPAIV shedding and less productive loNDV shedding via the cloaca. Additionally, co-infection affected loNDV and LPAIV OP shedding when these viruses were inoculated on the same day. Differences in fitness for replication sites and innate immune responses may explain some of these effects of co-infection. The minimal effects observed with loNDV and LPAIV co-infections in this study may reflect a long-term adaptation of both of these common waterfowl viruses to reduce competition in a shared host population, and with these minimal effects, it is unlikely that such competition would impact the maintenance and transmission of either virus in wild duck populations.

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Figure 4. Photomicrograph of the colon showing abundant NDV NP antigen in lamina propria macrophages in a mallard from the loNDV-single group at 2 d.p.i. Immunoperoxidase labelling, haematoxylin counterstain. Bar = 20 µm.

Acknowledgements The authors would like to acknowledge the histology technicians in the Department of Pathology at the University of Georgia, especially Abbie Butler and Patricia Rowe, for performing the immunohistochemistry for AIV. They also would like to thank Dr Mary Pantin-Jackwood and Dr Justin Brown for providing suggestions for this project and Benjamin Wilcox for his technical assistance. This work was funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN266200700007C. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.

References

Figure 5. Photomicrograph of the colon showing mild NDV NP antigen in enterocytes in a mallard from the loNDVxLPAIV-D5 group at day 1 post LPAIV inoculation (day 6 post loNDV inoculation). Immunoperoxidase labelling, haematoxylin counterstain. Bar = 20 µm.

due to destruction of sialic acid by the neuraminidase of the haemagglutinin–neuraminidase protein (Morrison & McGinnes, 1989). However, the neuraminidase also enhances viral replication by releasing these viruses from sialic acid receptors, and this may possibly explain the more productive LPAIV CL shedding seen in the loNDVxLPAIVD0 group. Additionally, both NDV and AIVs are capable inducers of interferon, which may affect replication kinetics and immune response in mixed infections. A previous study reported that another lentogenic NDV (La Sota strain) mildly induced interferon-α, interferon-β and interleukin-1β, and moderately induced interleukin-6 in splenocytes from chickens (Rue et al., 2011). A recent study reported that LPAIV significantly induced interleukin-2, interleukin-1β and interferon-γ, and activated the pattern recognition receptors’ tolllike receptor and retinoic acid inducible gene-I pathways in splenocytes from Pekin ducks (Maughan et al., 2013). Since NDV and LPAIV are recognized by toll-like receptors and retinoic acid inducible gene-I, the antiviral innate immune responses produced by these pattern recognition receptors may potentially limit viral replication in co-infected ducks.

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Co-infection of mallards with low-virulence Newcastle disease virus and low-pathogenic avian influenza virus.

Waterfowl are considered the natural reservoir of low-virulence Newcastle disease viruses (loNDVs) and low-pathogenic avian influenza viruses (LPAIVs)...
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