Co-infection of H9N2 influenza virus and Pseudomonas aeruginosa contributes to the development of hemorrhagic pneumonia in mink

Veterinary Microbiology 240 (2020) 108542

Contents lists available at ScienceDirect

Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic

Co-infection of H9N2 influenza virus and Pseudomonas aeruginosa contributes to the development of hemorrhagic pneumonia in mink

T

Zhang Bo-shuna,b,c, Li-juan Lia,b,c, Zhu Qiana,b,c, Wang Zhena,b,c, Yuan Penga,b,c, Zhou Guo-donga,b,c, Shi Wen-jiana,b,c, Chu Xue-feia,b,c, Shijin Jianga,b,c, Xie Zhi-jinga,b,c,* a

Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Agricultural University, Taian City, Shandong Province, 271018, China b College of Veterinary Medicine, Shandong Agricultural University, Taian City, Shandong Province, 271018, China c Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, Shandong Agricultural University, Taian City, Shandong Province, 271018, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Co-infection H9N2 IAV Pseudomonas aeruginosa Mink Hemorrhagic pneumonia

Influenza A virus (IAV) and bacteria co-infection can influence the host clinical conditions. Both H9N2 IAV and Pseudomonas aeruginosa (P. aeruginosa) are potential pathogens of respiratory diseases in mink. In this study, to clarify the effects of H9N2 IAV and P. aeruginosa co-infections on hemorrhagic pneumonia in mink, we carried out to establish the mink models of the two-pathogen co-infections in different orders. Compared with the single infections with H9N2 IAV or P. aeruginosa, the mink co-infected with H9N2 IAV and P. aeruginosa showed severe respiratory diseases, and exacerbated histopathological lesions and more obvious apoptosis in the lung tissues. H9N2 IAV shedding and viral loads in the lungs of the mink co-infected with H9N2 IAV and P. aeruginosa were higher than those in the mink with single H9N2 IAV infection. Furthermore, the clearance of P. aeruginosa in the co-infected mink lungs was delayed. In addition, the anti-H9N2 antibody titers in mink with P. aeruginosa coinfection following H9N2 IAV infection were significantly higher than those of the other groups. This implied that H9N2 IAV and P. aeruginosa co-infection contributed to the development of hemorrhagic pneumonia in mink, and that P. aeruginosa should play a major role in the disease. The exact interaction mechanism among H9N2 IAV, P. aeruginosa and the host needs to be further investigated.

1. Introduction The upper respiratory tract is continuously exposed to multiple potential pathogens, and viral-bacterial co-infection in the lung is more a common clinical manifestation than a rare curiosity (Jamieson et al., 2013; McArdle et al., 2018). In acute pneumonia cases, the mixed infection rate is as high as 27 % (Bello et al., 2014), and the most prevalent cases of co-infection involve the influenza virus (IAV) co-infection (Falsey et al., 2013). Although IAV is the pathogenic factor that determined the clinical conditions of the host, the co-infection of bacteria is the key factor leading to severe pneumonia (Cillóniz et al., 2012). The mechanism that facilitates the co-infection of IAV and bacteria has become a research focus for averting future pandemics (Cauley and Vella, 2015). Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous gram-negative rod growing in many environmental sites (Lovewell et al., 2014), and it is one of the bacteria commonly found in co-infection with IAV

⁎

(Cillóniz et al., 2012). A study showed that 14 % (6/42) of patients had P. aeruginosa co-infection with influenza A H1N1 infection (Cillóniz et al., 2012). A 39-year-old female patient co-infected with influenza A(H1N1)pdm09 and P. aeruginosa progressed to multifocal pneumonia with a fatal outcome (Su et al., 2019). Preceding A/PR/8/34 led to increased P. aeruginosa burden in the lung of mice (Lee et al., 2015). Hemorrhagic pneumonia was an acute and fatal disease of farmed mink caused by P. aeruginosa (Hammer et al., 2003). However, the report suggested that P. aeruginosa might be a commensal organism that only causes disease in certain circumstances (Long and Gorham, 1981). In agreement with this notion, a study confirmed that it was difficult to cause hemorrhagic pneumonia in mink challenged with P. aeruginosa, and there might be other causes of morbidity (Salomonsen et al., 2013). Mink were susceptible to H9N2 IAVs (Peng et al., 2015; Yong-Feng et al., 2017), which are lowly pathogenic and prevalent in wild birds, poultry, and mammals (Li et al., 2014). Although the mink experimentally infected with H9N2 IAV presented slight clinical signs, H9N2

Corresponding author at: College of Veterinary Medicine, Shandong Agricultural University, Taian, Shandong, 271018, China. E-mail address: [email protected] (X. Zhi-jing).

https://doi.org/10.1016/j.vetmic.2019.108542 Received 30 July 2019; Received in revised form 25 November 2019; Accepted 30 November 2019 0378-1135/ © 2019 Elsevier B.V. All rights reserved.

Veterinary Microbiology 240 (2020) 108542

Z. Bo-shun, et al.

The clinical symptoms were evaluated by a modified scoring system based on previous studies (Jirjis et al., 2004; Pomorska et al., 2014; Jaleel et al., 2017). The clinical symptoms were graded as follows, 0 no sign, 1 - mild, 2 - moderate, and 3 - severe. In detail, severity of depression in mink activity was visually characterized as 0 - no sign, 1 mild, 2 - moderate, or 3 - severe; inappetence, 0 - no sign, 1 - eating less, 2 - occasionally not eating, 3 - abolished appetite; ocular/nasal discharge, 0 - no sign, 1 - eyes appearing secretions, 2 - running nose or tears, 3 - running nose and tears; rhinoscopy dry, 0 - no sign, 1 - occasionally, 2 - often, 3 - dry cracking; cough, 0 - no sign, 1 - occasionally, 2 - often, 3 - frequently; respiratory distress, 0 - no sign, 1 heavy breathing, 2 - slight abdominal breathing, 3 - distinct abdominal breathing or screaming abnormally; diarrhea, 0 - no sign, 1 - less than three times, 2 - three times to five times, and 3 - five times or more. The mean clinical symptom score (CSS) was based on the sum of clinical symptom scores for symptoms of each group divided by the number of mink in each group, referring to the previous report (Jirjis et al., 2004).

IAV was prevalent in mink in China (Peng et al., 2015; Yong-Feng et al., 2017). Here, we questioned whether the co-infection of H9N2 IAV and P. aeruginosa induced hemorrhagic pneumonia in mink. We observed several differences between co-infections and single infections, implying that the interactions of P. aeruginosa and H9N2 IAV contributed to severe hemorrhagic pneumonia in mink, and that P. aeruginosa should play a major role in the diseases. 2. Materials and methods 2.1. Ethics statement The animal experiments were performed in accordance with regulatory standards and guidelines approved by the Shandong Agricultural University’s Animal Care and Use Committee (approval No. SDAUA-2018-44). 2.2. H9N2 IAV and P. aeruginosa

2.5. Histopathologic lesions and apoptosis in lung tissues

A/mink/Shangdong/F10/2013 (H9N2) (Mk/SD/F10/13) was isolated from the lung tissue of a mink exhibiting respiratory symptoms in our laboratory, and it had low pathogenicity to mink (Peng et al., 2015; Yong-Feng et al., 2017). H9N2 IAV was reproduced in 10-day-old SPF embryonic eggs, harvested and stored at - 80 ℃, and was titrated by 50 % egg infection dose (EID50) in 10-day-old SPF eggs at 37 ℃. The titer of Mk/SD/F10/13 was 107.0 EID50/mL. P. aeruginosa-CS-2, belonging to P. aeruginosa serotype G, was isolated from the lung tissue of the mink representing respiratory symptoms in our laboratory in 2016, and it was titrated by Colony-Forming Units (CFU) in NAC agar plates (Eiken Chemical Co., Ltd.) and agar plates with 5 % sheep blood using the spreading technique. The titer of P. aeruginosa-CS-2 was 106.0 CFU/mL.

To examine histopathologic lesions in lungs, parts of the lung samples collected on 3, 7 and 11 dpi were rapidly immersed in 10 % neutral formalin buffer to prevent autolysis, and then processed into paraffin, sectioned at 4 μm using the microtome Leica RM2235 (Leica Microsystems Co., Ltd.), and stained with hematoxylin-eosin (HE) for the detection of histological lesions by light microscopy. To demonstrate apoptosis in the lungs, paraffin sections of the lung tissues sampled at 7 dpi were stained by TUNEL kit (Roche Ltd; Shanghai, China), and then scanned and analyzed by Pannoramic Midi (3D HISTECH Ltd., Hungary). Five visual fields were randomly selected in each section with all of the cells counted in each field under the highpower microscope (400×). The percentage of apoptotic cells was calculated as the apoptotic index (AI) = apoptotic cell number/total cell number × 100 % (Dai et al., 2019).

2.3. Co-infection experiments designs Animal experiments were performed on 42 7-week-old healthy mink that were negative for IAV antigen, anti-IAV antibody, and P. aeruginosa using virus isolation, HI assays, and bacteria isolation. Forty-two mink were randomly divided into six groups on average. The animals were lightly anesthetized using Zoletil 50 and were intratracheally inoculated with H9N2 IAV and/or P. aeruginosa. Day 0 post-infection (dpi) was defined throughout the experiments as the day of the concurrent inoculation of H9N2 and P. aeruginosa in Group 3. The mink in Group 1 (H9 group) were challenged with of 5 × 106.0 EID50 H9N2 IAV on 0 dpi, using Mk/SD/F10/13, with PBS at day 3 before viral infection. The mink in Group 2 (P.A group) were inoculated with 5 × 105.0 CFU P. aeruginosa at 0 dpi, using P. aeruginosa-CS-2, with PBS at day 3 before bacterial infection. The mink in Group 3 (H9-P.A group) were inoculated with 5 × 106.0 EID50 H9N2 IAV combined with 5 × 105.0 CFU P. aeruginosa at 0 dpi, with PBS at day 3 before viral-bacterial infection. The mink in Group 4 (H9 + P.A group) were challenged with of 5 × 105.0 CFU P. aeruginosa at 0 dpi, with 5 × 106.0 EID50 H9N2 IAV at day 3 before bacterial infection. The mink in Group 5 (P.A + H9 group) were inoculated with 5 × 106.0 EID50 H9N2 at 0 dpi, with 5 × 105.0 CFU P. aeruginosa at day 3 before viral infection. The mink in Group 6 were inoculated with PBS twice at a 3-days interval, serving as control. The animals were raised separately and fed twice a day on a commercial meat-based diet. Water was available all day.

2.6. Viral shedding and distribution To determine the viral shedding, nasal swabs were collected from the experimental mink at 1, 3, 5, 7, 9 and 11 dpi. HA gene segments of H9N2 IAV in the swab elutes were examined by qRT-PCR. The specific primers were made available upon request. Briefly, viral RNA was extracted from tissues using Trizol reagent (TRANS Co., Ltd., Beijing, China), and cDNAs were synthesized by reverse transcription using Prime Script™ RT Master Mix (Takara Biomedical Technology (Beijing) Co., Ltd., China). HA gene segment was amplified by PCR. Then, the standard curves of HA was obtained by real-time PCR, using TB Green™ Premix Ex Taq™ (Takara Biomedical Technology (Beijing) Co., Ltd., China). The levels of viral nucleic acids were determined based on the standard curve, referring to the previous study (Zhang et al., 2010). At 3, 7 and 11 dpi, the lung, trachea, heart, liver, spleen, and kidney tissues were collected for virological examination, using qRT-PCR as previously described. The viral nucleic acid level (lg/ug total RNA) < 1 was defined as negative, because the sensitivity of the established qRTPCR was 10 copies/μL, that is, the virus copy number (lg/ug total RNA) = 1.

2.4. Clinical examination

2.7. Bacterial loads in the lungs

From post-infection onwards, clinical symptoms, including depression, inappetence, ocular/nasal discharge, rhinoscopy dry, coughing, respiratory distress, and diarrhea, were monitored and recorded daily for 14 days or until one mink in each group was euthanized for histopathologic, virological, and bacterial examinations at 3, 7 and 11 dpi.

To calculate bacterial loads in the lungs, the lung tissues sampled on 3, 7, and 11 dpi, were weighed and 0.1 g was homogenized in 1 mL PBS. The homogenates were serially diluted and cultured on NAC agar plates selective for P. aeruginosa at 37 ℃ for 16 h. Bacterial numbers in the tissues were calculated by CFU. 2

Veterinary Microbiology 240 (2020) 108542

Z. Bo-shun, et al.

The peak values of CSS of the mink co-infected with H9N2 IAV and P. aeruginosa appeared earlier and were higher than those of the mink infected with single H9N2 IAV or P. aeruginosa. The mink in the H9 + P.A and P.A + H9 groups showed a significantly higher CSS than those in the H9, P.A and H9-P.A groups at 1 dpi (P < 0.05). At 3 dpi, the CSS in H9-P.A, H9+P.A, and P.A+H9 groups peaked at significantly higher levels than in the H9 group (P < 0.05). The mink in the H9 and P.A groups had peak values of CSS at 5 dpi, and there was no significant difference with the other challenge groups (P > 0.05).

2.8. Detection of anti-H9N2 antibody titers For serological testing, serum samples were collected before inoculation and at 1, 3, 7, and 11 dpi, and were tested by haemagglutination inhibition (HI) assay, using MK/SD/F10/13, according to the World Health Organization manual on animal influenza diagnosis and surveillance. The positive and negative sera in this study were prepared and stored in our laboratory. 2.9. Cytokines assays

3.2. Co-infection aggravates lung histopathological lesions

The levels of the cytokine mRNA in the lungs and spleens collected at 3, 7, and 11 dpi, were detected using qRT-PCR. The specific primers for IFN-α, IFN-β, IFN-γ, and TNF-α were made available upon request. Briefly, total RNA were extracted from the lungs and spleens using Trizol reagent and cDNAs were obtained by reverse transcription. Then IFN-α, IFN-β, IFN-γ, and TNF-α and GAPDH genes were detected by qPCR. The fold change ratios between experimental and control samples for each gene were calculated and normalized against GAPDH using the ΔΔCt method (Schmittgen et al., 2000).

To examine histopathologic lesions in the lungs, the lung tissues sampled at 3, 7, and 11 dpi were analyzed using histopathological assays. As a result, mild histopathological lesions were found in the lung tissues of the mink with single H9N2 IAV infection (Fig. 1). Interstitial pneumonia in the mink infected with P. aeruginosa was observed at 3 and 7 dpi, with thickening of alveolar walls and infiltration of inflammatory cells. However, more severe histopathological lesions were found in the mink in the H9-P.A, H9 + P.A, and P.A + H9 groups. At 7 dpi, severe alveolar necrosis was observed in mink concurrently infected with H9N2 IAV and P. aeruginosa, with hemorrhage and a large number of exudates in the locally non-necrotic alveoli. The mink with P. aeruginosa co-infection following H9N2 IAV infection showed largearea alveolar collapse, complete loss of alveolar structure, and pulmonary parenchyma with a large number of inflammatory cells infiltration at 7 dpi, and lung lesions were alleviated at 11 dpi, representing part of alveolar fusion and small area bleeding. At 7 dpi, the pulmonary alveoli with unclear structure, a large amount of inflammatory cell infiltration and local hemorrhage with large area were observed in the mink with H9N2 IAV co-infection following P. aeruginosa infection. No histopathological lesions were observed in the control group.

2.10. Statistical analysis All of the data were expressed as mean ± SEM and statistically analyzed using SPASS version 22.0. The statistical significance of the findings was calculated using One-way analysis of variance (ANOVA) and Duncan’s multiple range tests were used. Graph-Pad Prism 5 software (Graph-Pad Software Inc., USA) was used to analyze the data. 3. Results 3.1. Co-infection leads to more severe clinical signs To elaborate the development of the diseases, the clinical signs of the experimental animals were daily observed and scored (shown in Table 1), which revealed differences among the co-infection groups and the single infection groups. The mink in the H9 group and P.A group appeared depressed, showed a slight inappetence at 1 dpi, moderate symptoms by the 4th and 5th day after inoculation, and the survived mink gradually recovered from the diseases. However, the mink with viral-bacterial co-infection were anorectic and weak at 1 dpi, and gradually developed rhinoscopy dry, ocular/nasal discharge, severe coughing, respiratory distress since 2 dpi. The survived animals in the co-infection groups were severely debilitated, but resumed eating and achieved clinical recovery. The mink in the negative control group showed no clinical signs.

3.3. Co-infection exacerbates apoptosis in the lung tissues To evaluate apoptosis in the lungs, the lung tissues collected at 7 dpi were examined using TUNEL assay. The lung tissues of the experimental mink appeared to be apoptotic. Compared with the H9 and P.A groups, the apoptotic cells in the lungs in the H9-P.A, H9 + P.A, and P.A + H9 groups were increased in number. As shown in Fig. 2, the mink lungs in the H9-P.A, H9 + P.A and P.A + H9 groups had significantly higher AI than those in the single infection groups (P < 0.05), and the AI of H9P.A group was the highest.

Table 1 Clinical signs scores (CSS) of the mink challenged with H9N2 AIV and/or P. aeruginosa. Days post infection (dpi)

−3 −2 −1 0 1 2 3 4 5 6 7 8 9 10 11

Groups 1

2

3

4

5

6

H9N2 – – – 0.00 ± 0.00c 0.43 ± 0.20cd 1.43 ± 0.29cd 2.71 ± 0.42c 4.00 ± 0.54b 4.40 ± 0.67a 3.60 ± 0.67a 3.00 ± 0.63a 2.33 ± 0.88a 2.00 ± 0.57a 1.33 ± 0.33b 0.67 ± 0.33bc

P.A – – – 0.00 ± 0.00c 0.86 ± 0.38bc 2.14 ± 0.34bc 4.43 ± 0.61bc 4.80 ± 0.58ab 5.20 ± 1.01a 4.60 ± 0.87a 3.75 ± 0.25a 2.67 ± 0.33a 2.67 ± 0.33a 2.00 ± 0.00ab 1.33 ± 0.33ab

H9N2-P.A – – – 0.00 ± 0.00c 1.42 ± 0.38b 3.14 ± 0.14b 7.85 ± 1.48a 7.20 ± 1.65a 6.25 ± 1.0a 5.75 ± 0.75a 4.75 ± 0.47a 3.50 ± 0.50a 3.00 ± 0.00a 2.50 ± 0.50a 2.00 ± 0.00a

H9N2 +P.A 0.00 ± 0.00 0.28 ± 0.18 1.28 ± 0.18 2.00 ± 0.30b 3.29 ± 0.75a 4.71 ± 0.89a 6.14 ± 1.05ab 5.00 ± 0.70ab 4.60 ± 0.67a 4.40 ± 1.50a 4.00 ± 1.04a 3.00 ± 1.00a 2.67 ± 0.88a 1.67 ± 0.33ab 1.00 ± 0.00b

P.A +H9N2 0.00 ± 0.00 0.57 ± 0.20 1.57 ± 0.20 3.42 ± 0.20a 3.71 ± 0.95a 6.14 ± 0.79a 8.29 ± 0.91a 6.80 ± 0.96a 6.25 ± 1.6a 5.00 ± 1.00a 4.33 ± 0.88a 3.50 ± 0.70a 3.00 ± 0.00a 2.00 ± 0.00ab 1.50 ± 0.50ab

Negative control – – – 0.00 ± 0.00c 0.00 ± 0.00d 0.00 ± 0.00d 0.00 ± 0.00d 0.00 ± 0.00c 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00c 0.00 ± 0.00c

3

Veterinary Microbiology 240 (2020) 108542

Z. Bo-shun, et al.

Fig. 1. Lung histopathological lesions in different groups. A, B, and C represent the lung histopathological lesions in H9 group at 3, 7, and 11 dpi, respectively. A, Part of alveolar fusion and part of alveolar consolidated with exudates; B, A large number of inflammatory cells and exudates in the bronchi, mucosal cilia shedding, and infiltration of inflammatory cells into lung tissues; C, The alleviated lung lesions with a small amount of exudates and inflammatory cells. D, E, and F show the lung histopathological lesions in P.A group at 3, 7, and 11 dpi, respectively. D, The thickened alveolar walls accompanied by mild interstitial pneumonia; E, The thickened alveolar wall with infiltration of inflammatory cells; F, The alleviated lung lesions accompanied by the infiltration of some inflammatory cells and local hemorrhage. G, H, and I show the lung histopathological lesions in H9-P.A group at 3, 7, and 11 dpi, respectively. G, The vasodilation of the lung, the presence of thrombus masses in the blood vessels, and a certain degree of bleeding; H, A large area of alveolar necrosis with hemorrhage, a large amount of exudates resulting from inflammation, and a small amount of macrophages; I, The alleviated lung lesions with part of alveolar fusion and mild hemorrhage. J, K, and L represent the lung histopathological lesions in H9 + P.A group at 3, 7, and 11 dpi, respectively. J, Part of alveolar fusion and local hemorrhage; K, The large-area alveolar collapse and pulmonary parenchyma accompanied by a large number of inflammatory cells infiltrated; L, Part of alveolar fusion and the small area of hemorrhage. M, N, and O show the lung histopathological lessions in P.A + H9 group at 3, 7, and 11 dpi, respectively. M, Part of alveolar fusion with a large area of hemorrhage; N, The alveoli with unclear structure with a large number of inflammatory cells infiltrated, and local tissue with a large area of hemorrhage; O, The local hemorrhage and a number of inflammatory cells infiltrated in alveolar. P, No significant histopathological lesions in the lung of the negative control group (HE, 200×).

viral nucleic acid levels in the lungs in the H9-P.A, H9 + P.A and P.A + H9 groups were higher than those in the H9 groups (P < 0.05), and the level in the H9-PA group was higher than those in other groups at 7 dpi (P < 0.05). At 11 dpi, the level in the mink lung in H9-P.A group decreased, but was significantly higher than those in other groups (P < 0.05). The levels in the tracheas were similar to those in the lung tissues. Although the levels of viral nucleic acid in the heart, liver, spleen, and kidney were different, the levels in the H9-PA and P.A+H9 groups were higher than those in the H9 group at 3 and 7 dpi. No viral nucleic acid was detected in the P.A group and the control group.

3.4. Co-infection contributes to viral shedding To define the effect of co-infection on viral shedding, the nasal swabs collected at 1, 3, 5, 7, 9 and 11dpi were examined by qRT-PCR. The viral nucleic acid levels in the swab elutes from the H9-P.A, H9 + P.A, and P.A + H9 groups were significantly higher than those from H9 group at 1 and 3 dpi (Fig. 3A). At 7, 9 and 11 dpi, the virus levels were the highest in the H9-P.A group, and the virus levels from the H9 + P.A group were the lowest. Furthermore, co-infection should contribute to earlier peak values of H9N2 IAV shedding in the co-infection groups than those in single H9N2 IAV infection group.

3.6. H9N2 IAV delays clearance of P. aeruginosa in the lungs 3.5. P. aeruginosa facilitates H9N2 IAV replication in the mink To determine whether H9N2 IAV infection delayed clearance of P. aeruginosa in lungs, the lung tissues sampled at 3, 7 and 11 dpi were tested using bacteria isolation and cultivation. Bacterial loads in the lung tissues are shown in Fig. 3 B. The bacterial loads in the lungs in the

To clarify whether P. aeruginosa facilitated H9N2 IAV replication in mink, the tissues sampled at 3, 7 and 11 dpi were detected using qRTPCR. Viral distribution in the mink is shown in Fig.3 C-H. On 3 dpi, the 4

Veterinary Microbiology 240 (2020) 108542

Z. Bo-shun, et al.

Fig. 4. HI antibody titers (Log2) to H9N2 of the experimental mink at 1, 3, 7, and 11 dpi. a-cBars with no common superscript were significantly different at P < 0.05.

Fig. 2. AI of TUNEL staining in the lung tissues of the experimental mink. Bars with no common superscript were significantly different at P < 0.05.

H9-P.A group were significantly higher than those in the P.A group at 3 and 11 dpi (P < 0.05), and the bacterial loads in the P.A + H9 group were higher than that in P.A group only at 11 dpi (P < 0.05). At 7 dpi, the bacterial loads in the lungs in the P.A and H9-P.A groups peaked, with a non-significant difference between them (P > 0.05). No bacteria were detected in the H9 group and the control group.

a-d

3.7. Co-infection influences seroconversion of anti-H9N2 antibody titers To investigate seroconversion of anti-H9N2 antibody titers in the

Fig. 3. Nasal H9N2 IAV shedding, the distribution and loads of H9N2 IAV virus and P. aeruginosa in the mink tissues. A, The viral levels in nasal swab elutes sampled from the experimental mink at 1, 3, 5, 7, 9, and 11 dpi. *, A P value of < 0.05 for the marked data point compared to all of the others, or for the data points connected by lines. B, The bacterial loads (colony-forming unit, CFU/g) in the lung tissues of the experimental mink at 3, 7, and 11 dpi. a-d Bars with no common superscript were signiificantly different at P < 0.05. C–H, Viral distribution and loads in the experimental mink at 3, 7, and 11 dpi. The virus copy number (lg/ug total RNA) < 1 was defined as negative. a-d Bars with no common superscript were significantly different at P < 0.05. 5

Veterinary Microbiology 240 (2020) 108542

Z. Bo-shun, et al.

Fig. 5. The fold mRNA expression changes of IFN-α (A), IFN-β (B), IFN-γ (C), and TNF-α (D) in the lungs of the experimental mink at 3, 7, and 11 dpi. If the fold mRNA expression change was less than 1, it revealed a downward trend. *, A P value of < 0.05 for the marked data point compared to all of the others, or for the data points connected by lines.

4. Discussion

experimental mink, serum samples collected before inoculation and at 1, 3, 7 and 11 dpi were tested by HI assay. As a result, the seroconversion of anti-H9N2 antibody in the mink in the H9, H9-P.A, H9 + P.A, and P.A + H9 groups were found. The peak of antibody titers in the H9 + P.A group appeared at 7 dpi, but the peaks in the H9, H9-P.A, and P.A + H9 groups were at 11 dpi (Fig. 4). At 1, 3 and 7 dpi, the HI titers in the H9 + PA and P.A + H9 groups were significantly higher than those in the H9 group (P < 0.05), and the titers in the H9+PA group were always the highest. The P.A group and the control group showed no anti-H9N2 antibody seroconversion.

P. aeruginosa was considered to be the cause of hemorrhagic pneumonia in mink that leads to high mortality among farmed mink. Usually without any prior clinical signs, the mink with hemorrhagic pneumonia often appear dead with blood around the nostrils and mouth (Hammer et al., 2003; Salomonsen et al., 2013). However, P. aeruginosa might be an opportunistic bacterial pathogen in mink (Long and Gorham, 1981; Salomonsen et al., 2013). H9N2 IAV was prevalent in mink and the experimental H9N2 IAV infection caused mild diseases in mink (Peng et al., 2015). In respiratory diseases, co-infection of IAVs and bacteria is very common, influencing the process and severity of infection (McCullers, 2014; Cauley and Vella, 2015). In this study, we investigated the effect of H9N2 IAV and P. aeruginosa co-infections on hemorrhagic pneumonia in mink. As a result, H9N2 IAV and P. aeruginosa co-infections contributed to the enhanced clinical symptoms and lung lesions in the mink, which is similar to co-infection with Escherichia coli and H9N2 IAV in chickens (Jaleel et al., 2017; Mosleh et al., 2017). Compared with the single infection, the apoptosis of the mink lungs in the co-infection groups was more severe. The severe diseases associated with H9N2 in the field were considered to be the result of environmental stress and co-infection (Bano et al., 2003). Mink with H9N2 IAV and P. aeruginosa co-infections had significantly higher viral loads in the lungs than that in the H9 group at 3dpi. Bacterial infection could promote the release of IAVs and exacerbated viral infections (Smith et al., 2013; Kiedrowski and Bomberger, 2018). The bacterial loads in the lungs in the H9-P.A and H9 + P.A groups were significantly higher than in the P.A group at 3 dpi, suggesting that co-infection of H9N2 IAV contributed to P. aeruginosa proliferation and delayed bacterial clearance in mink, as in the previous studies using the mouse models with A/PR/8/34 (H1N1) and P. aeruginosa (Lee et al., 2015; Ishikawa et al., 2016). The dual infection enhanced bacterial growth and impacted immunity to the virus (Cauley

3.8. Cytokine responses associated with co-infection To detect local cytokine responses during co-infection, the levels of the cytokine mRNA in the lungs and spleens collected at 3, 7 and 11 dpi were examined by qRT-PCR. The mRNA levels of IFN-α and IFN-γ in the lungs in H9 + PA group and P.A + H9 group were significantly higher than those in other groups at 3 and 7 dpi (P < 0.05) (Fig. 5). The mRNA levels of IFN-α and IFN-γ in H9-P.A group were higher than those in H9 group and P.A group at 3 dpi, but the difference was not significant, and the level of IFN-α in the H9-P.A group with a downward trend (fold change < 1) was significantly lower than those in other groups at 11 dpi. The levels in the H9 group and P.A group peaked at 11 dpi. The mRNA levels of TNF-α in the H9-PA group and H9 + PA group were significantly higher than those in other groups at 7 dpi (P < 0.05). The expressional profiles of cytokines in the spleens are shown in Fig. 6. At 3 dpi, the levels of IFN-α, IFN-γ, IFN-β, and TNF-α in the H9 + P.A group were significantly higher than those in the H9 and P.A groups (P < 0.05), revealing a rapid and strong up-regulation. However, at 11 dpi, the spleen in P.A+H9 group had significantly higher levels of IFNα, IFN-β, IFN-γ, and TNF-α than those in the other groups (P < 0.05).

6

Veterinary Microbiology 240 (2020) 108542

Z. Bo-shun, et al.

Fig. 6. The fold mRNA expression changes of IFN-α (A), IFN-β (B), IFN-γ (C), and TNF-α (D) in the spleens of the experimental mink at 3, 7, and 11 dpi. If the fold mRNA expression change was less than 1, it was a downward trend. *, A P value of < 0.05 for the marked data point compared to all of the others, or for the data points connected by lines.

5. Conclusion

and Vella, 2015). The anti-H9N2 antibody titers in the mink with P. aeruginosa coinfection following H9N2 IAV infection were significantly higher than those in the other groups, which seemed to conflict with the viral loads. The co-infection environment led to less predictable immune correlates of protection than single virus infection (Kenney et al., 2015); however, the exact mechanism needs to be further explored. In this study, the mRNA levels of IFN-α, IFN-β, and IFN-γ in the lungs and spleens of the mink with H9N2 and P. aeruginosa co-infection were higher than those infected with single H9N2 or P. aeruginosa, implying that co-infection influenced the cytokine profiles. Besides playing a central role in the host antiviral response (Theofilopoulos et al., 2005), the production of type I IFN during IAV infection in mice enhanced susceptibility to bacterial pneumonia via immunosuppression (Lee et al., 2015). TNF-α can induce mediators such as interferon to aggravate the inflammatory response (Singh et al., 2010). In the study, the mRNA levels of TNF-α in the lungs and spleens of the co-infected groups were higher in the early stage of infection, and the levels in the H9 + P.A and H9 + P.A groups were significantly higher than those in the single infection groups. In the late stage of infection, the expression levels of TNF-α in the lungs of the co-infected groups decreased, but they were higher than those in the single infection groups. This demonstrated that the degree of inflammation in the lungs is related to TNF-α expression. Interestingly, at 7 dpi, the levels of IFN-β and IFN-γ in the H9-P.A group were lower than those in the single infection groups, while the expression level of TNF-α was significantly higher than those in the single infection groups. The lungs in the H9-P.A group had the highest AI. The host immune response was complex, and co-infections can lead to unpredictable and highly variable alterations in the immune response (Kenney et al., 2015), as the conflicts between the cytokine levels and apoptosis might be due to immune stochasticity. Thus, the exact mechanism underlying these observations remains to be further investigated.

The findings presented here imply that H9N2 IAV and P. aeruginosa co-infection caused severe respiratory diseases in mink, exacerbating mink hemorrhagic pneumonia. This study facilitates the design of future preventive approaches for hemorrhagic pneumonia in mink, and contributes to understanding the co-pathogenesis of viral–bacterial pneumonia in humans. The complete interaction mechanism among H9N2 IAV, P. aeruginosa, and the host will undoubtedly need to be considered in the future. Declaration of Competing Interest The authors declare they have no conflicts of interest regarding the design or conduct of this study. Acknowledgments We thank the staff of College of Veterinary Medicine, Shandong Agricultural University, in Tai’an, Shandong Province, China. This work was supported by a Key Project of Chinese National Programs for Research and Development (2016YFD0501005), Shandong Modern Agricultural Technology & Industry System (SDAIT-21-07), Natural Science Foundation of Shandong Province (ZR2017MC054), and Funds of Shandong “Double Tops” Program. References Bano, S., Naeem, K., Malik, S.A., 2003. Evaluation of pathogenic potential of avian inflfluenza virus serotype H9N2 in chickens. Avian Dis. 47, 817–822. https://doi.org/ 10.1637/0005-2086-47.s3.817. Bello, S., Mincholé, E., Fandos, S., Lasierra, A.B., Ruiz, M.A., Simon, L., Paadero, C., Lapresta, C., Menendez, R., Torres, A., 2014. Inflammatory response in mixed viralbacterial communityacquired pneumonia. BMC Pulm. Med. 14, 123. https://doi.org/ 10.1186/1471-2466-14-123. Cauley, L.S., Vella, A.T., 2015. Why is coinfection with influenza virus and bacteria so difficult to control? Discov. Med. 19 (102), 33–40.

7

Veterinary Microbiology 240 (2020) 108542

Z. Bo-shun, et al.

Lovewell, R.R., Patankar, Y.R., Berwin, B., 2014. Mechanisms of phagocytosis and host clearance of Pseudomonas aeruginosa. Am. J. Physiol. Lung Cell Mol. Physiol. 306 (7), L591–603. https://doi.org/10.1152/ajplung.00335.2013. McArdle, A.J., Turkova, A., Cunnington, A.J., 2018. When do co-infections matter? Curr. Opin. Infect. Dis. 31 (3), 209–215. https://doi.org/10.1097/QCO. 0000000000000447. McCullers, J.A., 2014. The co-pathogenesis of influenza viruses with bacteria in the lung. Microbiol. 12, 252–262. https://doi.org/10.1038/nrmicro3231. Mosleh, N., Dadras, H., Asasi, K., Taebipour, M.J., Tohidifar, S.S., Farjanikish, G., 2017. Evaluation of the timing of the Escherichia coli co-infection on pathogenecity of H9N2 avian influenza virus in broiler chickens. IJVR. 18, 86–91. https://doi.org/10. 22099/ijvr.2017.4086. Peng, L., Chen, C., Kai-yi, H., Feng-xia, Z., Yan-li, Z., Zong-shuai, L., Xing-xiao, Z., Shi-jin, J., Zhi-jing, X., 2015. Molecular characterization of H9N2 influenza virus isolated from mink and its pathogenesis in mink. Vet.Microbiol. 176, 88–96. https://doi.org/ 10.1016/j.vetmic.2015.01.009. Pomorska, M.M., Kwit, K., Markowska, D.I., Kowalski, C., Pejsak, Z., 2014. Local and systemic immune response in pigs during subclinical andclinical swine influenza infection. Res. Vet. Sci. 97, 412–421. https://doi.org/10.1016/j.rvsc.2014.06.007. Salomonsen, C.M., Chrié, M., Jensen, T.H., Rangstrup-Christensen, L., Høiby, N., Hammer, A.S., 2013. Effect of infectious dose and season on development of hemorrhagic pneumonia in mink caused by Pseudomonas aeruginosa. Can. J. Vet. Res. 77, 221–225 PMID: 24101800. Schmittgen, T.D., Zakrajsek, B.A., Mills, A.G., Gorn, V., Singer, M.J., Reed, M.W., 2000. Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. Anal. Biochem. 285, 194–204. https://doi.org/10.1006/abio.2000.4753. Singh, U., Kumar, A., Sinha, R., Manral, S., Arora, S., Ram, S., Mishra, R.K., Gupta, P., Bansa, S.K., Prasad, A.K., Biswal, S., Parmar, V.S., Raj, H.G., 2010. Calreticulin transacetylase catalyzed modification of the TNF-α mediated pathway in the human peripheral blood mononuclear cells by polyphenolic acetates. Chem. Biol. Interact. 185, 263–270. https://doi.org/10.1016/j.cbi.2010.02.025. Smith, A.M., Adler, F.R., Ribeiro, R.M., Gutenkunst, R.N., McAuley, J.L., McCullers, J.A., Perelson, A.S., 2013. Kinetics of coinfection with influenza A virus and Streptococcus pneumoniae. PLoS Pathog. 9, e1003238. https://doi.org/10.1371/journal.ppat. 1003238. Su, I.C., Lee, K.L., Liu, H.Y., Chuang, H.C., Chen, L.Y., Lee, Y.J., 2019. Severe communityacquired pneumonia due to Pseudomonas aeruginosa coinfection in an influenza A(H1N1)pdm09 patient. J. Microbiol. Immunol. Infect. 52 (2), 365–366. https://doi. org/10.1016/j.jmii.2018.05.007. Theofilopoulos, A.N., Baccala, R., Beutler, B., Kono, D.H., 2005. Type I interferons (alpha/beta) in immunity and autoimmunity. Annu. Rev. Immunol. 23, 307–336. Yong-Feng, Z., Fei-Fei, D., Jia-Yu, Y., Feng-Xia, Z., Chang-Qing, J., Jian-Li, W., Shou-Yu, G., Kai, C., Chun-Yi, L., Xue-Hua, W., Jiang, S.J., Zhi-Jing, X., 2017. Intraspecies and interspecies transmission of mink H9N2 influenza virus. Sci. Rep. 7, 7429. https:// doi.org/10.1038/s41598-017-07879-1. Zhang, Y.L., Cheng, T., Cai, Y.J., Yuan, Q., Liu, C., Zhang, T., Xia, D.Z., Li, R.Y., Yang, L.W., Wang, Y.B., Yeo, A.E., Shih, J.W., Zhang, J., Xia, N.S., 2010. RNA Interference inhibits hepatitis B virus of different genotypes in vitro and in vivo. BMC Microbiol. 10, 214. https://doi.org/10.1186/1471-2180-10-214.

Cillóniz, C., Ewig, S., Menéndez, R., Ferrer, M., Polverino, E., Reyes, S., Gabarrús, A., Marcos, M.A., Cordoba, J., Mensa, J., Torres, A., 2012. Bacterial co-infection with H1N1 infection in patients admitted with community acquiredpneumonia. J. Infect. 65, 23–30. https://doi.org/10.1016/j.jinf.2012.04.009. Dai, J., Wei, R.J., Zhang, P.H., Kong, B.H., 2019. Overexpression of microRNA-195-5p reduces cisplatin resistance and angiogenesis in ovarian cancer by inhibiting the PSAT1-dependent GSK3β/β-catenin signaling pathway. J. Transl. Med. 1, 190. https://doi.org/10.1186/s12967-019-1932-1. Falsey, A.R., Becker, K.L., Swinburne, A.J., Nylen, E.S., Formica, M.A., Hennessey, A., Criddle, M.M., Peterson, D.R., Baran, A., Walsh, E.E., 2013. Bacterial complications of respiratory tract viral illness: a comprehensive evaluation. J. Infect. Dis. 208, 432–441. https://doi.org/10.1093/infdis/jit190. Hammer, A.S., Pedersen, K., Andersen, T.H., Jørgensen, J.C., Dietz, H.H., 2003. Comparison of Pseudomonas aeruginosa isolates from mink by serotyping and pulsed-field gel electrophoresis. Vet. Microbiol. 94, 237–243. https://doi.org/10. 1016/s0378-1135(03)00103-2. Ishikawa, H., Fukui, T., Ino, S., Sasaki, H., Awano, N., Kohda, C., Tanaka, K., 2016. Influenza virus infection causes neutrophil dysfunction through reduced G-CSF production and an increased risk of secondary bacteria infection in the lung. Virology. 499, 23–29. https://doi.org/10.1016/j.virol.2016.08.025. Jaleel, S., Younus, M., Idrees, A., Arshad, M., Khan, A.U., Ehtisham, U., Haque, S., Zaheer, M.I., Tanweer, M., Towakal, F., Munibullah, T.M.Y., Sohail, M.L., Umar, S., 2017. Pathological alterations in respiratory system during co-infection with low pathologenic avian influenza virus (H9N2) and Escherichia coli in broiler chickens. J. Vet. Res. 61, 253–258. https://doi.org/10.1515/jvetres-2017-0035. Jirjis, F.F., Noll, S.L., Halvorson, D.A., Nagaraja, K.V., Martin, F., Shaw, D.P., 2004. Effects of bacterial coinfection on the pathogenesis of avian pneumovirus infection in turkeys. Avian Dis. 48, 34–49. https://doi.org/10.1637/7017. Jamieson, A.M., Pasman, L., Yu, S., Gamradt, P., Homer, R.J., Decker, T., Medzhitov, R., 2013. Role of tissue protection in lethal respiratory viral-bacterial coinfection. Science. 340 (6137), 1230–1234. https://doi.org/10.1126/science.1233632. Kenney, L.L., Cornberg, M., Chen, A.T., Emonet, S., de la Torre, J.C., Selin, L.K., 2015. Increased immune response variability during simultaneous viral coinfection leads to unpredictability in CD8 t cell immunity and pathogenesis. J. Virol. 89https://doi.org/ 10.1128/JVI.01432-15. 10786-80. Kiedrowski, M.R., Bomberger, J.M., 2018. Viral-bacterial co-infections in the cystic fibrosis respiratory. Tract. Front. Immunol. 9, 3067. https://doi.org/10.3389/fimmu. 2018.03067. Lee, B., Robinson, K.M., McHugh, K.J., Scheller, E.V., Mandalapu, S., Chen, C., Di, Y.P., Clay, M.E., Enelow, R.I., Dubin, P.J., Alcorn, J.F., 2015. Influenza-induced type I interferon enhances susceptibility to gram-negative and gram-positivebacterial pneumonia in mice. Am. J. Physiol. Lung Cell Mol. Physiol. 309, L158–67. https:// doi.org/10.1152/ajplung.00338.2014. Li, X., Shi, J., Guo, J., Deng, G., Zhang, Q., Wang, J., He, X., Wang, K., Chen, J., Li, Y., Fan, J., Kong, H., Gu, C., Guan, Y., Suzuki, Y., Kawaoka, Y., Liu, L., Jiang, Y., Tian, G., Li, Y., Bu, Z., Chen, H., 2014. Genetics, receptor binding property, and transmissibility in mammals of naturally isolated H9N2 Avian Influenza viruses. PLoS Pathog. 10, e1004508. https://doi.org/10.1371/journal.ppat.1004508. Long, G.G., Gorham, J.R., 1981. Field studies: pseudomonas pneumonia of mink. Am. J. Vet. Res. Dec. 42 (12), 2129–2133.

8