Pathological lesions in the immune organs of ducklings following experimental infection with goose parvovirus Contents lists available at ScienceDirect Research in Veterinary Science journal homepage[.]
Research in Veterinary Science 125 (2019) 212–217 Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc Pathological lesions in the immune organs of ducklings following experimental infection with goose parvovirus T Hongmei Liu, Chengcheng Yang, Miaomiao Liu, Kui Ma, Xueting Huang, Yu Zhao, Dongmei Hu, ⁎ Kezong Qi Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, Anhui Agricultural University, Hefei 230036, PR China A R T I C LE I N FO A B S T R A C T Keywords: Goose parvovirus Ducklings Immune organ Pathological lesion Viral load Goose parvovirus (GPV) is the etiological agent of Derzsy's disease, with a natural reservoir consisting only of geese and Muscovy ducks However, the pathological changes in the immune organs of ducklings experimentally infected with GPV remain unknown In this study, 2-day-old Cherry Valley ducklings were intramuscularly injected with GPV Immune organs (e.g., thymus, bursa of Fabricius, spleen, Harderian gland, cecal tonsil, bone marrow, and peripheral blood lymphocytes [PBLs]) were collected 1, 3, 5, 7, 10, and 14 days post-infection (dpi) Pathological lesions were assessed by histology and the viral load was concurrently assessed using quantitative real-time polymerase chain reaction GPV antigen was detected via immunofluorescence staining and immunohistochemistry No clinical symptoms or death were observed in the infected ducklings from to 14 dpi; however, lesions with different degrees of hemorrhage and hyperemia were observed in the thymus, spleen and Harderian gland Lymphocyte necrosis was identified in the thymus and spleen In the immune organs, the highest viral loads were found in the spleen at dpi, followed by the bone marrow, PBLs, and cecal tonsil at dpi, and the bursa, Harderian gland, and thymus at dpi GPV antigen was primarily expressed in the cecal tonsil, spleen, and Harderian gland at dpi, as well as in the PBLs and bone marrow at dpi Our findings indicate widespread GPV replication and dissemination in the immune organs of Cherry Valley ducklings Introduction Goose parvovirus (GPV) infection is also widely known as Derzsy's disease (Derzsy, 1967), or gosling plague in China(Fang, 1962) Goslings (Anser anser domestica) and Muscovy ducklings (Cairina moschata) are considered to be the natural reservoir of GPV (Gough et al., 1981; Jansson et al., 2007; Woźniakowski et al., 2009) Since the 1960s, live and inactivated vaccines have been administered to prevent and control GPV in several countries, including China; however, the disease remains enzootic in some regions and is currently recognized to be a major disease affecting A a domestica (Tatár-kis et al., 2004; Shien et al., 2008; Wang et al., 2014) In 2015, a distinct GPV-related parvovirus found to be associated with beak atrophy and dwarfism syndrome (BADS) in Cherry Valley ducks (Anas platyrhynchos domesticus) emerged in northern China (Chen et al., 2015) Multiple reports have identified BADS to exhibit various levels of pathology in Cherry Valley and Mule ducklings, as well as common goslings and goose embryos (Zhu et al., 2010; Chen et al., 2015, 2016a, 2016b; Ning et al., 2018) These GPV-related parvoviruses ⁎ are similar to GPV isolates both genetically and antigenically, and epidemiological investigations have also found that GPV can silently circulate in commercial ducks through asymptomatic infection (Shehata et al., 2016; Niu et al., 2017; Wang et al., 2017; Li et al., 2018) However, there is currently no evidence to support the transmission of GPV among common ducks Ning was the first to report that GPV can infect and replicate in Pekin ducklings (Ning et al., 2017) Moreover, Niu found that GPV in infected Shaoxing ducklings was distributed in histopathological lesions of the cardiac muscle, bursa, duodenum, kidney, and skeletal muscle (Niu et al., 2018) Although no ducklings died during the experiment, these results indicate that GPVs are pathogenic in ducklings To control the introduction of GPVs into new hosts, it is critically important to determine the host range of GPV and the potential for host shifts However, to our knowledge, few studies have investigated the characteristics of GPV infection in the immune organs of ducklings (A p domesticus) (Zhu et al., 2010) In the present study, we established an experimental model of GPV infection to investigate the pathogenicity of GPV in Cherry Valley Corresponding author at: 130 West Changjiang Road, College of Animal Science and Technology, Hefei, Anhui 230036, PR China E-mail address: qkz@ahau.edu.cn (K Qi) https://doi.org/10.1016/j.rvsc.2019.06.002 Received 22 March 2019; Received in revised form 30 May 2019; Accepted 10 June 2019 0034-5288/ © 2019 Elsevier Ltd All rights reserved Research in Veterinary Science 125 (2019) 212–217 H Liu, et al 2.5 Detection of viral antigen in immune organs by IHC and IFA ducklings, particularly the immune organs In the infected group, the distribution of the GPV antigen was assessed at 1–14 dpi All collected tissues were routinely processed for histology and 4-μm-thick sections were stained with hematoxylin-eosin (HE) The stained sections were then examined by IHC to detect the presence and distribution of the GPV antigen IHC was performed using a 1:100 dilution of monoclonal antibody against GPV in an indirect immunoperoxidase staining procedure, involving the application of a streptavidin-biotin-based technique (SABC complex kit, Boster, China) on the sections following previously published protocols (Wen et al., 2018) Samples from the uninfected group were used as negative controls Smeared slide samples, including bone marrow cells and PBLs, were tested for the presence of GPV antigen using IFA The smeared slides were blocked for h using 1% bovine serum albumin (Sangon Biotech, China) and 1% Triton X-100 (Sangon Biotech) in PBS (blocking buffer) Next, the sections were incubated overnight at °C with anti-GPV mAbs diluted 1:200 in blocking buffer The sections were washed in PBS and subsequently incubated with fluorescein-isothiocyanate-labeled goat anti-mouse IgG (Sangon Biotech) diluted in blocking buffer, and 10 μg/ mL of 4′, 6-diamidino-2-phenylindole (DAPI; Sercicebio, Wuhan, China) for h, and finally washed in PBS Samples from the uninfected group were used as negative controls Materials and methods 2.1 Virus and birds The GPV WX3 strain (ELD50 = 10–4.21/0.2 mL, GenBank: MK333463) was preserved by Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, Anhui Agricultural University, China The GPV WX3 strain was isolated from goslings (Anser anser domestica) and displayed a high level of pathogenicity in these birds The mortality rate of goslings infected with 0.3 mL/gosling of the GPV WX3 strain (105 ELD50) was 100%, and every gosling displayed clinical symptoms of GPV (e.g., paralysis and intestinal embolism) to days post infection (dpi) Thirty-six healthy, 1-day-old Cherry Valley ducklings (Anas platyrhynchos domesticus) free of parvovirus-specific maternal antibodies were purchased from the Cherry Valley duckling hatchery in Anhui Province, China The Ethical Committee of the Faculty of Veterinary Science of Anhui Agricultural University approved this study Anti-GPV mAbs were kindly provided by Professor Aijian Qin, College of Veterinary Medicine, Yangzhou, China 2.2 Experimental design The 36 Cherry Valley ducklings were divided equally and randomly into two groups The control and infected group were denoted Group (n = 18) and Group (n = 18), respectively The birds in the infected group were intramuscularly injected with 0.3 mL/duckling of the GPV WX3 strain (105 ELD50) The birds in the control group were injected with 0.3 mL/duckling sterile phosphate-buffered saline (PBS) The two groups were housed in the separate isolated cages under consistent conditions Clinical symptoms were observed daily following infection Tissues were harvested at 1, 3, 5, 7, 10, and 14 dpi 2.6 Statistical analysis 2.3 Sampling Bone marrow, peripheral blood lymphocytes (PBLs), serum, liver, spleen, bursa of Fabricius (bursa), thymus, cecal tonsil, and Harderian gland were harvested from ducklings The tissues were immediately processed and stored at −80 °C until use in quantitative real-time PCR assay (qPCR) The tissues were immediately fixed by immersion in a 4% paraformaldehyde solution for histological examination and immunohistochemistry (IHC) Heparinized peripheral blood samples were collected and PBLs were isolated by Ficoll-Paque density centrifugation (Haoyang Co., Ltd., Tianjin, China) Bone (tibia) marrow was flushed out of the bone with PBS and washed marrow cells were collected PBLs and marrow cells were directly smeared onto slides to conduct an indirect immunofluorescence assay (IFA) The infected ducklings were depressed and lost their appetite between 24 and 48 h post-inoculation; however, these symptoms decreased and then disappeared at 3–5 dpi No clinical signs were identified in the infected group from to 14 dpi, and no ducklings died during the experiment However, the spleen, liver, and intestinal tracts of the majority of ducklings from the infected group were swollen, and the thymus and spleen exhibited blood spots compared with the control group in necropsy (Fig 1A–B) Microscopically, moderate to severe hemorrhaging was observed in the thymus at 3, 5, 7, and 10 dpi (Fig 1D), and in the spleen at 3, 5, 7, 10, and 14 dpi (Fig 1E) Hyperemia were observed in the Harderian gland at 1, 3, 5, and dpi (Fig 1F) Degeneration and necrosis of lymphocytes in the spleen and thymus at 3, 5, 7, and 10 dpi were observed by HE (Table 1; Fig 1C) 2.4 Quantification of the viral load in the immune organs by real-time PCR 3.2 Most immune organs exhibited a detectable viral load A pair of primers targeting the GPV VP3 gene were designed: forward, 5′-TCCATCCTTCTCCGAATCT-3′ and reverse: 5′-CACTTCTGGTG CACGTATT-3′ Viral DNA was extracted from tissue samples using a viral DNA kit (OMEGA, USA) according to the manufacturer's instructions, and the viral loads in the immune organs were quantified by qPCR using an Applied Biosystems® Real-Time PCR Instrument (Thermo Fisher Scientific, USA) The qPCR reaction volume was 20 μL, containing 0.4 μL 0.1 mmol/L forward primer, 0.4 μL 0.1 mmol/L reverse primer, 10 μL AceQ qPCR SYBR Green Master Mix (Vazyme Inc., Nanjing, China), 0.4 μL ROX Reference Dye 1, 6.8 μL ddH2O, and 2.0 μL of the template DNA The cycling conditions were 95 °C for min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s The viral load in the different tissues of the ducklings at 1, 3, 5, 7, 10, and 14 dpi was detected using qPCR A standard curve was used to quantify the number of viral copies (R2 = 0.9986) (Supplemental Fig S1) High viral loads were observed in all the infected animals in the spleen at dpi, in the bone marrow at dpi, and in the bursa and thymus at dpi (Fig 2) The number of viral DNA copies in the bursa, thymus, and Harderian gland peaked at dpi, then gradually reduced to the lowest values at 10 or 14 dpi The number of viral DNA copies in the serum peaked at dpi, then decreased to the lowest values at 5–14 dpi The number of viral DNA copies in the PBLs peaked at dpi, then decreased The control birds were not positive for viral DNA Statistical analysis was performed using Prism software The mean and standard error of the mean (SEM) values were calculated Results 3.1 Pathological lesions in the immune organs in experimentally-infected ducklings 213 Research in Veterinary Science 125 (2019) 212–217 H Liu, et al Fig Gross and histological lesions in infected ducklings A Blood spots on the thymus B Swelling and congested spleen C Degeneration and necrosis of lymphocytes in the spleen Bar =50 μm D Hemorrhage in thymus E Hemorrhage in spleen F Hyperemia in Harderian gland G Thymus from the control group H Spleen from the control group I Harderian gland from the control group All histological lesions were detected by HE staining Bar =20 μm nucleus of bone marrow cells and PBLs (Fig 3A, B and D, E) Strong positive GPV staining was observed in the cecal tonsil, spleen and Harderian gland, and weak-to-medium positive GPV staining was found in the thymus and bursa by IHC (Table 1; Fig 4A, C, E, G) No viral antigens were detected in any of the tissue samples from the control birds Table GPV antigen distribution and intensity in histological lesions in HE and IHCstained samples Organ Stain dpi dpi dpi dpi 10 dpi 14 dpi Bursa HE IHC HE IHC HE IHC HE IHC HE IHC + + ++ + + + + ++ − ++ + + +++ ++ +++ + + ++ + +++ + + +++ + +++ ++ + ++ + +++ + + +++ + +++ ++ + ++ + ++ − + +++ − +++ + − + + ++ − − ++ − ++ + − − + ++ Thymus Spleen Harderian gland Cecal tonsil Discussion In the present study, qPCR data revealed that the viral load in the bursa, thymus, and Harderian gland peaked at dpi of Cherry Valley ducklings with GPV; that in the bone marrow, lymphocytes, cecal tonsil, and serum peaked at dpi; and that in the spleen and liver peaked at dpi The viral loads in all the organs then decreased, reaching their lowest values at around 14 dpi Although no ducklings died, and no external diagnostic pathological lesions were visible, GPV WX3 caused pathological lesions in the spleen, thymus, and Harderian gland (e.g., blood spots in the thymus, swelling of the spleen and Harderian gland) These lesions were partially consistent with the observations of Ning et al (2017) in infected Shaoxing ducks Hemorrhage, degeneration, and lymphocyte necrosis in the spleen, hemorrhage in the thymus and spleen, and hyperemia in the Harderian gland were observed in the challenged ducklings, which may be attributed to −, no histological lesions/negative for GPV by IHC; +, mild histological lesions/few positive cells; ++, moderate histological lesions/frequent positive cells; +++, marked histological lesions/clusters of positive cells 3.3 Viral antigens were detected in the majority of the immune organ tissues Viral antigens were detected in the birds in the infected group at 1–14 dpi IFA detection revealed that the fluorescence was localized in the cell nucleus, illustrating the primary replication of GPV in the 214 Research in Veterinary Science 125 (2019) 212–217 H Liu, et al Fig Viral load in various tissues of the infected groups Viral load in the immune organs of infected ducklings at 1, 3, 5, 7, 10, and 14 dpi The CT value of each sample was entered into the standard curve formula to calculate the number of viral DNA copies, and converted to the viral copy number (copies/μL) All samples were analyzed in triplicate and data are expressed as the average ± standard deviation Fig Viral antigen detection by IFA A Bone marrow cells from infected ducklings B Nuclei of bone marrow cells were counterstained with DAPI C Bone marrow cells from the control group D PBLs from infected ducklings E Nuclei of PBLs were counterstained with DAPI F PBLs from the control group GPV antigens were observed by IHC staining in multiple immune organs and were abundant at 3–7 dpi Collectively, our findings demonstrate that GPV was widespread in the immune organs of Cherry Valley ducklings 1–7 dpi In general, GPV primarily affects young goslings and Muscovy ducklings, and does not appear to cause any clinical signs in other duckling species (Chen et al., 2016b; Ning et al., 2017; Wang et al., 2017) However, our data show that GPV was able to replicate the high viral load observed in these organs Our results showed that GPV strain WX3 was pathogenic to Cherry Valley ducklings, but the virulence was lower than toward goslings The viral load in the bone marrow cells and PBLs of Cherry Valley ducklings peaked at dpi and the viral load in the spleen and liver peaked at dpi, demonstrating that GPVs can replicate in bone marrow cells and subsequently enter the bloodstream Finally, GPVs accumulated in the spleen and liver, peaking at dpi, by which time few GPVs were observed in the serum 215 Research in Veterinary Science 125 (2019) 212–217 H Liu, et al Fig Viral antigen detection by IHC A Strong positive cells in the cecal tonsil at dpi C Moderately positive cells in the spleen at dpi E Moderately positive cells in the Harderian gland at dpi G Moderately positive cells in the thymus at dpi B, D, F and H from uninfected groups Bar =20 μm Declaration of Competing Interest efficiently in Cherry Valley ducklings and caused lesions in the immune organs, indicating that the classical GPV is well adapted to ducklings These pathological changes in the immune organs may cause decreased cellular functionality and immunity, resulting in enhanced opportunity for coinfection with other pathogens, and potentially lead to failure of immunization Moreover, ducklings infected with GPV may become carriers that excrete and spread the virus Several studies have demonstrated that entry into and replication in new host cells are key factors impacting pathogen host range and pathogenicity (Fan et al., 2017) Host shifts exhibited by pathogens are also related to gene evolution and selection pressures Pathogens crossing the species barrier and host immunity are of particular concern We speculate that some pathogens are more likely to be infectious in hosts that are closely related to their natural host (e.g., in geese and ducks) GPV-contaminated feed and water or GPV vaccine immune pressure on goslings may thus be of prime importance for GPV-infection of ducklings When GPVs invade ducklings, this may lead to histopathological lesions in immune organs and potential for infection with other pathogens This leads to synergistic pathopoiesis and more severe pathological lesions Therefore, our findings help further understanding of GPV pathogenesis in ducklings Supplementary data to this article can be found online at https:// doi.org/10.1016/j.rvsc.2019.06.002 The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article Acknowledgements The authors express their gratitude to Prof Qin Aijian for kindly providing anti-GPV monoclonal antibodies References Chen, H., Dou, Y., Tang, Y., Zhang, Z., Zheng, X., Niu, X., Diao, Y., 2015 Isolation and genomic characterization of a duck-origin GPV-related parvovirus from cherry valley ducks in China PLoS ONE 10, e0140284 Chen, H., Dou, Y., Tang, Y., Zheng, X., Niu, X., Yang, J., Diao, Y., 2016a Experimental reproduction of beak atrophy and dwarfism syndrome by infection in cherry valley ducklings with a novel goose parvovirus-related parvovirus Vet Microbiol 183, 16–20 Chen, S., Wang, S., Cheng, X., Xiao, S., Zhu, X., Lin, F., Yu, F., 2016b Isolation and characterization of a distinct duck-origin goose parvovirus causing an outbreak of duckling short beak and dwarfism syndrome in China Arch Virol 161, 2407–2416 Derzsy, D., 1967 A viral disease of goslings I Epidemiological, clinical, pathological and aetiological studies Acta Vet Acad Sci Hung 17, 443–448 Fan, W.F., Sun, Z.Y., Shen, T.T., Xu, D.N., Huang, K.H., Zhou, J.Y., Song, S.Q., Yan, L.P., 2017 Analysis of evolutionary processes of species jump in waterfowl parvovirus Front Microbiol (421) Fang, D.Y., 1962 Recommendation of GPV Chin Anim Husb Vet Med 8, 19–20 (in Chinese) Gough, R.E., Spackman, D., Collins, M.S., 1981 Isolation and characterisation of a parvovirus from goslings Vet Rec 108, 399–400 Jansson, D.S., Feinstein, R., Kardi, V., Mató, T., Palya, V., 2007 Epidemiologic investigation of an outbreak of goose parvovirus infection in Sweden Avian Dis Digest 51, 609–613 Li, P., Li, J., Zhang, R., Chen, J., Wang, W., Lan, J., Xie, Z., Jiang, S., 2018 Duck “beak atrophy and dwarfism syndrome” disease complex: interplay of novel goose parvovirus-related virus and duck circovirus? Transbound Emerg Dis 65 (2), 345–351 Ning, K., Wang, M., Qu, S., Lv, J., Yang, L., Zhang, D., 2017 Pathogenicity of Pekin duckand goose-origin parvoviruses in Pekin ducklings Vet Microbiol 210, 17–23 Ning, K., Liang, T., Wang, M., Dong, Y., Qu, S., Zhang, D., 2018 Pathogenicity of a variant goose parvovirus, from short beak and dwarfism syndrome of Pekin ducks, in goose embryos and goslings Avian Pathol 47 (4), 391–399 Niu, X., Wang, H., Wei, L., Zhang, M., Yang, J., Chen, H., 2017 Epidemiological investigation of h9 avian influenza virus, Newcastle disease virus, tembusu virus, goose Funding This work was supported by grants from the National Natural Science Foundation of China (Grant No 31872445 and 31772707) Author contributions HL and KQ designed the experiments, analyzed the data, and wrote the manuscript CY, ML, KM, XH, YZ, and DH performed the experiments HL and KQ performed the histologic analysis All authors reviewed the draft of this manuscript 216 Research in Veterinary Science 125 (2019) 212–217 H Liu, et al a goose parvovirus vaccine strain SYG61v and rescue of infectious virions from recombinant plasmid in embryonated goose eggs J Virol Methods 200, 41–46 Wang, S., Cheng, X.X., Yu, B., Xiao, S.F., Chen, S.L., Zhu, X.L., Yu, F.S., Lin, S., Chen, S.Y., Lin, F.Q., Wu, N.Y., Wang, J.X., Huang, M.Q., Zheng, M., 2017 A simple, polymerase chain reaction and restriction fragment length polymorphismaided diagnosis method for short beak and dwarfism syndrome in ducklings Infect Genet Evol 53, 85–88 Wen, Y., Huang, Q., Yang, C., Pan, L., Wang, G., Qi, K., Liu, H., 2018 Characterizing the histopathology of natural co-infection with Marek's disease virus and subgroup J avian leucosis virus in egg-laying hens Avian Pathol 47 (1), 83–89 Woźniakowski, G., Kozdru, W., Samorek-Salamonowicz, E., 2009 Genetic variance of Derzsy's disease strains isolated in Poland J Mol Genet Med 3, 210–216 Zhu, L.Q., Ding, X.Y., Tao, J., Wang, J.Y., Zhang, X.J., Wang, X.B., Hu, Y., Li, H.F., Chen, K.W., Zhu, G.Q., 2010 Identification of target cells for goose parvovirus infection in the immune system organs Acta Virol 54 (3), 211–215 parvovirus and goose circovirus infection of geese in China Transbound Emerg Dis 65 (2), 304–316 Niu, Y.N., Zhao, L.L., Liu, B.H., Liu, J.L., Yang, F., Yin, H.C., Huo, H., Chen, H.Y., 2018 Comparative genetic analysis and pathological characteristics of goose parvovirus isolated in Heilongjiang, China Virol J 15, 27 Shehata, A.A., Gerry, D.M., Heenemann, K., Halami, M.Y., Tokarzewski, S., Wencel, P., Vahlenkamp, T.W., 2016 Goose parvovirus and circovirus coinfections in ornamental ducks Avian Dis 60, 516–522 Shien, J.H., Wang, Y.S., Chen, C.H., Shieh, H.K., Hu, C.C., Chang, P.C., 2008 Identification of sequence changes in live attenuated goose parvovirus vaccine strains developed in Asia and Europe Avian Pathol 37, 499–505 Tatár-kis, T., Mató, T., Markos, B., Palya, V., 2004 Phylogenetic analysis of Hungarian goose parvovirus isolates and vaccine strains Avian Pathol 33, 438–444 Wang, J., Duan, J., Meng, X., Gong, J., Jiang, Z., Zhu, G., 2014 Cloning of the genome of 217 ... Competing Interest efficiently in Cherry Valley ducklings and caused lesions in the immune organs, indicating that the classical GPV is well adapted to ducklings These pathological changes in the immune. .. identified in the infected group from to 14 dpi, and no ducklings died during the experiment However, the spleen, liver, and intestinal tracts of the majority of ducklings from the infected group... respectively The birds in the infected group were intramuscularly injected with 0.3 mL/duckling of the GPV WX3 strain (105 ELD50) The birds in the control group were injected with 0.3 mL/duckling sterile