Porcine viral diarrhea disease still seriously endangers the development of the pig industry, and leads to significant economic losses for pig farmers worldwide . Clinically, the complexity of the disease has increased. In some cases, multiplex infections with two or more viruses are common, which seriously interfere with the clinical diagnosis [8–12]. It has been speculated that the incidence of diarrhea would decline due to the vaccine prevention of PEDV, TGEV and PoRV triplets. However, diarrhea continued to threaten pig farms. Expect for these three major porcine viral diarrhea pathogens (PEDV, TGEV and PoRV), other viral diarrhea pathogens have also been reported in recent years [13–20]. In particular, the situation of multiplex infections has become more serious, resulting in increased pressure in the prevention and control of porcine diarrhea. Although the correlation between emerging viruses and diarrhea has not been clearly discussed, these co-infections have indeed enhanced the severity of diarrhea in the present study. Therefore, in order to accurately differentiate the infections in clinical specimens and prevent the transboundary spread of porcine viral diarrhea disease, it is necessary to conduct pathogen monitoring in clinical production.
Currently, PCR-based methods have been proven to be convenient and highly sensitive for detecting porcine diarrhea-associated viruses [2, 21]. The multiple PCR method for testing 4 or 7 kinds of diarrhea pathogens was established in the laboratory of the investigators, and were applied for clinical detection [21, 39]. However, complicated infections require a more accurate detection method. Therefore, we further developed Luminix xTAG high-throughput detection method for viral diarrhea pathogens in pigs, which has the advantages of high flux, wide range of detection, and small sample size. Furthermore, this is suitable for the large-scale screening of clinical samples, and is especially suitable for the multiplex infection detection of samples. Using Luminix xTAG technology, the standard curves for the above 11 diarrheal pathogens were established, and the content of each pathogen was calculated by measuring the MFI value of each sample. By analogy, the viral load can be determined for each sample, and finally, the pathogen with the highest risk of infection in each pig farm was analyzed, which would help guide the formulation of immunity and control measures in pig farms. In addition, this can also intuitively identify the maximum level of pathogens in each sample based on the cluster analysis software.
It was considered that animals co-infected with more than one enteric virus experienced increased intestinal epithelium damage and/or viral replication, which results in more severe diarrhea . Forty samples of diarrhea in piglets in Sichuan Province were tested and five samples (12.5%) of multiplex infections of PKoV, PAstV and PToV were identified . Chang Tiecheng et al.  tested 165 samples obtained from 42 pig farms, and reported that 2 of 42 pig farms were infected with PEDV and TGEV, accounting for 4.76%. Furthermore, seven pig farms were infected with PEDV and PoRV, accounting for 16.67%, and two pig farms were infected with three viruses, accounting for 4.76%. This was consistent with the present results, in which there were serious multiplex infections in these pathogens, and seven pathogens were even detected in a sample. However, no new vaccines for diarrhea pathogens had been developed and applied to pig farms. Furthermore, there have been instances of co-infections in sows, even though these are usually asymptomatic. This may explain the persistence of viruses within the herd, and facilitation of vertical transmission.
Since PKoV has the highest infection rate and co-infection rate, further investigations should be conducted to research its characteristics and pathogenic mechanism. Since the first report of PKoV in Hungary and China , it has been confirmed that PKoV was widely present in several countries, and plays an important role in diarrhea outbreak in pigs [24–28]. The statistical analysis of the PKoV positive rate between diarrheic and healthy pigs, as well as a survey for other enteric pathogens in diarrheic pigs, suggested that PKoV may play a role as a causative agent of gastroenteritis in pigs. Recent studies have revealed the genetic diversity and possible pathogenic role of PKoV in conjunction with other pathogens in piglets [13, 25].
PAstV is widely distributed worldwide, and is highly prevalent among piglets with or without diarrhea. PAstV was first identified by electron microscopy from the feces of piglets with diarrhea in 1980 . A survey of PASTV infection in pig farms in Japan revealed that the positive rate of PAstV was 82.9% . Similarly, the high prevalence (79.6%) of the astroviruses in pigs between 2005 and 2007 was reported in Canada . In South Korea, 25/129 (19.4%) domestic pigs and 1/146 (0.7%) wild boar fecal samples were tested positive for PAstV . Multiple PAstV types were identified in many cities in China [32–37]. The co-infection of different genotypes in the same pig was commonly observed, while in an individual pig a high genetic diversity was observed for viruses that belong to the same PAstV genotype . PAstV exists in at least five distinct lineages (PAstV1-PAstV5) within the genus Mamastrovirus. The phylogenetic analysis revealed that PAstV1, PAstV2 and PAstV3 were more closely correlated to AstVs from humans and other animals, when compared to each other, indicating the past cross-species transmission and zoonotic potential of these PAstV types [30, 38].
The pathogen composition of different farms varied. Therefore, clinical control should be based on pathogen monitoring. Although infection was frequent in the winter season on farms in the temperate climate, diarrhea infection occurred throughout the year in porcine farms. Furthermore, the pathogen composition was more complex, and multiple infections more frequently emerged. Therefore, seasonal prevention measures are more important to control the diarrhea. The etiology of porcine diarrhea is complex. This might be influenced by bacteria, in addition to viral factors, and the interaction between bacteria and viruses might also contribute to the complexity of the disease. A single pathogen might not be the main cause of diarrhea, however, the unique relationship among different pathogens need further research. The specific mechanisms of the diarrhea pathogens for porcine diarrhea diseases remain unknown. However, the potential hazards can not be ignored, and these should be detected and prevented at an early stage.