Bertran et al. Vet Res (2019) 50:89 https://doi.org/10.1186/s13567-019-0704-5 RESEARCH ARTICLE Open Access Pathobiology and innate immune responses of gallinaceous poultry to clade 2.3.4.4A H5Nx highly pathogenic avian influenza virus infection Kateri Bertran1,2, Mary J. Pantin‑Jackwood1, Miria F. Criado1, Dong‑Hun Lee1,3, Charles L. Balzli1,4, Erica Spackman1, David L. Suarez1 and David E. Swayne1* Abstract In the 2014–2015 Eurasian lineage clade 2.3.4.4A H5 highly pathogenic avian influenza (HPAI) outbreak in the U.S., backyard flocks with minor gallinaceous poultry and large commercial poultry (chickens and turkeys) operations were affected. The pathogenesis of the first H5N8 and reassortant H5N2 clade 2.3.4.4A HPAI U.S. isolates was investi‑ gated in six gallinaceous species: chickens, Japanese quail, Bobwhite quail, Pearl guinea fowl, Chukar partridges, and Ring-necked pheasants. Both viruses caused 80–100% mortality in all species, except for H5N2 virus that caused 60% mortality in chickens. The surviving challenged birds remained uninfected based on lack of clinical disease and lack of seroconversion. Among the infected birds, chickens and Japanese quail in early clinical stages (asymptomatic and listless) lacked histopathologic findings. In contrast, birds of all species in later clinical stages (moribund and dead) had histopathologic lesions and systemic virus replication consistent with HPAI virus infection in gallinaceous poultry. These birds had widespread multifocal areas of necrosis, sometimes with heterophilic or lymphoplasmacytic inflam‑ matory infiltrate, and viral antigen in parenchymal cells of most tissues. In general, lesions and antigen distribution were similar regardless of virus and species. However, endotheliotropism was the most striking difference among species, with only Pearl guinea fowl showing widespread replication of both viruses in endothelial cells of most tis‑ sues. The expression of IFN-γ and IL-10 in Japanese quail, and IL-6 in chickens, were up-regulated in later clinical stages compared to asymptomatic birds. Introduction America was a reassortant H5N2 with five Eurasian The H5 A/goose/Guangdong/1/1996 (Gs/GD) line- avian influenza (AI) virus gene segments (including age of highly pathogenic avian influenza (HPAI) virus the H5 clade 2.3.4.4 hemagglutinin) and three North has spread across multiple continents, affecting wild American wild bird lineage low pathogenic AI (LPAI) birds, poultry, and humans [1]. In 2014, Gs/GD line- virus gene segments [3, 4] detected in November 2014 age clade 2.3.4.4 Group A, also described as Buan2-like in British Columbia, Canada. Concurrently, an H5N8 or icA, spread across Asia, Europe, and North America HPAI virus with all 8 gene segments of Eurasian origin [2]. The initial detection of this viral lineage into North and the reassortant H5N2 HPAI virus were detected in a captive-reared gyrfalcon (Falco rusticolus) and a wild Northern pintail duck (Anas acuta), respectively, in *Correspondence: david.swayne@usda.gov Washington state, U.S. Over the next 7 months, the U.S. 1 Exotic and Emerging Avian Viral Diseases Research Unit, Southeast poultry industry experienced an unprecedented out- Poultry Research Laboratory, U.S. National Poultry Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, GA break caused by these H5 HPAI viruses, with more than 30605, USA 7.5 million turkeys and 42.1 million chickens having died Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bertran et al. Vet Res (2019) 50:89 Page 2 of 14 or culled during the control program [5] and widespread several studies have proven that HPAI viruses are able to bans on exports of U.S. poultry and poultry products [6]. infect and cause lesions and death in many gallinaceous During the outbreak, virus was detected in 21 back- species under experimental conditions [19, 37, 38]. Col- yard flocks that included: (i) minor gallinaceous poul- lectively, these findings highlight the relevance of avian try species (quail, guinea fowl, pheasants, partridges, species other than chickens, turkeys, and domestic ducks and grouse) on the same premise; (ii) different breeds of in the epidemiology of AI in small farming operations, chickens and turkeys; and (iii) domestic ducks and geese village poultry, and LPMs. [5, 7]. Minor gallinaceous poultry are commonly raised Recently, we experimentally confirmed that the first in small commercial outdoor operations and backyards, U.S. Eurasian H5N8 and reassortant H5N2 clade 2.3.4.4A or are sometimes marketed in temporary or permanent HPAI viruses lacked adaptation to chickens, i.e. 4.4 and live poultry markets (LPMs) [8], which highlights their 5.7 ­log10 mean bird infectious doses (­ BID50), respectively importance as intermediary hosts in virus transmis- [39], but were more adapted to minor gallinaceous poul- sion under poor biosecurity conditions. Globally, two try, i.e. < 3.7 ­log10 ­BID50 for direct infection [40]. In addi- facts support their importance in AI epidemiology: (i) tion, these higher ­ BID50 required to produce chicken outdoor-raised systems often have higher potential of AI infections also resulted in greatly reduced contact trans- virus exposure from wild waterfowl, contributing to the mission. Here we present the pathology results from our increase of AI outbreaks and their impact [9]; and (ii) previously published infectivity studies [39, 40]. Specifi- LPMs, with tendency for prevalent AI virus [10], contain cally, we examined the severity and distribution of gross a wide variety of live poultry and non-poultry species, and microscopic lesions in experimentally infected birds providing the ideal environment for introduction, main- and identified the organs and cell types with AI virus tenance, and adaptation of viruses, as well as potential replication. In addition, we analyzed the innate immune conditions for zoonotic transmission [11–13]. Although response in chickens and Japanese quail at different clini- there is no direct epidemiological link between minor cal stages. gallinaceous poultry from backyard flocks and chickens and turkeys from commercial farms in these U.S. clade Materials and methods 2.3.4.4A virus outbreaks [14], minor poultry have been Viruses identified or suggested as key link species in other out- The influenza A isolates A/Gyrfalcon/Washing- breaks [15–18]. Moreover, the game bird poultry indus- ton/40188-6/2014 (H5N8) and A/Northern pintail/Wash- try and backyard flocks in both developed and developing ington/40964/2014 (H5N2) were used as challenge viruses. countries are known to suffer from HPAI epidemics [19]. These were the first two HPAI isolates from the U.S. out- Gallinaceous species infected with clade 2.3.4.4 viruses break and they are considered representative of the initial generally exhibit clinical disease, mortality, and patho- AI viruses from wild waterfowl introduction of both the logical features that are indicative of HPAI virus infection Eurasian lineage H5N8 viruses and the reassortant Eura- [2], although several studies have pointed out that clade sian/North American lineage H5N2 viruses, respectively 2.3.4.4 H5 reassortants have reduced virulence compared [40]. The viruses were propagated and titrated by allantoic to the parental Gs/GD H5N1 virus [2, 4, 20]. Lee et al. sac inoculation of 9–10 day-old embryonated chicken eggs examined the pathogenicity of clade 2.3.4.4 viruses in by standard methods [41]. Japanese quail and showed severe clinical disease, virus shedding, and contact transmission following challenge Birds and housing with 6 ­log10 ­EID50 of Korean Group A H5N8 virus [21]. Six species of the order Galliformes were utilized: specific Prior to the emergence of clade 2.3.4.4 viruses, numer- pathogen free White Leghorn chickens (Gallus domes- ous outbreaks of both LPAI and HPAI viruses had been ticus; Southeast Poultry Research Laboratory [SEPRL], reported in species such as Japanese quail, Pearl guinea Athens, GA, USA), Japanese quail (Coturnix c. japonica; fowl, and Ring-necked pheasants [19, 22]. Some studies McMurray Hatchery, Webster City, IA, USA), Bobwhite suggest that certain gallinaceous species like Ring-necked quail (Colinus virginianus; M&M Quail Farm Inc., Gills- pheasants and Japanese quail are more susceptible to ville, GA, USA), Pearl guinea fowl (Numida meleagris; LPAI viruses from free-living aquatic birds than chickens McMurray Hatchery), Chukar partridges (Alectoris chu- and turkeys [19, 23–28]. Others show that Japanese quail kar; McMurray Hatchery), and Ring-necked pheasants and European quail (Coturnix c. coturnix) may support (Phasianus colchicus; McMurray Hatchery). All birds the replication of almost all LPAI virus subtypes [26, 29]. were inoculated at 4 weeks of age. Prior to inoculation, Japanese quail are recognized as mixing vessels for avian 30–50% of birds were randomly sampled and confirmed and mammalian viruses [30–33] and facilitate the adap- negative for current infection with or previous exposure tation of duck viruses to chicken [34–36]. In addition, to AI virus [42, 43]. Each experimental group was housed Bertran et al. Vet Res (2019) 50:89 Page 3 of 14 separately in negative-pressure isolators with HEPA- from chickens and Japanese quail necropsied at dif- filtered inlet air. Birds had ad libitum access to feed and ferent clinical stages of infection. The FFPE tissue water. All procedures were performed according to the sections and RNA extraction were performed as pre- requirements of the protocol approved by the Institu- viously described [49] with modifications. Briefly, tional Laboratory Animal Care and Use Committee. ten 10-μm-thick sections were collected and depar- affinized, total RNA was extracted using the RNe- Experimental design and sampling asy FFPE Kit (Qiagen, Germantown, MD, USA), and Bird inoculation and sampling were performed as previ- RNA concentration and purity were measured on a ously described [39, 40]. Briefly, each species was divided NanoDrop 1000 Spectrophotometer (Thermo Fisher into three groups: H5N2 virus inoculated group, H5N8 Scientific). The cDNA was synthetized with the High virus inoculated group, and sham-inoculated group (10 Capacity cDNA Reverse Transcription Kit (Thermo to 17 birds/virus group and 5 birds/sham group). Birds Fisher Scientific) using approximately 500 ng of RNA were inoculated intrachoanally with approximately 6 and following the manufacturer’s protocol and ther- ­log10 mean egg infectious doses (­ EID50) of H5N2 or H5N8 mal cycling conditions. The mRNA expressions of the virus, or sterile allantoic fluid. This challenge dose was aforementioned genes were quantified by quantitative necessitated for comparison with previous HPAI patho- real time PCR (qRRT-PCR) using gene specific prim- genesis studies [21, 25, 44–48]. Clinical signs were moni- ers and conditions previously described [50, 51] with tored twice a day during the first 4 days post-challenge modifications. Briefly, qRRT-PCR was performed on (dpc) and daily thereafter. Two birds from each species a 7500 FAST Real-time PCR System (Applied Biosys- exposed to each virus and showing severe clinical signs tems, Foster City, CA, USA). The qRRT-PCR reac- or found dead were necropsied at 2 and 3 dpc, except for tion mixture contained 1.0 μL of sample cDNA, 0.1 chickens and Japanese quail that were necropsied at four to 0.5 μL of forward and reverse primers (1 to 5 μM time points based on clinical progression, as previously each, standardized for efficient detection of target described [39, 40]: asymptomatic (twice, at 18 and 24 h gene and absence of dimers) (Additional file 1), 5 μL post-challenge [hpc]), listless (showing mild to moderate of KAPA SYBR FAST MasterMix (KAPA Biosystems, clinical signs), and moribund or dead. One sham-inocu- Wilmington, MA, USA), and nuclease free water for a lated bird of each species was euthanized and necropsied final reaction volume of 10.0 μL. The thermal profile at the first and the last necropsy time points. At time of consisted of one cycle for 3 min of polymerase activa- necropsy, portions of nasal cavity, brain, thymus, trachea, tion at 95 °C, followed by 45 cycles of PCR at 95 °C for lung, proventriculus, duodenum, pancreas, jejunum- 10 s and specific annealing temperature of 60 °C for ileum, spleen, kidney, adrenal gland, gonad, liver, skeletal 30 s. After the completion of amplification step, the muscle, comb, and heart were collected in 10% buffered dissociation (melting) curve was performed at 95 °C formalin (Thermo Fisher Scientific, Waltham, MA, USA) for 10 s and 60 °C for 30 s. The relative expression of for histopathologic evaluation. Severely sick birds were each target gene was normalized using the housekeep- euthanized. At 10 dpc, surviving birds were bled to evalu- ing gene β-actin (Additional file 1). The expression of ate antibody titers and euthanized. β-actin was constant within each species regardless of clinical stage and tissue. The relative quantification of Histopathology and immunohistochemistry gene expression was done by the 2 − ΔΔct formula and Tissues in 10% formalin were processed for routine expressed as fold change in infected birds compared to hematoxylin and eosin (HE) staining and immunohisto- sham (negative control) birds. Results from H5N2 and chemical (IHC) staining using a mouse-derived mono- H5N8 virus infected birds were pooled due to similar clonal antibody (P13C11, developed at SEPRL) specific mRNA expression levels for all the genes. Similarly, for type A influenza virus nucleoprotein [39, 44]. results from moribund and dead birds were pooled due to similar mRNA expression levels for all the genes. Quantification of innate immune response genes After normalization with β-actin, gene expression The mRNA expression of genes representative of dif- results in infected birds were analyzed based on spe- ferent innate pathways, including type 1 interferon cies, tissue, and clinical stage and compared to sham (IFN) (IFN-α), type 2 IFN (IFN-γ), Th1-type cytokine birds. Our data had a non-parametric distribution and (interleukin (IL)-12, IL-18), Th2-type cytokine (IL- was analyzed with Kruskal–Wallis test and Dunn’s 10), pro-inflammatory cytokine (IL-6), and toll-like Multiple Comparison Test using Prism 7 (GraphPad receptor (TLR-7), was quantified from formalin-fixed software, San Diego, CA, USA). A p value of < 0.05 was paraffin-embedded (FFPE) lung and spleen tissues considered to be significant. Bertran et al. Vet Res (2019) 50:89 Page 4 of 14 Results cloacal bursa, the opposite was observed. A remark- Clinical signs, mortality, and gross lesions able difference among species was observed in the kid- Previously, we determined that intrachoanal inoculation ney: Chukar partridges and Ring-necked pheasants of 6 l­og10 ­EID50 of either H5N2 or H5N8 virus caused (Figure 1H) displayed mild to moderate nephrosis and 80–100% mortality in the six gallinaceous species [39, widespread staining, while chickens, Japanese quail, and 40], with the exception of H5N2 virus that caused 60% Bobwhite quail had generally no lesions or staining, and mortality in chickens [39] (Table 1). Mean death times lesions and staining in Pearl guinea fowl tissues were in (MDTs) ranged from 2.5 to 5.2 days and were not signifi- between in extent. Another difference among species cantly different among them [39, 40] (Table 1). The sur- was found in nasal cavity, which was especially affected viving birds were considered uninfected based on lack in Bobwhite quail (Figures 1B–E) and Pearl guinea fowl. of clinical disease and lack of HA antibodies at the end Virus staining in vascular endothelial cells was infre- of the experiment [39, 40]. Among the infected birds, quent in all the species examined except for Pearl guinea clinical signs and gross lesions are described in detail fowl, which showed extensive endothelial cell staining for chickens [39] and the other gallinaceous species [40] with both viruses. In particular, vascular endothelial cells elsewhere. expressed virus antigen in the following tissues: sheath arterioles of spleen of H5N2 virus inoculated Japanese Microscopic findings quail; comb of H5N8 and nasal cavity of H5N2 virus Multifocal areas of necrosis, sometimes accompanied by inoculated Bobwhite quail; systemically in Pearl guinea heterophilic or lymphoplasmacytic inflammatory infil- fowl (Figure 1F); ovary of H5N8 virus inoculated Ring- trate, with viral antigen were widespread in the parenchy- necked pheasants; spleen and proventriculus of H5N2 mal cells of most tissues (Tables 2, 3, 4 and Figure 1). In and brain of H5N8 virus inoculated Chukar partridges. general, asymptomatic or listless chickens and Japanese quail did not show significant histopathological lesions or Immune gene expression profiles antigen staining, with some exceptions like severe vacuo- The mRNA expression of innate immune response lation and necrosis of the pancreatic acinar epithelium of genes is summarized in Figure 2. The mRNA levels of a listless H5N2 virus infected Japanese quail (Figure 1A) IFN-γ were significantly up-regulated in lung of mori- and nucleoprotein-positive pancreatic acinar cells (Fig- bund/dead Japanese quail compared to asymptomatic ure 1D). More severe lesions and widespread viral stain- birds. Similarly, the mRNA levels of IL-10 were signifi- ing were observed in moribund and dead birds, especially cantly up-regulated in both lung and spleen of Japanese in lung, heart, brain, pancreas, spleen, and adrenal gland. quail in later clinical stages compared to earlier clinical Collectively, similar type and severity of histological stages. In chickens, IL-6 was the only gene with signifi- lesions and antigen staining were observed with all spe- cant variation among clinical stages, as its mRNA levels cies and both viruses, although some differences between were significantly up-regulated in spleen of moribund/ the two viruses were observed (Tables 2 and 3). In the dead chickens compared to asymptomatic ones. Several pancreas, lesions and staining were mild and infrequent genes were down-regulated in some clinical stages when in H5N2 virus infected birds, but were severe and wide- compared to shams, like IFN-α, IL-12, and IL-18 for both spread in H5N8 virus infected birds; in the thymus and Table 1 Summary results of gallinaceous poultry challenged with 6 log10 EID50 of A/northern pintail/ Washington/40964/2014 (H5N2) and A/gyrfalcon/Washington/40188-6/2014 (H5N8). Data from [39, 40] Species H5N2 H5N8 a b c Mortality MDT Log10 ­BID50 Mortalitya MDTb Log10 ­BID50c Chicken 3/5 (60%) 3.0 5.7 5/5 (100%) 4.1 4.4 Japanese quail 4/5 (80%) 2.8 3.7 4/5 (80%) 2.5 3.2 Bobwhite quail 7/7 (100%) 4.7 <2 8/8 (100%) 4.9 <2 Pearl guinea fowl 5/5 (100%) 2.8 3.0 5/5 (100%) 3.8 3.0 Chukar partridge 8/8 (100%) 4.1 3.6 7/8 (87.5%) 5.2 3.6 Ring-necked pheasant 8/8 (100%) 4.7 3.4 8/8 (100%) 4.8 3.0 a #dead birds/total (%) b MDT, mean death time: #dead birds × dpc/total dead birds (expressed as dpc). MDTs were not statistically different among species or between viruses c BID50, mean bird infectious dose Bertran et al. Vet Res (2019) 50:89 Page 5 of 14 Table 2 Microscopic lesions and viral antigen distribution in gallinaceous inoculated with A/Northern pintail/ Washington/40964/2014 (H5N2) HPAI virus Chicken Japanese quail Bobwhite quail Pearl guinea fowl Chukar partridge Ring-necked pheasant Clinical Asymptomatic Asymptomatic 2d 2d 2d 2d stage or Listless Listless 2d 2d 2d 2d day post- Moribund/dead Moribund/dead 3d 3d 3d challengea 3d 3d 3d Tissue Histo IHC Histo IHC Histo IHC Histo IHC Histo IHC Histo IHC ­scoreb ­scorec ­scoreb ­scorec ­scoreb ­scorec ­scoreb ­scorec ­scoreb ­scorec ­scoreb ­scorec Nasal cavity − − − − ++ +++ − +++ − − − − + + − − − +++ − +++ − − − − ++ + − − ++ +++ − − − − +++ +++ − ++ − + Trachea − − − − − − − − − − − − − − − − nt nt nt nt − + − − − − − − − − − − nt nt − − − − ++ + Lung − − − − − +++ +++ +++ − − − − − − ++ +++ − + +++ +++ ++ +++ ++ +++ ++ ++ − + − +++ +++ +++ ++ +++ − + +++ +++ ++ +++ Comb − − nt nt − +++ − +++ − − − − + + nt nt − +++ − +++ − − − − +++ +++ nt nt − − − − − + − − − ++ − + Heart − − − − + +++ − ++ − − − − − + − − + ++ − ++ + +++ + +++ +++ +++ ++ +++ ++ +++ + +++ ++ +++ + ++ ++ +++ +++ +++ Brain − − − − − +++ − +++ − − − − − − − − − ++ − +++ − ++ + + ++ +++ + +++ − +++ − ++ − +++ − ++ + +++ + +++ Proventricu‑ − − nt nt − − − − − − − − lus − − nt nt − − nt nt − + − − + + nt nt − − − − − − − − − + + ++ Intestine − − − − − − − ++ − − − − − − − − − − − + − − − − + + − − + + − − − − − − − − − ++ Pancreas − − − − − − − ++ − − − − − − + ++ − − − + − + − − + − +++ +++ ++ +++ − ++ − + − − + ++ +++ +++ Liver − − − − − − − +++ − − − − − + − − − − − ++ − ++ − +++ − + − − + +++ − − − − − − − + − ++ Spleen − − − − +++ ++ − +++ − − − − + + + + ++ +++ − +++ + +++ ++ +++ ++ ++ − + + ++ − − + +++ nt nt + ++ ++ +++ Thymus − − − − − − − +++ − − − − + − − − + + − ++ − + + ++ ++ ++ nt nt + ++ − +++ + ++ + ++ + ++ + +++ Cloacal − − + − +++ ++ nt nt − − − − bursa − − + − +++ + + + − + ++ − ++ ++ + − ++ +++ − + ++ ++ ++ ++ + − ++ +++ Bertran et al. Vet Res (2019) 50:89 Page 6 of 14 Table 2 (continued) Tissue Histo IHC Histo IHC Histo IHC Histo IHC Histo IHC Histo IHC ­scoreb ­scorec ­scoreb ­scorec ­scoreb ­scorec ­scoreb ­scorec ­scoreb ­scorec ­scoreb ­scorec Kidney − − − − − − − ++ − − − − − − − − − − − + − ++ − − + ++ − − − − − ++ + +++ − − + ++ + +++ Gonad − − − − − + nt nt − − nt nt − − − − nt nt nt nt + +++ nt nt − + + +++ nt nt nt nt nt nt nt nt + ++ nt nt Adrenal − − − − − − nt nt − − − − gland − − − − nt nt nt nt nt nt nt nt + ++ + +++ nt nt nt nt +++ +++ nt nt +++ +++ nt nt Skeletal − − − − − − − ++ − − − − muscle − − − − − − − + − − − ++ − − − − − + − − nt nt − − − − nt nt nt: no tissue. a For chicken and Japanese quail, scores represent the mean of the birds sampled at each clinical stage: asymptomatic (18 and 24 hpc), listless, and moribund/dead. b Histopathology score of lesions in HE staining: −, no lesions; +, mild; ++, moderate; +++, severe. c Immunohistochemical (IHC) staining: −, no antigen staining; +, infrequent; ++, common; +++, widespread. species; IFN-γ and IL-10 for chickens; and TLR-7 for Jap- In the present study, chickens and Japanese quail were anese quail. necropsied at four time-points based on clinical pro- gression. Similar type and severity of histological lesions Discussion and antigen staining were observed in both species and Since 2014, Gs/GD H5 HPAI clade 2.3.4.4 viruses have for both viruses at each clinical stage, but differences spread rapidly and globally by migratory aquatic birds were observed between clinical stages. Asymptomatic and have evolved through reassortment with prevailing and listless chickens and Japanese quail (necropsied from local LPAI viruses [2]. Under both experimental and nat- 18 hpc to 2–3 dpc) lacked histopathological lesions or ural conditions, a wide range of avian species including antigen staining in the tissue samples tested, while mori- wild and domestic waterfowl, domestic poultry, and even bund or dead birds (necropsied from 2 to 5 dpc) displayed zoo birds appear to be permissive for infection by and/or more severe lesions and widespread viral staining in transmission with these viruses [2]. In the present study, known HPAI virus-target tissues and cells [47]. Although the pathobiology of the first U.S. clade 2.3.4.4A HPAI rapidity of clinical progression is highly dependent on the viruses in the 2014–2015 outbreak was investigated in HPAI virus strain [47], our findings are in line with previ- six gallinaceous species. In addition, innate immune ous studies showing lack of antigen staining when clinical responses to infection with these viruses were analyzed signs are absent, but severe histopathological lesions and in chickens and Japanese quail. widespread antigen staining in moribund or dead gallina- We previously confirmed that these two viruses were ceous birds [44, 52]. Our observations suggest that there poorly adapted to chickens, based on high mean chicken is a short time window between the initial virus replica- infectious dose (i.e. 4.4 and 5.7 l­og10 ­BID50) and poor tion in epithelial cells of the respiratory tract and the sub- transmissibility [39], but were more adapted to minor gal- sequent viremia and dissemination to multiple organs, linaceous poultry (< 3.7 ­BID50) [40]. As expected with 6 where the virus replicates in parenchymal cells [53]. The ­log10 ­EID50 challenge dose, some chickens did not become other four species were necropsied when showing severe infected, as evident by only 60% mortality with H5N2, clinical signs or found dead (at 2 and 3 dpc), with both and the survivors were confirmed as not being infected viruses showing similar type and severity of histological because they lacked clinical disease, virus shedding, and lesions and antigen detection in tissues across species. HA antibodies [39]. By contrast, high morbidity and mor- Among the few discrepancies observed in virus rep- tality rates and severe pathobiology upon challenge with 6 lication in tissues of different species, the vascular ­log10 ­EID50 confirmed that these viruses were highly path- endothelium showed the most remarkable variations. We ogenic for minor gallinaceous poultry [40]. observed that chickens, Japanese quail, Bobwhite quail, Bertran et al. Vet Res (2019) 50:89 Page 7 of 14 Table 3 Microscopic lesions and viral antigen distribution in gallinaceous inoculated with A/Gyrfalcon/ Washington/40188-6/2014 (H5N8) HPAI virus Chicken Japanese quail Bobwhite quail Pearl guinea fowl Chukar partridge Ring-necked pheasant Clinical stage Asymptomatic Asymptomatic 2d 2d 2d 2d or day post- Listless Listless 2d 2d 2d 2d challengea Moribund/dead Moribund/dead 3d 3d 3d 3d 3d 3d 3d Tissue Histo IHC Histo IHC Histo IHC Histo IHC Histo IHC ­scorec Histo IHC ­scorec ­scoreb ­scorec ­scoreb ­scorec ­scoreb ­scorec ­scoreb ­scorec ­scoreb ­scoreb Nasal cavity − − − − ++ +++ − +++ − − − − − − − ++ ++ +++ − +++ + + − − + + + ++ ++ +++ − ++ − + − ++ + +++ − − − ++ Trachea − − − − − − − − − − − − − − − − − ++ − − + − − − + + − − − − − − − + + ++ − − − − − + Lung − − − − − + +++ +++ +++ +++ − ++ − − ++ +++ ++ +++ +++ +++ + ++ − − ++ + + ++ + ++ +++ +++ ++ ++ ++ +++ − + + ++ + ++ Comb − − nt nt − ++ − ++ − + nt nt − − nt nt − ++ − ++ − − − − +++ +++ nt nt + +++ + ++ − ++ nt nt + ++ − − nt nt Heart − − − − + − − +++ + ++ + ++ − − + +++ + +++ − +++ + ++ − ++ +++ +++ +++ +++ + +++ + +++ ++ +++ ++ +++ ++ +++ ++ +++ ++ +++ Brain − − − − − − − +++ − ++ − + − − + +++ − + − +++ − + − ++ + +++ +++ +++ + +++ − +++ − +++ ++ +++ − +++ − ++ ++ +++ Proventricu‑ − − nt nt − − − ++ − − − − lus − − nt nt − + − +++ − − − − − + nt nt − − − − − − − ++ − − − − − − Intestine − − − − − − nt nt − + − − − − − − − − − − − − − − − − − − − − − − − − − ++ − − − − − − Pancreas − − − − − − − − + ++ − − − − ++ +++ − − − + − − − − + + +++ +++ ++ ++ ++ + +++ +++ +++ +++ + + +++ +++ + ++ Liver − − − − − − − + + ++ − − − − − − − + − ++ − − − − − + − − − + − − − − − ++ − − − − ++ − Spleen − − − − + − ++ +++ + − + − − − ++ ++ + ++ + +++ − − + − + + + − ++ +++ + ++ − + − +++ + ++ − − − − Thymus − − − − + − − − − + − + − − nt nt − − − − − − − − + + − − + − + + + + − +++ nt nt − − − +++ Cloacal bursa − − − − + − + ++ ++ + + − − − nt nt − − − − − − − − ++ + ++ − ++ + + − + − ++ ++ ++ ++ − + ++ − Bertran et al. Vet Res (2019) 50:89 Page 8 of 14 Table 3 (continued) Tissue Histo IHC Histo IHC Histo IHC Histo IHC Histo IHC ­scorec Histo IHC ­scorec ­scoreb ­scorec ­scoreb ­scorec ­scoreb ­scorec ­scoreb ­scorec ­scoreb ­scoreb Kidney − − − − − − − ++ + ++ − − − − − − nt nt − ++ − ++ + ++ + + − - − + + ++ + ++ ++ +++ − − + ++ ++ +++ Gonad − − − − nt nt nt nt − − − − − − − − nt nt nt nt − − nt nt + + ++ ++ nt nt nt nt − − − ++ nt nt − − + ++ Adrenal − − − − nt nt nt nt ++ +++ ++ +++ gland − − + +++ nt nt nt nt nt nt ++ +++ ++ + ++ +++ nt nt nt nt +++ +++ +++ +++ nt nt +++ +++ +++ +++ Skeletal − − − − − − − + − − − − muscle − − − − − − − + − − − + − + + − − + − + − − + +++ − − − − + ++ nt: no tissue. a For chicken and Japanese quail, scores represent the mean of the birds sampled at each clinical stage: asymptomatic (18 and 24 hpc), listless, and moribund/dead. b Histopathology score of lesions in HE staining: −, no lesions; +, mild; ++, moderate; +++, severe. c Immunohistochemical (IHC) staining: −, no antigen staining; +, infrequent; ++, common; +++, widespread. Table 4 Microscopic lesions and viral antigen distribution in gallinaceous inoculated with clade 2.3.4.4 H5Nx HPAI virus Tissue Lesions Cell types expressing virus antigen Nasal cavity Epithelial cell necrosis and desquamation, rhinitis, sinusitis, Vascular endothelial cells, nasal epithelial cells, nasal gland epi‑ mononuclear cell infiltrate, heterophilic rhinitis thelium, mononuclear cells, bone marrow, autonomic nerves, buccal stratified epithelium, skeletal jaw muscle, feather pulp Trachea Focal necrosis with mild lymphoplasmacytic inflammatory Pseudostratified epithelial cells, vascular endothelial cells, ster‑ infiltrate notrachealis muscle Lung Severe interstitial pneumonia, edema, congestion, necrosis, Epithelium of air capillaries, mononuclear cells, necrotic debris monocytic infiltrate Comb Edema, hemorrhages, necrosis Vascular endothelial cells, mononuclear cells, necrotic debris, feather follicle epithelium, nerves in dermis, gland basilar cells, pili muscles Heart Focal necrosis of myocardiocytes Myocardiocytes Brain Neuronal necrosis, gliosis, chromatolysis of Purkinje cell layer Neurons, Purkinje cells, ependymal cells, glial cells, vascular endothelial cells Proventriculus Focal necrosis Glandular and surface epithelium, nerves Intestine Lymphohistiocytic infiltration in submucosa Mononuclear cells in lymphoid associated tissue, capillary endothelium and serosa Pancreas Degeneration of individual pancreatic acinar cells Pancreatic acinar cells, duct cells, capillary endothelium Liver Focal necrosis with lymphoplasmacytic inflammatory infiltrate Kupffer cells, hepatocytes, vascular endothelial cells, macrophages Spleen Multifocal areas of necrosis, hemorrhages, lymphoid depletion, Mononuclear cells, periarteriolar lymphoid sheaths, mononuclear hyperplasia of macrophage-phagocytic cells phagocytic system Thymus Focal necrosis, lymphocyte depletion, apoptotic lymphocytes Mononuclear cells, thymic epithelium in medullary area Cloacal bursa Lymphocyte necrosis and apoptosis, lymphocyte depletion, Mononuclear cells, medullary support cells, nerve ganglia phagocytic hyperplasia Kidney Focal necrosis of tubular epithelium with lymphoplasmacytic Tubular epithelial cells, glomerular cells inflammation Gonad Necrosis, focal interstitial necrosis with heterophilic infiltration Tegument/interstitial tissue Adrenal gland Necrosis with mononuclear inflammatory infiltrate, heterophilic Corticotrophic and corticotropic cells infiltrate Skeletal muscle Scattered necrotic fibers Myocytes Bertran et al. Vet Res (2019) 50:89 Page 9 of 14 Figure 1 Histological lesions in gallinaceous following experimental infection with clade 2.3.4.4A HPAI viruses. A/Northern pintail/ Washington/40964/2014 (H5N2); A/Gyrfalcon/Washington/40188-6/2014 (H5N8). Magnification ×40. Japanese quail, listless (3 dpc), H5N2 virus, pancreas, HE staining (A) and IHC staining (D). Bobwhite quail, 3 dpc, H5N2 virus, respiratory epithelium, HE staining (B) and IHC staining (E). Pearl guinea fowl, 2 dpc, H5N8 virus, lung (endothelium), HE staining (C) and IHC staining (F). Ring-necked pheasant, 3 dpc, H5N2 virus, cerebrum, IHC staining (G). Ring-necked pheasant, 3 dpc, H5N2 virus, kidney, IHC staining (H). Chukar partridge, 3 dpc, H5N8 virus, adrenal gland, IHC staining (I). Chukar partridges, and Ring-necked pheasants lacked have shown that endotheliotropism is common in gal- widespread virus replication in capillary endothelial linaceous poultry infections with HPAI viruses [46, 54]. cells at any clinical stage, with consequent lack of severe Such tropism has been extensively studied in chickens edematous and hemorrhagic lesions. In contrast, Pearl infected with early H5N1 HPAI Gs/GD viruses, which guinea fowl presenting severe clinical signs or found dead typically show widespread virus replication in vascular had endothelial cell staining for both viruses in capil- endothelium alongside edematous, hemorrhagic, and lary endothelium of almost all tissues. Previous studies necrotic cutaneous lesions [44, 45, 48]. Noteworthy, Bertran et al. Vet Res (2019) 50:89 Page 10 of 14 IFNa-Ck IFNa-JQ 6 6 5 5 Type I IFN Fold change Fold change 4 4 3 3 2 2 1 1 0 0 Lung Spleen Lung Spleen IFNg-Ck IFNg-JQ 12 12 Type II IFN 10 10 Fold change Fold change 8 8 6 6 * 4 4 2 2 0 0 Lung Spleen Lung Spleen IL12-Ck IL12-JQ 3 3 Fold change Fold change 2 2 1 1 Th1 cytokine 0 0 Lung Spleen Lung Spleen IL18-Ck IL18-JQ 3 3 Fold change Fold change 2 2 1 1 0 0 Lung Spleen Lung Spleen IL10-Ck IL10-JQ 12 12 * * Th2 cytokine 10 10 * Fold change 8 Fold change 8 6 6 4 4 2 2 0 0 Lung Spleen Lung Spleen IL6-Ck IL6-JQ Pro-inflammatory 3 3 * Fold change Fold change 2 2 1 1 0 0 Lung Spleen Lung Spleen TLR7-Ck TLR7-JQ 3 3 Receptor Fold change Fold change 2 2 1 1 0 0 Lung Spleen Lung Spleen asymptomatic listess morbid/dead Figure 2 Relative RNA expression of innate immune genes in chicken (Ck) and Japanese quail (JQ). The fold change was calculated relative shams). Blue (*) shows significant differences between clinical stages ● asymptomatic, ■ listless, and ▲ moribund/dead. Significance p < 0.05. to levels of mRNA expression in infected birds compared to sham birds. Data plotted in green represents down-regulation (significantly lower than Bertran et al. Vet Res (2019) 50:89 Page 11 of 14 these early H5N1 HPAI Gs/GD viruses are well adapted chickens infected with Gs/GD H5 HPAI virus clade 1 to and remarkably virulent for chickens [38, 55]. Peracute [66]. In the present study, chickens appeared to elicit HPAI virus infections tend to be more endotheliotropic weaker innate immune responses than Japanese quail, than subacute infections, with subacute infections having suggesting that the lower infectivity and replication of more extensive virus replication in parenchymal cells of these viruses in chickens may trigger weaker antiviral visceral organs [38, 53]. Therefore, it is unclear why the immune responses. It is worth mentioning that, ini- clade 2.3.4.4A HPAI viruses tested in the present study tially, the innate immune gene expression analysis was replicated systemically and extensively in endothelial performed separately for moribund and dead birds. All cells of Pearl guinea fowl but had infrequent endothe- genes analyzed had similar mRNA expression levels in lial replication and in a limited number of tissues in the moribund and dead birds within each species, confirm- other gallinaceous species, even in peracute infections. ing that tissue necrosis did not alter cytokine expres- Interestingly, Pearl guinea fowl not only showed some of sion. Also, it is worth emphasizing that quantification the lowest ­BID50 among the gallinaceous species tested, of innate immune genes was performed on FFPE tis- but also some of the shortest MDTs [40], indicating that sues. Despite the improved isolation methods, RNA the virus was well adapted to this host. Further stud- obtained from FFPE tissues can be of lower quality and ies are needed to elucidate the mechanisms that deter- smaller size (less than 200 bp) than fresh tissues due mine either restrictive or permissive replication of clade to formalin-induced cross-linking and deterioration 2.3.4.4A viruses in endothelial cells of different gallina- of RNA during fixation and storage [67, 68]. Conse- ceous species. quently, smaller amplicon sizes are needed for optimal Several studies have shown that different avian spe- sensitivity [67], with targets in the 70–150-bp being the cies display differential innate immune responses to AI ideal range [69]. Yet, FFPE tissues have been commonly infection [51, 56–60], and that these responses tend to used for the analysis of RNA expression because of the correlate with different pathobiology outcomes [57, 60, high availability of FFPE-preserved clinical samples and 61]. Here, the innate immune responses of chickens and the direct correlation of these analyses with clinical Japanese quail infected with two H5Nx HPAI viruses data [67, 70]. Therefore, although many of our results were compared in order to elucidate any potential link corresponded with previous data, they should be inter- between cytokine responses and clinical or pathological preted with caution. progression of infection. Type II IFN-γ and Th2-type In conclusion, although the first U.S. clade 2.3.4.4A cytokine IL-10 in Japanese quail, and pro-inflamma- HPAI viruses in the 2014–2015 outbreak were differently tory IL-6 in chickens, were up-regulated in later clini- adapted to the six gallinaceous species studied here [39, cal stages compared to asymptomatic birds, probably in 40], we observed similar type and severity of histopatho- response to widespread virus replication in parenchy- logical lesions and antigen distribution in those birds mal cells. Previously, Uno et al. found up-regulation of that became infected, regardless of virus and species. IFN-γ, IL-10, and IL-6 genes in peripheral blood mono- Asymptomatic or listless infected chickens and Japanese nuclear cells of Japanese quail collected 24 h after Gs/ quail lacked microscopic findings, emphasizing the risk GD H5N1 HPAI virus challenge and before showing of unrecognized virus spread if only passive surveillance severe neurologic signs around 3 dpc [51]. This sup- is practiced. These viruses appear to have high mortality ports the idea that the pro-inflammatory cytokine in minor gallinaceous poultry without prior adaptation, IL-6 is produced early after infection as part of the supporting the relevance of minor poultry species in the induced innate immune response and has been asso- epidemiology of HPAI as intermediate hosts between ciated with the recruitment of inflammatory cells and wild waterfowl and major commercial gallinaceous poul- severe pathology [58, 62, 63]. Similarly, IL-6 was up- try. The striking endotheliotropism in Pearl guinea fowl regulated in lung and spleen collected from H5 or H7 but not the other species calls for further investigation. HPAI virus inoculated chickens [57, 58]. Besides IL-6, innate responses of infected chickens were similar or Supplementary information down-regulated compared to shams, which differs from Supplementary information accompanies this paper at https​://doi. other studies that find up-regulation in the expres- org/10.1186/s1356​7-019-0704-5. sion of IFN-α, IFN-γ, and IL-12 in lung and spleen of chickens infected with H5N1 HPAI viruses, H7 HPAI Additional file 1. Primers used for the qRRT-PCR of innate immune response genes. and LPAI viruses, or H9N2 LPAI [56–58, 60, 64, 65]. However, in line with our findings, TLR-7 remained stable in lung of chickens infected with H7 HPAI virus Abbreviations AI: avian influenza; BID50: mean bird infectious dose; dpc: day post-challenge; [57], and IL-10 remained stable in lung and spleen of EID50: mean egg infectious dose; FFPE: formalin-fixed paraffin-embedded; Gs/ Bertran et al. Vet Res (2019) 50:89 Page 12 of 14 GD: A/goose/Guangdong/1/1996; HE: hematoxylin and eosin; HPAI: highly 5. United States Department of Agriculture, Animal and Plant Health pathogenic avian influenza; IFN: interferon; IL: interleukin; IHC: immunohis‑ Inspection Service (2015) Highly Pathogenic Avian Influenza Infected tochemistry; LPM: live poultry market; LPAI: low pathogenic avian influenza; Premises 2014–2015. https​://www.aphis​.usda.gov/anima​l_healt​h/anima​ MDT: mean death time; nt: no tissue; qRRT-PCR: quantitative real-time RT-PCR; l_dis_spec/poult​r y/downl​oads/hpai-posit​ive-premi​ses-2014-2015.pdf SEPRL: Southeast Poultry Research Laboratory; TLR: toll-like receptor. 6. United States Department of Agriculture, Food Safety and Inspection Service (2015) Export library—requirements by country. http://www.fsis. Acknowledgements usda.gov/wps/porta​l/fsis/topic​s/inter​natio​nal-affai​rs/expor​ting-produ​ The authors gratefully acknowledge Kira Moresco, Roger Brock, Jerry Damron, cts/expor​t-libra​r y-requi​remen​ts-by-count​ry Keith Crawford, Suzanne DeBlois, Marisela Rodriguez, Diane Smith, Mar Costa- 7. World Organisation for Animal Health (OIE) (2017) Update on avian influ‑ Hurtado, and Eric DeJesus for their excellent technical assistance. The authors enza in animals (types H5 and H7). http://www.oie.int/en/anima​l-healt​ also thank Dr Alba for statistical assistance. h-in-the-world​/updat​e-on-avian​-influ​enza/2017/ 8. Sims LD, Weaver J, Swayne DE (2016) Epidemiology of avian influenza Authors’ contributions in agricultural and other man-made systems. In: Swayne DE (ed) Animal MPJ, ES, DLS, and DES conceived this project. KB, MFC, DHL, CLB, and MPJ Influenza. Blackwell Publishing, Ames conducted the animal experiments and sample processing. KB, MFC, and DHL 9. Alexander DJ (2007) An overview of the epidemiology of avian influenza. analyzed the data. KB, MFC, MPJ, and DES drafted the manuscript. All authors Vaccine 25:5637–5644 read and approved the final manuscript. 10. Ni X, He F, Hu M, Zhou X, Wang B, Feng C, Wu Y, Li Y, Tu J, Li H, Liu M, Chen H, Chen S (2015) Investigation of avian influenza virus in poultry and wild Funding birds due to novel avian-origin influenza A(H10N8) in Nanchang City, This research was supported by USDA/ARS Research Project 6612-32000- China. Microbes Infect 17:48–53 066-00D, by the NIAID-funded Center of Excellence in Influenza Research 11. Lee SS, Wong NS, Leung CC (2013) Exposure to avian influenza H7N9 in and Surveillance (CEIRS; Contract HHSN272201400008C), by the ARS/Animal farms and wet markets. Lancet 381:1815 and Plant Health Inspection Service (APHIS) interagency agreement Project 12. Lai S, Qin Y, Cowling BJ, Ren X, Wardrop NA, Gilbert M, Tsang TK, Wu P, 60-6040-6-005, and by the DELTA-FLU Project 58-6040-7-012FN. The funders Feng L, Jiang H, Peng Z, Zheng J, Liao Q, Li S, Horby PW, Farrar JJ, Gao GF, had no role in study design, data collection and interpretation, or the decision Tatem AJ, Yu H (2016) Global epidemiology of avian influenza A H5N1 to submit the work for publication. Its contents are solely the responsibility of virus infection in humans, 1997-2015: a systematic review of individual the authors and do not necessarily represent the official views of the USDA case data. Lancet Infect Dis 16:e108–e118 or NIH. Mention of trade names or commercial products in this publication is 13. World Organisation for Animal Health (OIE) (2018) Cumulative number solely for the purpose of providing specific information and does not imply of confirmed human cases of avian influenza A(H5N1) reported to WHO. recommendation or endorsement by the USDA. http://www.who.int/influ​enza/human​_anima​l_inter​face/H5N1_cumul​ ative​_table​_archi​ves/en/ Availability of data and materials 14. United States Department of Agriculture, Animal and Plant Health The datasets generated and/or analyzed during the current study are included Inspection Service (2015) Epidemiologic and other analyses of HPAI- in this published article and its supplementary information files, or are avail‑ affected poultry flocks, September 2015. https​://www.aphis​.usda.gov/ able from the corresponding author on reasonable request. anima​l_healt​h/anima​l_dis_spec/poult​r y/downl​oads/Epide​miolo​gic- Analy​sis-Sept-2015.pdf Competing interests 15. 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