In 2010, a novel Tembusu virus (TMUV) that caused a severe decrease in the egg production of ducks was isolated in southeast China. Given the novelty of this duck pathogen, little information is available regarding its pathogenesis. Here, we systematically investigated the replication kinetics of TMUV PTD2010 in adult male and female ducks. We found that PTD2010 was detectable in most of the parenchymatous organs as well as the oviduct and intestinal tract from days 1 to 7 after inoculation. Viral titers were maintained at high levels for at least 9 days in the spleen, kidney, bursa of Fabricius, brain, and ovary. No virus was detected in any of these organs or tissues at 18 days after inoculation. PTD2010, thus, causes systemic infections in male and female ducks; its replication kinetics show similar patterns in most organs, with the exception of the ovaries and testes.
A novel Tembusu virus (TMUV), a member of the genus
Duck TMUV has been frequently detected in ducks and other animals in China since the first reported outbreak. This virus has been isolated from mosquitoes in Shandong Province, China [
Because TMUV was only recently identified as a duck pathogen, little information is available regarding its etiological characteristics and pathogenesis. To date, the many reports published on this new pathogen have focused on viral identification [
TMUV strain PTD2010, isolated from ducks in the Fujian province of China, was propagated for four passages in 9-day-old specific pathogen-free (SPF) embryonated duck eggs. Allantoic fluids were collected and stored at −70°C for further use. Stock titers were detected in duck embryo fibroblasts (DEFs). DEFs were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) plus 100
A total of 120 8-month-old SPF adult male and female (1 : 1) Shaoxing ducks were provided by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Harbin, China). Of these, 30 male and 30 female ducks were subcutaneously inoculated with a median tissue culture infective dose (TCID50) of 103 of PTD2010 in phosphate-buffered saline (PBS). The remaining 30 male and 30 female ducks served as controls. Three ducks from each group were euthanized on days 1, 3, 5, 7, 9, 18, and 35 after infection, and the parenchymatous organs (brain, heart, liver, spleen, lung, kidney, and ovary/testis), digestive tracts (duodenum, jejunum, ileum, caecum, and rectum), and oviducts of the female ducks (infundibulum, magnum, isthmus, shell grand pouch, and vagina) were harvested for viral titration. For histopathological and immunohistochemical analyses of the ovaries and testes, samples were fixed in 10% neutral buffered formalin solution. The remaining ducks were observed daily for signs of sickness until 40 days after inoculation (dpi).
After dehydration, tissue blocks of organs were embedded in paraffin and then cut into 4
Each tissue sample (0.2 g) was homogenized in 1 mL of PBS, freeze-thawed three times, and centrifuged at 10,000 ×g at 4°C. The supernatant was serially diluted 108-fold from 100 to 10−8 with DMEM. DEFs (104) were grown in 96-well plates and infected with 50
After inoculation with PTD2010, food intake by all infected male and female ducks began to decline at 3 dpi, and green-colored feces was found from 5 dpi. The egg production of infected female ducks declined rapidly from 3 dpi and egg production ceased from 5 dpi (Figure
Daily egg production rate after PTD2010 inoculation.
Necropsies of the infected female ducks clearly showed severe ovarian lesions from 5 dpi, including ovarian hemorrhage, ovaritis, and peritonitis caused by rupturing of the ovarian follicles. Vitellose in the abdominal cavity was completely absorbed by 18 dpi; the ovaries remained in the resting stage and were characterized by small abnormal follicles. Newly formed small ovarian follicles were observed at 35 dpi. However, no obviously identical lesions were found in the testes of the male ducks.
Histopathological analysis of the infected ducks showed that the pathological changes in the ovaries began at 3 dpi and developed to severe changes at 5 dpi (Figures
Histopathological analysis of ovaries and testes. Histopathological analysis of the ovary at 5 dpi: mock-infected control (a), ruptured follicles (b), interstitial hemorrhage (c), lymphocytic infiltration (d), smooth muscle degeneration (e), and small vessel hyperplasia (f) can be seen. Pathological changes in the testes at 5 dpi, mock-infected control (g), reduced sperm production, mild interstitial fibrous hyperplasia, spermatocytic swelling, vacuolar degeneration, and desquamation (h) can be seen. The pathological changes progressed, becoming more severe at 9 dpi; focal lymphocytic infiltration was observed (i). Images (a), (d), (e), (g), (h), and (i) were taken at ×200 magnification; Images (b), (c), and (f) were taken at ×100 magnification.
To investigate the replication of PTD2010 in different parenchymatous organs of male and female ducks, we titrated the tissues collected on days 1, 3, 5, 7, 9, 18, and 35 after inoculation by using DEFs. As shown in Figure
Replication kinetics of PTD2010 in the parenchymatous organs and blood of infected ducks. Data from thespleen (a), bursa of Fabricius (b), lung (c), kidney (d), heart (e), liver (f), brain (g), blood (h), and ovary/testis (i) are shown. Each time point represents the mean viral titer ± SD obtained from three ducks. The black dashed line indicates the limit of detection.
Significant differences in PTD2010 replication in male and female ducks were detected in the ovaries and testes (Figure
Immunohistochemical analysis of the ovary and testis at 3 dpi. Mock-infected testis (a) and mock-infected ovary (b) are shown; viral antigen was detected in the testis (black arrow) (c) and in the ovary (black arrow) (d). Images (a)–(d) were taken at ×200 magnification.
In this study, we also investigated the replication of PTD2010 in the intestinal tracts of male and female ducks. As shown in Figure
Replication kinetics of PTD2010 in the intestinal tract and oviduct of infected ducks. Data from the digestive tract (a) and oviduct (b) are shown. Each time point represents the mean viral titer ± SD obtained from three ducks. The black dashed lines in (a) and (b) indicate the limit of detection.
The
In summary, PTD2010 caused systemic infections in female and male ducks and was detected in most parenchymatous organs as well as the oviducts and intestinal tracts from 1 to 7 dpi. Viruses were detectable in only four organs of female and three organs of male ducks at 9 dpi. No virus was detected in any of the examined organs or tissues after 18 dpi. The viruses showed similar replication kinetics in female and male ducks, except in the ovary and testis, where the replication kinetics differed only slightly. Although the viral titer in the testes was significantly lower than that in the ovaries at 5 dpi, the pathological effects were very severe, causing total destruction of sperm generation. This study is the first to systematically assess the viral load and tissue distribution of TMUV in both male and female adult ducks. Our results provide further insights into the pathogenesis of TUMV in ducks.
The authors have no conflict of interests.
Li Wu and Jinxiong Liu contributed equally to this work.
The authors thank Susan Watson for editing the paper. This work was supported by the NSFC (31302064).