Porcine viral diarrhea disease 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. Beside these three traditional 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 was applied for clinical detection [21, 39]. However, complicated multiplex infections require a more accurate detection method. Therefore, we further developed Luminex 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 Luminex 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 be helpful to 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.
PEDV has become the most important intestinal pathogen in swine in China . Many studies of the mechanism of PEDV infection and effective vaccines have been published. However, the variations of the virus and its co-infections with other enteric viruses, had contributed to the poor control of PEDV infection. In order to better understand the prevalence of the co-infection in southern China, an epidemiological investigation of porcine diarrhea viruses was carried out in this study. It was reported that the co-infection of PEDV and PBoV was more prevalent in diarrhea samples than non-diarrhea samples . A recent study showed that 27% of samples had PEDV infection alone, whereas the remaining 73% of samples exhibited two to nine pathogens . According to this survey, single infection with PEDV occurred in only 6.56% of samples (34 out of 518), while most of the samples involved co-infections.
Except for the co-infections with PEDV, various other types of co-infections existed in the study. 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. Among the 11 enteric viruses, 50% (91/182) of samples had at least two pathogens, and 32.97% (60/182) of samples had three different pathogens, while the positive rate of quadruple- and quintuple-infection was 13.19% and 2.20%, respectively. Notably, co-infection with six viral diarrhea pathogens was identified in three samples (1.65%). 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.
PKoV can infect pigs of all ages and varieties with prevalence of ranges from 19.3–99.0% in different countries . 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 [27–31]. 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, 28]. PKoV has also been linked to porcine diarrhea although its pathogenesis remains unclear (Barry et al., 2011; Chen et al., 2013a). It was reported that piglets with diarrhea shed more Kobuvirus than healthy individuals during the late-nursing stage (6–21 days old) , which accounted for the high positive rate of PKoV. Based on the rates of infection documented in this study, PKoV exhibited the highest infection rate (40.35%), and high co-infection rates ranging from 2.20–32.97%, suggesting a high prevalence of co-infections in the sampled regions. Since PKoV has the highest infection rate and co-infection rate, further investigations should be conducted to research its characteristics and pathogenic mechanism. Interestingly, co-infections with PEDV and PKoV accounted for 31.87% (29/91) of the PEDV positive samples, which was similar with the frequency of infection with PEDV alone (37.36%, 34/91). These data suggested that PKoV had a potential role in PEDV-induced diarrhea symptoms, which was consistent with the previous study . The high prevalence of co-infection, particularly PKoV and PEDV, is a cause for concern and should be seriously considered.
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 needs further research. The specific mechanisms of the diarrhea pathogens for porcine diarrhea diseases remain unknown. However, the potential hazards cannot be ignored, and these should be detected and prevented at an early stage.