Epidemiological analysis of Porcine Viral Diarrhea Pathogens in Local Area

Porcine viral diarrhea can cause great damage to the pig industry and high mortality to piglets. Furthermore, multiple pathogen infections and synergistic infections commonly existed in clinic. This has resulted in great difficulties in determining the main pathogenic factors, which would delay the prevention and control of diseases. A total of 518 porcine stool specimens were collected from 9 pig herds in Shanghai, China from 2015 to 2017 and used for pathogen detection. 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.


Introduction
In recent years, viral diarrhea in pig herds has led to serious problems with clinical symptoms of diarrhea, vomiting, and dehydration, thereby affecting pig growth and leading to huge economic loss [1]. This disease increases the infection rate to piglets and mortality, which could reach up to 100% [2]. Furthermore, multiplex infection and synergistic infection, which are commonly observed in clinic, pose a new challenge to disease diagnosis and control. The viruses that cause porcine diarrhea disease are diverse, including porcine epidemic diarrhea virus (PEDV) [3][4][5], porcine deltacorona virus (PDCoV), porcine transmissible gastroenteritis virus (TGEV) [6], porcine teschvirus (PTV), clinical symptoms, including severe diarrhea and dehydration, and in some cases, multiplex infections of these pathogens are common. However, it remains difficult to distinguish these in clinic [4,[16][17][18].
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 need to be investigated, in order to identify the dominant viruses. Thus, the surveillance of porcine is warranted to better understand the evolution in the field. 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 specificity 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, specificity 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 and the genetic diversity of the virus 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 and extent of genetic diversity in these circulating strains, in order to develop vaccination programs and establish a surveillance system.
The present study focuses on the epidemiology of porcine viral diarrhea, using multiplex RT-PCR method to investigate the prevalence of multiple diarrhea viruses and identify the dominant ones. Furthermore, the multiplex infection situation was analyzed, which will pave the way to improving strategies to prevent and control 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

Establishment of Luminex xTAG multiplex detection method
Based on a single detection system, multiple detection systems were multiplex to establish a Luminex xTAG multiplex detection method for the simultaneous detection of 11 diarrheal viruses. The optimal of hybridization system and reaction conditions were as follows: 20µL microsphere working solution, 5µL PCR amplification product, and 75µL SAPE report buffer; the results of optimal hybridization temperature was 42 °C, and the best hybridization time was 30 min. The Luminex xTAG assay method specificity was test, which showed that each primer pair had good specificity and there was no cross-reaction between the primer pairs. The sensitivity test of the Luminex xTAG detection method showed that the minimum detection lines of this method were: PTV of 3.12 × 10 3 copies/ µL, PKOV of 2.92 × 10 3 copies/µL, PDCoV of 2.79 × 10 3 copies/µL, PSV of 3.37 × 10 3 copies/ µL, PSaV of 2.7 × 10 3 copies/µL, PAstV of 3.02 × 10 3 copies/µL, PToV of 2.65 × 10 3 copies/ µL, PoRV of 2.57 × 10 3 copies/µL, PEDV of 1.74 × 10 3 copies/µL, BVDV of 2.41 × 10 3 copies/ µL, TGEV of 2.75 × 10 3 copies/µL. The minimum detection rate was at least 10 times higher than the traditional multiplex PCR method. Specific tests showed that each primer pair had good specificity and there was no cross-reaction between the primer pairs. Viral pathogens infected in diarrhea stools were diversified A total of 518 porcine stool specimens from five districts in Shanghai were detected using multiplex RT-PCR method. Results revealed that all 11 viral diarrhea pathogens could be   (Table 2). High positive rate (11.58%) of tripleinfection was also observed, while the positive rate of quadruple-and quintuple-infection was 4.63% and 0.77%, respectively. Most surprisingly was that six (0.58%) or seven viral diarrhea pathogens (0.58%) were simultaneously detected from one diarrhea stool, indicating a complex diarrhea pathogen infection pattern and pathogenesis in clinic. As we can see (Fig. 2), dual-infection with PKOV and PASTV had the highest positive rate

Viral infection spectrum in one 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 (Fig. 3). In 2015, PEDV had the highest positive rate of 45.83%, while the second highest was PKoV (33.33%). However, in 2016, PKoV became the most popular pathogen with a very high positive rate of 68.67%, and this was closely followed by PAstV (32%) and PSV (24%). The PEDV positive rate significantly decreased, which was merely 12%. In 2017, PAstV had the highest detection rate of pathogens (52.50%), followed by PKoV (40%) and PEDV (17.5%).
It indicates that diarrhea disease is no longer mainly caused by three major pathogens (PEDV, TGEV and PoRV), which hints that other pathogens may play synergistic roles in the pathogenesis of diarrhea disease.
According to the present surveillance results, the farm strengthened its prevention and control of infectious diseases. As observed in the recent years, there was a decrease in enteric pathogens, proving the great importance of epidemiological surveillance and the guidance effect to clinical production.

Different farms show variable infection spectrum
The composition of enteric pathogens among different farms was analyzed (Fig. 4& Fig. 5).
In farm A, eleven kinds of pathogens were detected including multiple co-infections (dual-,  . 6). Therefore, the pathogen content in each sample could be estimated intuitively.
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. The quantitative analysis speculated that the five viruses (PoSaV, PoRV, PAstV, PToV, and PEDV) were the predominant viruses in the multiplex infection sample. They not only could be detected in multiple co-infected samples, but also were relatively high in co-infected samples. 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.

Discussion
Porcine viral diarrhea disease still seriously endangers the development of the pig industry, and leads to significant 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. 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][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 were applied for clinical detection [21,39]. However, complicated infections require a more accurate detection method. Therefore, we further developed Luminix xTAG high- 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]. 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 [12]. Chang Tiecheng et al. [13] 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 [22]and China [23], it has been confirmed that PKoV was widely present in several countries, and plays an important role in diarrhea outbreak in pigs [24][25][26][27][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 [25]. 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 [11]. A survey of PASTV infection in pig farms in Japan revealed that the positive rate of PAstV was 82.9% [29]. Similarly, the high prevalence

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

Competing interests
The authors declare no competing financial interests.      The Heatmap for quantitative results of some samples. 173 outbreaks of diarrhea in swine from 2017 were tested.by the Luminex xTAG multiplex detection method.
Only part data were present in the figure.