The Multiple Infections of Porcine Diarrhea Viruses in Local Area Based on A Luminex xTAG Multiplex Detection Method

A Luminex xTAG multiplex detection method was developed for the detection of 11 viral diarrhea pathogens, which allows for the simultaneous qualitative and quantitative detection of viral diarrhea pathogens in clinical samples. A total of 518 porcine stool specimens were collected from 9 pig herds in Shanghai, China from 2015 to 2017, and the pathogen spectrums and co-infections were analyzed. PEDV was still the key pathogen and multiple pathogens synergistically complicated the infection status in southern China, suggesting that controlling porcine diarrhea might be more complex than previously thought. A better understanding of viruses that cause diarrhea in piglets will aid in better preventing and controlling epidemics of viral porcine diarrhea.

Although regular vaccine immunization has been strictly conducted, the high morbidity of diarrhea remains as a serious problem, which needs to be solved in time. Therefore, the epidemiology of diarrhea viruses needs to be investigated, in order to identify the dominant viruses. Thus, the surveillance of porcine is warranted to better understand the evolution in the eld. Laboratory detection methods include virus isolation, enzyme linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). However, virus isolation needs long period and complicated operations, and ELISA is mainly used for epidemiological survey. Therefore, it is important and urgent to establish a detection method with high speci city and high sensitivity, which can meet the needs of multiple pathogen detection at the same time. As the multiplex reverse transcription-polymerase chain reaction (RT-PCR) method has high sensitivity, speci city and could simultaneously detect a variety of pathogen, it is more suitable for rapid diagnosis of multiplex infection. Therefore, in this study, a multiple detection method for viral diarrhea was developed.
The precise data about the prevalence of multiple infections in porcine and wild boars have only been reported in a limited number of countries [10,11]. Moreover, the prevalence and multiplex infections of porcine diarrhea in Shanghai have never been studied. Therefore, the epidemiology is needed to determine the prevalence in these circulating strains, in order to develop vaccination programs and establish a surveillance system.
Co-infection with various viruses makes preventing and curing diarrhea in pigs more complex. Therefore, we performed an epidemiological study to identify the frequency of these viruses in cases of porcine viral diarrhea in Southern China. Furthermore, the multiplex infection situation was analyzed, which will pave the way to improving the strategies in preventing and controlling virus infection in swine farms.

Specimens
A total of 518 porcine stool specimens were collected from 9 pig herds in Shanghai, China from 2015 to 2017. Pigs of all ages were sampled and 1 to 3-week-old piglets were particularly collected. Antibiotic treatment was invalid for all sampled pigs. Each sample was suspended in phosphate-buffered saline (PBS) containing 1,000 U/ml penicillin and 1,000 U/ml streptomycin and centrifuged at 12,000 r.p.m. for 10 minutes. A portion of the suspension was used for RNA extraction, while the remaining supernatants were stored at -70 °C.

Establishment of Luminex xTAG multiplex detection method
According to the conserved sequences in GenBank, DNAStar and Oligo7 software were used to design the PCR primer pairs of 11 pathogens (Table 1). The M gene of PToV, M gene of PDCoV, RDRP gene of PAstV, 3D gene of PKoV, RDRP gene of PSV, 3D gene of PTV, RDRP gene of PsaV, 5' URT gene of BVDV, M gene of PEDV, N gene of TGEV and VP6 gene of PoRV were as target genes. Spacer C12 was added between the 5' end of all upstream primers and the 3' end of the anti-TAG sequence, and the 5' end of all downstream primers was biotinylated (Biotin-) tag; selecting TAG microspheres complementary to anti-TAG sequence; hybridizing biotinylated tagged   ampli cation product with TAG microspheres, and obtaining the hybridization product for liquid-phase chip detection, and detecting the pathogen according to the MFI value. The speci city and sensitivity of Luminex xTAG assay method were analyzed. And the hybridization system and reaction conditions were also optimized. instructions and sampling materials were provided to the farmer. A dry cotton rectal swab was collected from each individual pig with diarrhea and was placed immediately in 2 ml of viral transport medium (phosphate buffered saline containing 1,000 U/ml penicillin and 1,000 U/ml streptomycin) and stored at -20 °C. The farmer was asked to mark the tube of each sample for diarrheic signs. Every week, samples were collected from the farm and transported to the laboratory for pathogen detection.

Establishment of Luminex xTAG multiplex detection method
Based on a single detection system, a Luminex xTAG multiplex detection method for the simultaneous detection of 11 diarrheal viruses was established. The optimal hybridization system and reaction condition were as follows: 20μL microsphere working solution, 5μL PCR ampli cation product, and 75μL SAPE report buffer; the results of optimal hybridization temperature was 42°C, and the best hybridization time was 30 min.
The pathogen composition in each sample could be estimated intuitively. The concentration of different pathogens in each sample was presented as different colors in a heat-map ( Figure 2). Therefore, Different colors represent different concentrations of pathogens. The closer the color was to black, the lower concentration was present; and the closer the color was to red, the higher concentration was present in the sample. In addition the predominant viruses in the multiplex infection samples could be speculated according to quantitative analysis. Therefore, in virtue of the quantitative analysis, the detection results will be more clearly visible and targeted prevention or therapy may be carried out in the pigsty, paving the way to instructing the clinical production.  Figure 3). Therefore, complicated pathogen composition existed in clinic, which emphasized the importance of monitoring the uctuant infection spectrum to guide the clinical production.  Among the samples positive for the 11 enteric viruses, PKoV exhibited high co-infection rates, ranging from 2.20% to 32.97% ( Figure 4B)

Viral infection spectrum in a speci c farm
In tracking the annual viral diarrhea tests in one farm from 2015 to 2017, it was observed that the prevalence of the viral diarrhea pathogens also changed over time (Figure 7). In 2015, PEDV had the highest positive rate of 45.83%, while the second highest was PKoV (33.33%). Accordingly, high diarrhea rate (52%) and mortality (7.1%) were observed. In 2016, PKoV became the most popular pathogen with a particularly high positive rate of 68.67%, and this was closely followed by PAstV (32%) and PSV (24%). The PEDV positive rate was merely 12%, accordingly, diarrhea rate decreased to 38% and mortality to 5.5%. In 2017, PAstV had the highest detection rate of pathogens (52.50%), followed by PKoV (40%) and PEDV (17.5%), while the diarrhea rate and mortality became 42% and 6.8%, respectively. Taken together, these results do indicate that the uctuations of diarrhea rate and mortality were accordance with the tendency of PEDV positive rate, while high rate of PKoV or PAstV did not necessarily correspond to high diarrhea rate and mortality.  16.07% (9/56) and 5.36% (3/56), respectively. PKoV was always one of the co-infecting viruses; however, the viral load was not extremely high according to the quantitative analysis.  The morbidity and mortality rate of the farm during sampling was 57% and 5.23%; even vaccinated suckling piglets were not spared. The infected piglets predominantly showed yellow watery stools, weight loss, and death from dehydration. Interestingly, single PEDV infection or co-infection of PEDV with other diarrhea viruses was identi ed in all the dead piglets. One piglet infected with 6 kinds of diarrhea viruses including PEDV, nally died. While another piglet infected with six other viruses was still alive with great weight loss. The same situation also applied to other multiple infections. All in all, PEDV is still the key pathogen which was closely related to death of diarrhea piglets. Other pathogens might play synergistic roles in the pathogenesis of diarrhea disease.

Longitudinal investigation showed dominant infection agents
According to the present surveillance results, relative proposals and solutions were put forward and carried out to control the emerging diarrhea and to prevent later potential occurrence.

Discussion
Porcine viral diarrhea disease seriously endangers the development of the pig industry, and leads to signi cant economic losses for pig farmers worldwide [7]. 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][9][10][11][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][14][15][16][17][18][19][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 ux, 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 nally, 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 [22]. 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 [23]. A recent study showed that 27% of samples had PEDV infection alone, whereas the remaining 73% of samples exhibited two to nine pathogens [24]. 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 [10]. PKoV can infect pigs of all ages and varieties with prevalence of ranges from 19.3-99.0% in different countries [16]. Since the rst report of PKoV in Hungary [25]and China [26], it has been con rmed that PKoV was widely present in several countries, and plays an important role in diarrhea outbreak in pigs [27][28][29][30][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 [28]. 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) [32], 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 coinfection 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 [32]. 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 in uenced 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 speci c 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.

Conclusions
In summary, here we provided a Luminex xTAG multiple detection method for viral diarrhea pathogen infection in clinical, which was more sensitive and speci c than general multiplex PCR method. Furthermore, the surveillance con rmed high infection rate of PKoV, but PEDV was still the key pathogen and multiple pathogens synergistically complicated the infection status in southern China, suggesting that controlling porcine diarrhea might be more complex than previously thought. A better understanding of viruses that cause diarrhea in piglets will aid in better preventing and controlling epidemics of viral porcine diarrhea. Furthermore, attention should be given to the relevant relevant vaccines, and the expansion of the vaccine reserve, in order to prepare for the outbreak of a new round of porcine diarrhea disease in advance.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analyzed during this study are included in this published article [and its supplementary information les.

Competing interests
The authors declare no competing nancial interests.  The speci city and sensitivity detection of the Luminex test. A. The speci city detection. B. The sensitivity detection.

Figure 2
The Heatmap for quantitative results of some samples. The porcine stool specimens were tested.by the Luminex xTAG multiplex detection method. Only part data were present in the gure. The co-infections of different farms. Different co-infections were analyzed in different farms.