| In 2010, a newly infectious disease caused by Tembusu virus(TMUV) was obeserved in many egg-laying and breeder duck farms in China, resulting in sharply egg production drop in affected egg-laying and breeder ducks and neurological symptoms(ataxia and paralysis) in ducklings. TMUV infection occurred in almost all duck breeder areas and led to serious economic loss. TMUV belongs to Ntaya virus group of family Flaviviridae, genus Flavivirus.In the study, possible vertical transmission of TMUV was investigated, providing reliably epidemiological foundation to control TMUV infection.1. Detection of TMUV from naturally affected ducks, eggs, embryos and ducklingsIn 2013, TMUV infection was sequentially observed in four breeder duck farms in Liaocheng, Taian and Guangrao of Shandong province. 125 eggs produced by affected breeder ducks were collected. Vitelline membrane samples of 35 eggs selected randomly from all were collected. The rest of 90 eggs were incubated according to the incubation criterion of Cherry Valley eggs. Simultaneously, 20 pure Cherry Valley eggs were incubated as control.Eggs from affected ducks and control eggs were incubated individually. All eggs incubated were checked once a day. Non-fertilized eggs and dead embryos were picked out and vitelline membrane, brain, liver and spleen were collected. All 1-day-old ducklings were killed and brain, liver and spleen were collected. All samples collected were saved at-80oC. All samples were disposed normally and inoculated into the allantoic sacs of 9 day-old pure duck eggs.Collected allantoic fluid was used for virus detection by nested RT-PCR. Consequently,fertility rate and hatching rate of affected eggs were 82.22%(74/90) and 62.16%(46/74),respectively. Corresponding data of control eggs were 95%(19/20) and 84.21%(16/19),respectively. 28 embryos died during incubation with hyperemia, hemorrhage and edema in brains and hemorrhage in hearts and livers. As a result, the detection rate of TMUV in eggs,dead embryos and ducklings were 51.43%(18/35) 、 60.71%(17/28) and 10.87%(5/46).Additionally, 2 of 16 non-fertilized eggs were detected TMUV positive. Overall, 42 of 125(33.6%) samples of affected eggs were positive for TMUV. TMUV was negative in controleggs. A virus strain, designated as TMUV-SDDE, was isolated from one dead duck embryos with 10-2.50ELD50/0.2 mL. E gene of TMUV-SDDE was amplified and sequenced. The results of phylogenetic analysis showed that E gene of TMUV-SDDE virus was closely related to other TMUV strains isolated in China in 2010-2013. TMUV-SDDE was injected intramuscularly in 1-day-old ducklings and isolated again. The findings provide evidence of possible vertical transmission of TMUV from breeder ducks to ducklings.2 Establishment of Experimental Infection Model of TMUV infectionAccording to the results of above-mentioned, experimental infection model of TMUV infection was established to investigate the possible vertical transmission and pathogenicity of TMUV for breeder ducks. 38-week-old TMUV negative Cherry Valley breeder ducks were divided into 5 groups. Group A, B and C were comprised of 15 female ducks and 4 male ducks. Female ducks in group A and male ducks in group B were inoculated by intravenous(i.v.) with 2.5 mL TMUV-SDSG strain(ELD50=10-2.37/0.2 mL). Male ducks in group A and female ducks in group B were bred without any treatment. Ducks in group C were injected by i.v. with 2.5 mL sterile normal saline as control. Group D and E were comprised of 20 female ducks. Ducks in group D were injected by intravenous(i.v.) with 2.5 mL TMUV-SDSG strain.Ducks in group E were injected with 2.5 mL sterile normal saline as control. Each group was bred separately. Eggs of every group were collected and incubated. Partial 1 day-old ducklings were killed and samples(e.g., brain, liver, spleen) were collected. Other ducklings were bred in SPF isolators and killed at 15 days-old with samples collection. Samples of dead ducklings were collected timely. All samples collected were treated normally and inoculated into duck embryos for virus isolation and detection by nested RT-PCR later. As a result,fertility rate and hatching rate of eggs from group A and group B were 82.2%(74/90), 62.2%(46/74) and 78.0%(67/86), 65.7%(44/67), respectively. Corresponding data of group C were94%(94/100), 85.1%(80/94), respectively. 43 eggs from group A and 37 eggs from group B were found dead during different stages of incubation. At necropsy, cutaneous hemorrhage,malabsorption in yolk, thin yolk and hemorrhage in liver or heart were found. Moreover,edema and hemorrhage in neck and edema, hyperemia in brain were found in several deadembryos. 4 weak ducklings in group A and 7 in group B died with edema or hemorrhage in brain and malabsorption in yolk were found. Results of virus detection showed that 60% eggs,67.44% dead embryos, 35.48% 1-day-old ducklings, 16.67% 15-day-old ducklings and 100%dead ducklings in group A were TMUV positive. Corresponding data in group B were 50%,51.35%, 46.88%, 10% and 71.43%, respectively. However, all samples from group C were found TMUV negative. Sequence analysis results showed that positive fragments shared98.7%-100% nucleotide homology with TMUV-SDSG strain. In a word, TMUV was detected in eggs, embryos and ducklings of experimentally infected breeder ducks, indicating that TMUV could pass on from infected ducks to offspring via eggs. In other words, TMUV can transmit vertically in ducks.10 ducks in group D and E were marked for sampling regularly. Blood and cloacal swab samples were collected at 1, 4, 7, 10, 13, 16 and 19 day post infection(dpi). Antibody against TMUV was detected by indirect ELISA. IFN-γ and IL-4 were detected by cell factor detection kit. Virus discharge through cloaca was detected by nested RT-PCR. 2 ducks chose randomly from another 10 ducks in each group were killed at 3, 7, 11, 15 and 19 dpi. Brain, liver, spleen,theca folliculi and e.g. were collected for the detection of viral load in samples were carried out by real time fluorescent quantitation RT-PCR. Consequently, egg production drop(from about 80% to 60%) and white loose stools were checked in infected ducks. Antibody against TMUV, IFN-γ and IL-4were detected positive from 1 dpi on. And their expression levels reached peak at 10 dpi, 13 dpi and 16 dpi, respectively. Levels of antibody and cell factor were detected changeless in control group. TMUV was detected positive in clocal swab from4 dpi till the experimental end(19dpi). The main pathologic changes observed were hemorrhage in lungs, glandular stomach and intestinal tract; hemorrhage in theca folliculi,transformation or rupture in follicle. Detection results of viral load in tissues showed that brain, pancreas and theca folliculi were detected TMUV positive at 3 dpi; theca folliculi, brain,pancreas, spleen, liver and duodenum were positive at 7 dpi; all samples were positive at11-19 dpi. It is noteworthy that viral load in theca folliculi, brain, pancreas and liver was higher than other tissues. All these results indicated that TMUV infection could stimulatehumoral immunity and cellular immunity, and mainly damaged follicle, brain, pancreas, liver and e.g.. In addition, TMUV could be discharged via cloaca for a long time, promoting horizontal transmission of TMUV in ducks. |
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