Isolation and Characterization of A Novel Fowl Adenovirus Serotype 8a Strain from China.

Since 2012, the clinical cases of inclusion body hepatitis showed an increasing trend in China, causing considerable economic losses to the poultry industry. In this study, a fowl adenovirus strain CH/GDLZ/201801 was isolated from a chicken flock experiencing inclusion body hepatitis and analyzed by complete genome sequencing. The pathogenicity of the new virus strain was examined by experimental infection of specific pathogen free chickens. The isolate was identified by immunofluorescence and the virions presented typical icosahedral particles under transmission electron microscopy. The full genome of the isolate was 44,329 nucleotides in length with 58% G+C content. Phylogenetic analysis, based on the whole genome, revealed that the new isolate was closest to serotype 8a from the species Fowl aviadenovirus E (FAdV-E). Recombination analysis and phylogenetic analysis showed that the new isolate is a recombinant strain between FAdV-8a and FAdV-8b. In infection experiments, three infected chickens showed clinical signs and one chicken died on day 7 post infection, corresponding to 5% mortality. Macroscopic and microscopic lesions in the liver were observed, and viral antigen could be detected in the livers by immunohistochemical staining and TEM. Taken together, our study describes the genomic characteristics and pathogenicity of a FAdV-8a strain in China. It would lay a solid foundation for further study of the pathogenic mechanism and vaccine development of the virus.

Epidemiological investigation of fowl adenovirus infections in poultry in China during 2015-2018.

BACKGROUND: Fowl adenoviruses (FAdVs) are associated with many diseases, resulting in huge economic losses to the poultry industry worldwide. Since 2015, outbreaks of FAdV infections with high mortality rates have been reported in China. A continued surveillance of FAdVs contributes to understand the epidemiology of the viruses. RESULTS: We isolated 155 FAdV strains from diseased chickens from poultry in China between 2015 and 2018. PCR analysis determined that 123 samples were FAdV species C, 27 were FAdV species E, and five contained two different FAdV strains. The phylogenetic analysis demonstrates that these sequences of hexon regions were clustered into three distinct serotypes: FAdV-4 (79.4%, 123/155), FAdV-8a (13.5%, 21/155) and FAdV-8b (3.9%, 6/155), of which FAdV-4 was the dominant serotype in China. CONCLUSIONS: The characterization of newly prevalent FAdV strains provides valuable information for the development of an effective control strategy for FAdV infections in chickens.

A Newly Isolated Bacillus subtilis Strain Named WS-1 Inhibited Diarrhea and Death Caused by Pathogenic Escherichia coli in Newborn Piglets.

Bacillus subtilis is recognized as a safe and reliable human and animal probiotic and is associated with bioactivities such as production of vitamin and immune stimulation. Additionally, it has great potential to be used as an alternative to antimicrobial drugs, which is significant in the context of antibiotic abuse in food animal production. In this study, we isolated one strain of B. subtilis, named WS-1, from apparently healthy pigs growing with sick cohorts on one Escherichia coli endemic commercial pig farm in Guangdong, China. WS-1 can strongly inhibit the growth of pathogenic E. coli in vitro. The B. subtilis strain WS-1 showed typical Bacillus characteristics by endospore staining, biochemical test, enzyme activity analysis, and 16S rRNA sequence analysis. Genomic analysis showed that the B. subtilis strain WS-1 shares 100% genomic synteny with B. subtilis with a size of 4,088,167 bp. Importantly, inoculation of newborn piglets with 1.5 × 1010 CFU of B. subtilis strain WS-1 by oral feeding was able to clearly inhibit diarrhea (p < 0.05) and death (p < 0.05) caused by pathogenic E. coli in piglets. Furthermore, histopathological results showed that the WS-1 strain could protect small intestine from lesions caused by E. coli infection. Collectively, these findings suggest that the probiotic B. subtilis strain WS-1 acts as a potential biocontrol agent protecting pigs from pathogenic E. coli infection. Importance: In this work, one B. subtilis strain (WS-1) was successfully isolated from apparently healthy pigs growing with sick cohorts on one E. coli endemic commercial pig farm in Guangdong, China. The B. subtilis strain WS-1 was identified to inhibit the growth of pathogenic E. coli both in vitro and in vivo, indicating its potential application in protecting newborn piglets from diarrhea caused by E. coli infections. The isolation and characterization will help better understand this bacterium, and the strain WS-1 can be further explored as an alternative to antimicrobial drugs to protect human and animal health.

Isolation and characterization of a highly pathogenic strain of Porcine enteric alphacoronavirus causing watery diarrhoea and high mortality in newborn piglets.

Porcine enteric alphacoronavirus (PEAV) was first discovered in China in February 2017, and the origin and virulence of this novel porcine coronavirus were not fully characterized. Here, we isolated a strain of PEAV, named GDS04 that is identified by immunofluorescence and typical crown-shaped particles observed with electron microscopy. Genomic analysis reveals that PEAV GDS04 shares a close relationship with SADS-CoV and SeACoV. Furthermore, newborn piglets orally challenged with PEAV GDS04 developed typical clinical symptoms as watery diarrhoea in neonatal piglets. Viral RNA was detected in faeces and various tissues of the infected piglets. Moreover, macroscopic and microscopic lesions in whole intestinal tract were observed, and viral antigen could be detected in the small intestines by immunohistochemical staining and electron microscopy. Importantly, the mortality rate of inoculated-newborn piglets was 100% and half of the cohabiting piglets died. Collectively, we demonstrate that PEAV is highly pathogenic in newborn piglets.

Recombinant fiber-2 protein protects Muscovy ducks against duck adenovirus 3 (DAdV-3).

As a novel duck adenovirus 3 (DAdV-3) infection caused significant economic losses to the poultry industry in China, there is an urgent need to develop a safe and effective vaccine. In the research, fiber-1 and fiber-2 proteins were expressed and purified, respectively. To evaluate the immunogenicity of the two recombinant proteins, we investigated the IgY antibodies and virus-neutralizing antibodies in duck sera. The protective efficacy was evaluated by mortality, virus shedding and histopathological examinations after challenged with the DAdV-3. Results showed that the IgY antibody levels of the fiber-2 group was significantly higher than that of the fiber-1 group and inactivated vaccine group. Ducks vaccinated with fiber-2 group provided full protection with no mortality, no virus shedding and no histological lesions, superior to other groups. These results suggest that the fiber-2 protein can be an ideal candidate for subunit vaccine against the disease.

Porcine deltacoronavirus induces TLR3, IL-12, IFN-α, IFN-ß and PKR mRNA expression in infected Peyer's patches in vivo.

Porcine deltacoronavirus (PDCoV) is a newly identified swine enteropathogenic coronavirus that causes watery diarrhea in piglets and results in significant economic losses to the pig industry. Currently there are no effective treatments or vaccines for PDCoV. In particular, the pathogenesis of PDCoV infection is still largely unknown. In this study, we reported that inoculating conventional weaned piglets with 1 × 109 TCID50 of the PDCoV CHN-GD-2016 strain by oral feeding could cause severe diarrhea. Virus RNA was detected in rectal swabs from 1 to 7 days post inoculation. In addition, microscopic lesions in small intestine were observed, and viral antigen also detected in the small intestines with PDCoV immunohistochemical staining. Importantly, PDCoV significantly induced mRNA expression of TLR3, IL-12, IFN-α, IFN-ß, and PKR, the genes involved in modulation of the host immune responses, in infected Peyer's patches at 3 d.p.i., indicating that Peyer's patches play an important role in PDCoV immune responses in vivo. Collectively, our findings suggest that the observed gene expression profile might help explain immunological and pathological changes associated with PDCoV infection.

Oral administration of coated PEDV-loaded microspheres elicited PEDV-specific immunity in weaned piglets.

Porcine epidemic diarrhea virus (PEDV) infects pigs of all ages by invading villous epithelial cells of the small intestine causing severe diarrhea with high mortality rate in suckling piglets. Mucosal immunity is believed to play an important role in PEDV control and mucosal delivery of vaccines induces mucosal immunity more efficiently than parenteral vaccination. In this study, coated PEDV-loaded microspheres with the size range of 700-900 µm in diameter were developed by centrifugal granulation-fluidized bed coating and demonstrated as an effective oral delivery system to protect PEDV antigens against the complex gastrointestinal environment by detecting the live virus particles in microspheres after the simulated gastric fluid treatment and the PEDV RNA in fecal swabs collected from all weaned piglets (100%) orally inoculated with coated PEDV-loaded microspheres. Weaned piglets orally immunized with coated PEDV-loaded microspheres developed higher levels of PEDV-specific antibodies (IgG and IgA) in their sera and saliva than those negative control groups (p < 0.001 or p < 0.01). Furthermore, neutralization assays demonstrated that serum antibodies in coated PEDV-loaded microspheres groups could significantly inhibit virus infection in Vero cells, compared to PEDV only group (p < 0.05). Overall, our results indicate that the coated PEDV-loaded microspheres might serve as an effective way to induce PEDV-specific mucosal immunity in pigs against PEDV.

A Highly Pathogenic Strain of Porcine Deltacoronavirus Caused Watery Diarrhea in Newborn Piglets.

Porcine deltacoronavirus (PDCoV) is a newly identified virus that causes watery diarrhea in newborn piglets and results in significant economic losses to the pig industry. Since first reported in Hong Kong in 2012, PDCoV has been subsequently detected in USA, South Korea, Thailand, and mainland China. Here we isolated a strain of PDCoV, named CHN-GD-2016, from the intestinal content of a diseased newborn piglet with severe diarrhea in a pig farm in Guangdong, China. PDCoV CHN-GD-2016 could be identified by immunofluorescence with PDCoV specific rabbit antisera, and typical crown-shaped particles with spiky surface projections of this PDCoV were observed with electron microscopy. Genomic analysis showed that the PDCoV CHN-GD-2016 was closely related to other Chinese PDCoV strains, with the highest sequence similarity with the strain CHN/Tianjin/2016. Importantly, inoculation of newborn piglets with 1 × 105 TCID50 of CHN-GD-2016 by oral feeding successfully reproduced clear clinical symptoms, including vomiting, dehydration, and severe diarrhea in piglets. In addition, the virus RNA in rectal swabs from 1 to 7 days post inoculation was detected, macroscopic and microscopic lesions in small intestine were observed, and viral antigen was also detected in the small intestines with immunohistochemical staining. Collectively, the data show in this study confirms that PDCoV is present in Guangdong, China and is highly pathogenic in newborn piglets.