Abstract
Avian influenza virus (AIV) usually infects wild birds and domestic poultry; however, this virus could be transmitted to mammals and humans. The previous studies reported that the farmed mink could be infected with the H5 AIV and H9 AIV, indicating that the farmed fur–bearing animals may be susceptible to AIV. Here, we report the serological evidence of infection of H7 AIV and co-infection of H7 and H9 AIV in healthy framed fur–bearing animals. We collected serum specimens from healthy farmed fur–bearing animals (farmed mink and farmed fox) and make an investigation of serological surveillance of clade 2.3.2 H5 AIV, clade 7.2 H5 AIV, clade 2.3.4.4 H5 AIV, H7 AIV, and H9 AIV. We did not find the hemagglutination inhibition (HI) antibodies against clade 2.3.2 H5 AIV, clade 7.2 H5 AIV, or clade 2.3.4.4 H5 AIV in the serum specimens of farmed fur–bearing animals. However, we found that both farmed mink and farmed fox possess HI antibodies against H7 AIV or H9 AIV; furthermore, we found that some serum specimens possess both anti-H7 AIV antibodies and anti-H9 AIV HI antibodies, suggesting that one farmed fur–bearing animal can be infected with two different subtype AIVs and may play an important role in the reassortment course of the novel avian influenza viruses. Taken together, our data suggested that the enhanced surveillance of AIV in farmed fur–bearing animals and humans or animals in close contact with them is needed.
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Keywords: Avian influenza virus, H5, H7, H9, Farmed fox, Farmed mink
Avian influenza virus (AIV) is an important zoonotic pathogen [10]. AIV could be divided into 16 hemagglutinin (HA) subtype, and H5, H7, and H9 subtype AIVs have caused human infection for many times and pose an important threat to public health and safety. For example, since the human infection with a novel avian influenza A (H7N9) virus was reported in eastern China in 2013 [2], this novel H7N9 virus has caused 1567 cases of human infections, with about 40% fatality rate (http://www.who.int/influenza/human_animal_interface/HAI_Risk_Assessment/en/).
The natural hosts of AIV are the migratory birds [5]; however, it is worth noting that this virus could also be transmitted to humans and animals in close contact with humans such as farmed animals [7–9]. Farmed fur–bearing animals are important economics animals. The farmed mink and farmed fox as the important members of the farmed fur–bearing animal family in eastern China possess an enormous population. It is worth noting that there have been some reports about the infection of AIV in farmed mink in the past several years [7, 8]. Here, we make a serological investigation about H5, H7, and H9 AIV in healthy farmed fur–bearing animals including farmed mink and farmed fox, and we found the serological evidence of the natural infection of H7 AIV and H9 AIV in these animals, suggesting that the enhanced surveillance in farmed fur–bearing animals and humans or animals in close contact with them is needed. It is worth noting that this is the first study evaluating the seroprevalence of avian influenza viruses in foxes kept in fur-bearing farms, and we found that farmed fox can be infected with H7 and H9 subtypes of influenza virus.
From December 2016 to November 2017, we collected 347 healthy farmed mink serum specimens and 195 healthy farmed fox serum specimens at farms in eastern China. These animal serum specimens were friendly provided by the animal husbandry and veterinary station of song village in Weihai city for scientific research. We used commercial H5 (clade 2.3.2), H5 (clade 7.2), H5 (clade 2.3.4.4), H7, and H9 AIV antigens (HARVAC, a high-tech enterprise wholly owned by Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences) as diagnosis antigen in the course of the serological investigation. There are many genetic branches (clade) of H5 subtype influenza virus. In this study, we selected three H5 subtype clades (clade 2.3.2, clade 7.2, and clade 2.3.4.4) which are relative frequent found in Shandong Province in recent years as representative of H5 antigens to detect H5 virus antibody in serums. Serum specimens were pretreated as previously described, and HI antibody titers were shown as the reciprocal of the highest serum dilution preventing the hemagglutination of the chicken red erythrocytes [9]. Briefly, 25 μl serum was serially diluted twofold in phosphate-buffered saline (PBS), and 25 μl of the undiluted and serially diluted serum was mixed with 25 μl of test antigen containing four hemagglutination units in each well of a microplate. Antigen and serum were incubated at room temperature for 30 min, and 50 μl of 0.5% chicken erythrocytes was added to each well. Hemagglutination inhibition (HI) antibody titers were read and recorded after incubation at room temperature for 30 min, and HI antibody titers are the reciprocal of the highest serum dilution that prevented antigen-mediated hemagglutination. HI inhibition test was not repeated. Specific pathogen-free chicken serum was used as negative control.
When reference serum specimens known to contain HI antibodies against each of the viral subtypes were evaluated for potential cross-reactivity against the other influenza subtypes, we observed no apparent cross-reactivity among H5 (clade 2.3.2) AIV, H5 (clade 7.2) AIV, H5 (clade 2.3.4.4) AIV, H7 AIV, and H9 AIV (Table 1).
Table 1.
Cross-reactivity among H5, H7, and H9 AIV*
| Antigens | Serums | ||||
|---|---|---|---|---|---|
| H5 (clade 2.3.2) | H5 (clade 7.2) | H5 (clade 2.3.4.4) | H7 | H9 | |
| H5 (clade 2.3.2) | 160 | < 10 | < 10 | < 10 | < 10 |
| H5 (clade 7.2) | < 10 | 160 | < 10 | < 10 | < 10 |
| H5 (clade 2.3.4.4) | < 10 | 10 | 320 | < 10 | < 10 |
| H7 | < 10 | < 10 | < 10 | 320 | < 10 |
| H9 | < 10 | < 10 | < 10 | < 10 | 320 |
*The H5, H7, and H9 AIV antigens and serums were purchased from HARVAC, which is a high-tech enterprise wholly owned by Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences
We then analyzed the serological prevalence of H5 AIV, H7 AIV, and H9 AIV in farmed mink (347 serum specimens). None mink serum specimens (0%) has detectable HI antibody titers (HI titers < 10) against H5 (clade 2.3.2) AIV, H5 (clade 7.2) AIV, or H5 (clade 2.3.4.4) AIV (Table 2). However, we found that twenty-three mink serum specimens (6.63%) have detectable HI antibody titers against the H7 AIV (HI titers range from 10 to 80) (Table 2; Supplemental Table 1). Furthermore, the hemagglutinin inhibition titer of about 43% H7 AIV positive serum specimens is 10, the hemagglutinin inhibition titer of about 13% H7 AIV positive serum specimens is 20, the hemagglutinin inhibition titer of about 30% H7 AIV positive serum specimens is 40, and the hemagglutinin inhibition titer of about 13% H7 virus-positive serum specimens is 80. We also found that three hundred and thirty-four mink serum specimens (96.25%) have detectable HI antibody titers against the H9 AIV (HI titers range from 10 to 640) (Table 2; Supplemental Table 1). Furthermore, the hemagglutinin inhibition titer of about 9% H9 AIV positive serum specimens is 10, the hemagglutinin inhibition titer of about 8% H9 AIV positive serum specimens is 20, the hemagglutinin inhibition titer of about 26% H9 AIV positive serum specimens is 40, the hemagglutinin inhibition titer of about 37% H9 AIV positive serum specimens is 80, the hemagglutinin inhibition titer of about 16% H9 AIV positive serum specimens is 160, the hemagglutinin inhibition titer of about 2% H9 AIV positive serum specimens is 320, and the hemagglutinin inhibition titer of about 1% H9 AIV positive serum specimens is 640. Interestingly, we found that twenty-three mink serum specimens possess both anti-H7 AIV HI antibody and anti-H9 AIV antibody (Fig. 1; Supplemental Table 1). Taken together, the above data indicated that H7 AIV and H9 AIV spread in farmed mink herds, and farmed mink can be co-infected with H7 AIV and H9 AIV.
Table 2.
HI antibody titers of farmed fur–bearing animals serum specimens
| Species | Antigens | No. serum specimens per HI antibody titers | Range of HI antibody titers of positive serum specimens (no. serum specimens with HI antibody titers ≥ 10) | No. positive serum specimens/no. serum specimens collected | No. positive serum specimens | No. serum specimens collected | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| < 10 | 10 | 20 | 40 | 80 | 160 | 320 | 640 | ||||||
| Mink | H5 (clade 2.3.2) | 347 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0% | 0 | 347 | |
| H5 (clade 7.2) | 347 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0% | 0 | |||
| H5 (clade 2.3.4.4) | 347 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0% | 0 | |||
| H7 | 324 | 10 | 3 | 7 | 3 | 0 | 0 | 0 | 10–80 | 6.63% | 23 | ||
| H9 | 13 | 30 | 26 | 88 | 124 | 54 | 8 | 4 | 10–640 | 96.25% | 334 | ||
| Fox | H5 (clade 2.3.2) | 195 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | \ | 0% | 0 | 195 |
| H5 (clade 7.2) | 195 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0% | 0 | |||
| H5 (clade 2.3.4.4) | 195 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0% | 0 | |||
| H7 | 163 | 4 | 10 | 10 | 1 | 4 | 2 | 1 | 10–640 | 16.41% | 32 | ||
| H9 | 175 | 3 | 7 | 6 | 1 | 3 | 0 | 0 | 10–160 | 10.26% | 20 | ||
Fig. 1.
HI titers of positive serum specimens against H7 virus and H9 virus in farmed mink. A total of 347 serum specimens were tested by HI assay of which 23 samples had detectable HI antibody titers against the H7 virus and 334 samples had detectable HI antibody titers against the H9 virus. Circle indicates that a sample had detectable HI antibody titers against the H7 virus. Square indicates that a sample had detectable HI antibody titers against the H9 virus. A sample that had both anti-H7 antibody titer and anti-H9 antibody titer is shown in the same color. The geometric mean and 95% confidence intervals are indicated by long and short horizontal lines, respectively
A total of 195 serum specimens collected from farmed fox were analyzed for serological evidence of H5 AIV, H7 AIV, and H9 AIV exposure. None fox serum specimens (0%) has detectable HI antibody titers (HI titers < 10) against H5 (clade 2.3.2) AIV, H5 (clade 7.2) AIV, or H5 (clade 2.3.4.4) AIV (Table 2). Thirty-two fox serum specimens (16.41%) have detectable HI antibody titers against the H7 AIV (HI titers range from 10 to 640) (Table 2; Supplemental Table 2). Furthermore, the hemagglutinin inhibition titer of about 13% H7 AIV positive serum specimens is 10, the hemagglutinin inhibition titer of about 31% H7 AIV positive serum specimens is 20, the hemagglutinin inhibition titer of about 31% H7 AIV positive serum specimens is 40, the hemagglutinin inhibition titer of about 3% H7 AIV positive serum specimens is 80, the hemagglutinin inhibition titer of about 13% H7 AIV positive serum specimens is 160, the hemagglutinin inhibition titer of about 6% H7 AIV positive serum specimens is 320, and the hemagglutinin inhibition titer of about 3% H7 AIV positive serum specimens is 640. Additionally, we found that twenty fox serum specimens (10.26%) have detectable HI antibody titers against the H9 AIV (HI titers range from 10 to 160) (Table 2; Supplemental Table 2). Furthermore, the hemagglutinin inhibition titer of 15% H9 AIV positive serum specimens is 10, the hemagglutinin inhibition titer of 35% H9 AIV positive serum specimens is 20, the hemagglutinin inhibition titer of 30% H9 AIV positive serum specimens is 40, the hemagglutinin inhibition titer of 5% H9 AIV positive serum specimens is 80, and the hemagglutinin inhibition titer of 15% H9 AIV positive serum specimens is 160. Interestingly, we found that ten fox serum specimens possess both anti-H7 AIV HI antibody and anti-H9 AIV antibody (Fig. 2; Supplemental Table 2). Taken together, these data indicated that H7 AIV and H9 AIV spread in farmed fox herds, and farmed fox can be co-infected with H7 AIV and H9 AIV.
Fig. 2.
HI antibody titers of positive serum specimens against H7 virus and H9 virus in farmed fox. A total of 195 serum specimens were tested by HI assay of which 32 samples had detectable HI antibody titers against the H7 virus and 20 samples had detectable HI antibody titers against the H9 virus. Circle indicates that a serum specimen had detectable HI antibody titers against the H7 virus. Square indicates that a serum specimen had detectable HI antibody titers against the H9 virus. Anti-H7 HI antibody titers and anti-H9 antibodies. A serum specimen that detected both anti-H7 and anti-H9 antibodies is shown in the same color. The geometric mean and 95% confidence intervals are indicated by long and short horizontal lines, respectively
The majority of the influenza virus cross-species risk is known to be caused by influenza A virus, and human influenza A viruses usually stem from AIV. Furthermore, cross-species transmission of AIV poses a potential threat to animal husbandry or public health [1, 2, 6, 10, 11]. Hence, AIV is a global threat to food animal production and distribution systems, as well as human health [4].
Farmed mink (Mustela vison) and farmed fox (Alopex lagopus) are important farmed fur–bearing animals that possess high economic value in eastern China. The previous study suggested that farmed mink can be infected with H9N2 avian influenza virus [12], which is the internal gene donor for the novel H7N9 virus [2, 8]. Those authors evaluated the percentage of sera positive for H9N2 virus antibodies in 560 sera collected from mink farms in five different areas of Shandong Province in China. Furthermore, they also evaluated the virulence of H9N2 virus in minks. These findings suggested that farmed fur–bearing animals could play a role in the transmission of AIV and enhanced surveillance is needed. Here, we found that farmed fur–bearing animals can infect with H7 and H9 AIV in eastern China and make a potential threat to public health. However, we did not assess what (which) is (are) the neuraminidase of the H7 and H9 viruses that had infected the farmed fur–bearing animals in this study, and that is a limitation of our study. Our findings suggest that the farmed fur–bearing animals may be an important animal host of AIV. In this study, we found for the first time that H7 subtype influenza virus infects fur-bearing animals; furthermore, we found that the same fur-bearing animal can be naturally infected with H7 virus and H9 virus, combined with fur-bearing animals can be infected with different subtypes of influenza virus [3, 12], suggesting that fur-bearing animals may become a potential influenza mixer.
In this study, H7- and H9-specific seroconversion was detected in farmed mink and farmed fox at farmed fur–bearing farms in eastern China; furthermore, we found that these animals can co-infect with H7 AIV and H9 AIV, suggesting that farmed fur–bearing animals may play an important role in the reassortment course of the novel avian influenza viruses. However, we did not find the serological evidence of H5 virus in farmed fur–bearing animals, although H5 virus infection in farmed mink was reported in a previous study [3]. We found that some mink serum specimens and some fox serum specimens possess both anti-H7 AIV HI antibody and anti-H9 AIV antibody in this study. Because we found that there was no cross antigenicity between H7 virus and H9 virus in our previous study [11], we speculated that animals may be sequentially infected with these two different viruses by eating food containing viruses or contacting utensils carrying viruses. In the future, it is necessary to determine which of these two ways (simultaneous infections or sequential infections) of infection is more likely to cause one animal possess two different viral antibodies in its serum by animal infection experiments. Our findings suggest that the farmed fur–bearing animals may be a potential important host of influenza viruses. Further study should pay attention to the persistent monitor for H7 AIV, H9 AIV, and other influenza viruses in farmed fur–bearing animals and humans or animals in close contact with them.
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Acknowledgments
We thank Jizhen Zou at animal husbandry and veterinary station of song village for his help in collecting animal serum specimens.
Funding information
This work was supported by the Shandong Provincial Natural Science Foundation (ZR2018QC005), the High-Level Talents and Innovative Team Recruitment Program of the Shandong Academy of Agricultural Sciences, the High-level Talent Projects (ts201511069; W03020496), the Construction of Subjects and Teams of Institute of Poultry Science (CXGC2018E11), and the Shandong Provincial Key Laboratory of Special Construction Project (SDKL201810).
Compliance with ethical standards
Conflict of interest
The authors declare that there are no conflicts of interest.
Ethics statement
The protocol of the study was conducted in accordance with guidelines of animal welfare of World Organization for Animal Health, and all animal experiments were approved from an ethics committee of Poultry Institute, Shandong Academy of Agricultural Sciences.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Zhijun Yu, Kaihui Cheng and Jiaqiang Wu contributed equally to this work.
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