Goose parvovirus (GPV) infection can cause a fatal infectious waterfowl disease affecting goslings (Anser domestica) and Muscovy ducks (Cairina moschata), which is usually known as the classical Derzsy’s disease from the early 1960 s to the 21th century (Isidan et al., 2021, Kisary et al., 1978, Woźniakowski et al., 2012). Since the outbreak of Derzsy’s disease in a vaccinated Muscovy duck flock in Fujian Province in 1997, several evolutions and mutations of classical GPV (C-GPV) have been observed, resulting in emergence of Muscovy duck-origin GPV (MDGPV) PT-like recombinant variants (Wang et al., 2013, Shen et al., 2015). Additionally, especially since 2015, novel goose parvovirus, a distinct lineage of the classical Derzsy's disease viruses, which was reported to cause short beak and dwarfism syndrome (SBDS) in Mule, Pekin, Sheldrake, and Muscovy ducklings in China (Chen et al., 2016, Wang et al., 2021, Zhu et al., 2022). These diversified strains of GPVs can be transmitted both vertically and horizontally, which belong to the same species Anseriform dependoparvovirus 1, within the genus Dependoparvovirus of the family Parvoviridae (Kapgate et al., 2018). In contrast to the high morbidity and mortality of classical Derzsy's disease, the mortality rate of SBDS ranged from 2% to 6% in the affected duck flocks, but the losses were especially linked to high morbidity which was on average 20% or higher in some regions (Chen et al., 2021, Matczuk et al., 2020). Noteworthily, Derzsy's disease-endemic areas overlap with SBDS related GPVs (SBDS-GPVs)-affected areas and both the classical and novel GPVs are transmitted by the susceptible host, Muscovy duck – thereby increasing opportunities of co-infection by both viruses (Chen et al., 2016, Liu et al., 2014, Wang et al., 2013, Wang et al., 2021).

Waterfowl-originated reoviruses (WRVs) are members of the genus Orthoreovirus of the Reoviridae family, which are non-enveloped icosahedral double-capsid double-stranded RNA viruses (Varga-Kugler et al., 2022). WRV infections cause considerable economic losses to the duck and goose industry in Europe and Asia (Farkas et al., 2014). Reoviruses isolated from Muscovy ducks were first reported in France in 1972 and designated as classical Muscovy duck reovirus (MDRV) (Malkinson et al., 1981). MDRV infections have since emerged in China from 1997, and the most frequent clinical characteristics associated with leg weakness and the main macrodissected lesions present as the liver and spleen swollen and covered small white necrotic foci (Chen et al., 2018). Since 2005, a number of virulent field reovirus strains have been isolated from dead duckings and goslings. These viruses are economically significant pathogens that cause a wide variety of diseases in the different species of ducks and geese, including enteric and respiratory problems, stunting syndrome, necrotic hepatitis and malabsorption syndrome (Wang et al., 2015). These strains caused serious hemorrhage and necrosis in the liver and spleen of the sick birds, and were traditionally designated as novel duck reoviruses (NDRVs) to distinguish them from the previously reported MDRVs (Peng et al., 2023). According to the previously reported genomic characterizations and phylogenetic analyses, the MDRVs and NDRVs undergo mutations, segment recombination, and genome reassortment inside the naturally susceptible host species at an incessant rate that allows them to elude the immune response (Wang et al., 2020, Zhang et al., 2007).

Goose astroviruses (GAstVs) are small, non-enveloped, single-stranded positive RNA viruses that belong to the genus Avastrovirus and the family Astroviridae (Zhu et al., 2022), and can infect different domesticated waterfowl species causing a range of enteric disease signs such as diarrhea, enteritis, interstitial nephritis and have been linked with runting-stunting syndrome and visceral gout (Xu et al., 2023). Recent studies have reported that the infection and mortality rates of GAstV in goslings and ducklings can reach 80% and 50%, respectively (Zhu et al., 2022). GAstVs have been reported to inhibit the host oxidation-reduction and antiviral responses, which provides vital conditions for other pathogens to infect (Liu et al., 2023).

GPV, WRV, and GAstV infections have been observed in commercial Muscovy ducks with hepatitis, enteritis or/and kidney disease, especially if subclinical infections occur, making it difficult to distinguish among GPV, WRV, and GAstV without further testing (Chen et al., 2021, Woźniakowski et al., 2014, Xiao et al., 2017). While a single assay only can detected an individual virus target in each reaction and is time-consuming, laborious, and costly, especially when large numbers of samples need to be tested for mixed infections (Hue-Roye et al., 2008). Therefore, it is necessary to simultaneously and differentially detect these pathogens by laboratory techniques. These limitations can be overcome by employing a multiplex PCR, which can amplify simultaneously more than one target conserved sequences of different viruses by several primer pairs in one signal reaction, offering a significant time and cost-saving advantage (Markoulatos et al., 2002). However, there have been no reports of the application of routine PCR for the simultaneous detection and differentiation of GPV, WRV, and GAstV. In the present study, a sensitive and specific multiplex PCR method for the detection of GPV, WRV, and GAstV has been developed. The multiplex PCR DNA products consisted of three specific fragments of 493 bp (GPV), 300 bp (WRV), and 156 bp (GAstV) that can be visualized by gel electrophoresis. This novel method allows for these common enteric viruses in the routine surveillance samples from clinically affected waterfowl species to be detected and differentiated in one reaction.