Seroprevalences of Classical Swine Fever Virus and Porcine Reproductive and Respiratory Syndrome Virus in Pigs in Hunan Province, China

Background: Several infectious diseases including classical swine fever (CSF) and porcine reproductive and respiratory syndrome (PRRS) are responsible for major economic losses and represent a threat to the swine industry worldwide. Hunan is a province in south-central China, serological statistics will have certain effect on local breeding industry. The present study investigated the seroprevalences of classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV) in pigs from eight cities of Hunan province from 2017 to 2019. The samples were divided into two groups based on whether the hosts were immunized or not, and analysed by enzyme-linked immunosorbent assay (ELISA). Results: The seropositivity of CSFV and PRRSV between different parts of Hunan province in China was statistically signicant. In unvaccinated group, our ndings showed CSFV antibody in piglets' sera decreases gradually with increase of piglets' age whereas PRRSV-specic antibodies may appear in various growth stages. In immunized group, the average seropositive rate of CSFV antibody was 82.2%, while the rate of PRRSV antibody was relatively higher (84.4%). Conclusions: Results from this study suggested that a serological investigation on these two major infectious diseases (CSF and PRRS) among various growth stages of pigs will provide a basis for further development of more effective preventions and control measures.

statistically signi cant. In unvaccinated group, our ndings showed CSFV antibody in piglets' sera decreases gradually with increase of piglets' age whereas PRRSV-speci c antibodies may appear in various growth stages. In immunized group, the average seropositive rate of CSFV antibody was 82.2%, while the rate of PRRSV antibody was relatively higher (84.4%).
Conclusions: Results from this study suggested that a serological investigation on these two major infectious diseases (CSF and PRRS) among various growth stages of pigs will provide a basis for further development of more effective preventions and control measures.

Background
Classical swine fever (CSF) is listed as a major noti cation disease by the World Organization for Animal Health (OIE) and listed as a Class A animal epidemic disease in China [1]. Classical swine fever virus (CSFV) is a small RNA virus with envelope in the Flaviviridae family, and the genome is approximately 12.3 kb in length [2]. Importantly, CSF is a highly infectious disease that occurs in both piglets and adult pigs by contact, and causes persistent infections as CSFV can evade the host immune surveillance [3].
CSF has been endemic in China since the early 20th century, and the prevention and control of this disease mainly depends on prophylactic vaccination, such as the Chinese lapinized vaccine [4]. However, compared to the classic strain, some newly emerging CSFV isolates could also be epidemic in many immunized pig farms or sporadically isolated from immunized pig farms, and showed distinct virulence and clinical manifestations [5,6].
Porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped and single positive-stranded RNA virus, belongs to a member of the family Arteriviridae [7]. It has been determined the key etiologic agent responsible for porcine reproductive and respiratory syndrome (PRRS) since the early 1990s [8]. PRRSV has been epidemic more than 20 years in China, and administration of PRRSV vaccines has provided an e cient protection from the virus infection by reducing clinical signs, decreasing viremia and shortening duration of viral shedding [9]. To date, the existing commercial vaccines, mostly known as modi ed live attenuated viruses, provide the greatest levels of protection [10]. However, the attenuated vaccines fall short in a variety of areas [11]. Modi ed live attenuated viruses may cause chronic or persistent infections and have the potential to revert to virulence, bringing new challenges to PRRSV prevention and control in China In order to accelerate the eradication of CSFV and PRRSV, the serology of both virus infections in the context of the current immunization programs should be surveyed and vaccine administrations also need to be evaluated to e ciently manage these virus-associated diseases on farms.
In this study, CSFV and PRRSV antibodies were tested in serum samples from immunized and unvaccinated pigs in different parts of Hunan province of China from 2017 to 2019, to evaluate vaccine e cacy and serological prevalence of both viruses.

Results
Seroprevalence-speci c rates of unvaccinated pigs.
A total of 129 unvaccinated pigs from eight regions were sampled for testing CSFV antibody, and 77.5% (100/129) were positive for CSFV (Table 1). Furthermore, 0 (0/10) and 100% (4/4) in the regions of Chenzhou and Yueyang were the positive rates for CSFV as a minimum and maximum, respectively, and the seropositivity of CSFV between the sample locations (n = 8) was statistically signi cant by using chisquare test (χ 2 = 43.648, df = 7, p < 0.001). The results showed that no CSFV infection on fattening pigs (0/10), whereas all the positive CSFV samples were from the piglets (100/100). It is worth to note that CSFV antibody in piglets decreased gradually with increase of piglets' age ( Fig. 2A). For detecting PRRSV antibody, 636 serum samples from pigs without vaccination against PRRSV were detected by ELISA, and the average positive rate of PRRSV-speci c antibody was 48.1% (Table 1). PRRSV appeared in the six regions of Hunan province, and the highest positive rate was 94.3% in Yiyang, whereas the lowest was 37.4% in Changde ( Table 1). The seropositivity of PRRSV between the sample locations (n = 6) was also statistically signi cant by using chi-square test (χ 2 = 54.095, df = 5, p < 0.001). Moreover, the highest positive rate in different growth stages was 77.8% for boars, and the lowest was 18.9% for fattening pigs (Fig. 2B). Unlike CSFV, PRRSV antibody in piglets rst increased from one week old to two weeks old, and then decreased gradually with increase of piglets' age ( Fig. 2B).
Seroprevalence-speci c rates of immunized pigs.
Vaccination e cacies against CSFV has been studied in Hunan province, and the positive rates of CSFV antibody was 82.2% by detecting 1320 serum samples in Hunan province (Table 1). By analysis of regional differences, the vaccination e cacy in Yongzhou was best with the sera-positive rate is 96.9%, but the positive rates of four regions (Changsha, Shaoyang, Changde, and Chenzhou) were below the average (Table 1). Further statistical analysis, the seropositivity of CSFV between the sample locations (n = 8) was statistically signi cant by using chi-square test (χ 2 = 17.074, df = 7, p = 0.017). The vaccination e cacy was not uniform at different growth stages, the positive rate from high to low was boars (92.6%), sows (90%), piglets (75.9%), fattening pigs (69.3%), and nursery pigs (50.3%) ( Table 2). Vaccination e cacies against PRRSV were also studied in Hunan province, the positive rate of PRRSV antibody in sera was 84.4% by detecting 935 serum samples (Table 1). About the regional difference, the highest positive rate was 95.8% in Yiyang with the best vaccination e cacy, and the lowest was 74% in Hengyang ( Table 1). The seropositivity of PRRSV between the sample locations (n = 8) was also signi cantly different using chi-square test (χ 2 = 20.309, df = 7, p = 0.005). Among various growth stages, the positive rate of sows was highest (88.1%) implying that the vaccination e cacy reached a high level, the lowest rate was 28.6% in piglets, which indicated a poor vaccination e cacy (Table 3).

Discussion
The transportations of people and pigs are apparently different now from that in the past, and this has led to an increasing concern about viruses, such as CSFV or PRRSV, spreading to the pathogen-free areas, leading not only to animal infections, but also to economic loss in pig industry [12]. Hunan is the thirdlargest swine-raising province in China, and serological statistics of CSFV and PRRSV antibodies in Hunan province have certain effect on the whole breeding industry.
The prevalence of 77.5% is a serological indication that CSF may exist in this study area without of using CSFV vaccine. However, Chen at al. similarly reported that only 4 pigs were solely infected with CSFV among 159 diseased pigs in China from 2016 to 2018 [13]. Thus, our prevalence (100/129, 77.5%) tested by ELISA is signi cantly higher than the reported positive percentage of CSFV (4/159, 2.52%) detected by real-time PCR. It is possible that all the positive CSFV samples were from the unvaccinated piglets while their mothers were immunized against CSFV, therefore the serological level of CSFV antibody in the nonvaccinated pigs comes from the maternally derived antibodies (MDA), and it decreases with the age of piglets. It is worth noting that CSFV vaccination should be administered when residual MDA are minimal and before pigs become naturally infected, avoiding potential interference from MDA at the time of vaccination [14].
What distinguishes CSFV from PRRSV? The answer is that PRRSV antibody in the unvaccinated piglets (their mothers were immunized against PRRSV) increases rst and then reduces with the ages. Further, more unvaccinated pigs from different growth stages (nursery pigs, sows, and boars) were used to detect PRRSV antibody which can re ect the prevalence of PRRSV in corresponding region. The presence of the virus in each growth stage (except piglets) presented obvious different positive rates: 71.7% for nursery pigs, 18.9% for fattening pigs, 53.4% for sows, and 77.8% for boars. High positive of boars in this study, may explain that PRRSV was widespread in pig farms.
Vaccination e cacies against CSFV or PRRSV have also been studied in Hunan province. Only the serapositive rate of CSFV antibody in Yongzhou was above 90%, showing an ideal vaccination e cacy, but other areas in Hunan province need enhance the e cacy of vaccines. On the different growth stage of CSFV-immune pigs, sows and boars achieved a high level of vaccination e cacy, while others (piglets, fattening pigs, and nursery pigs) were not satisfactory yet. It is considered to be the ideal immune effect when the positive rate of PRRSV antibody in immune pig farms is above 70%. Our study showed that most PRRSV-immune pigs from areas in Hunan province under investigation got the antibody (84.4%). However, the positive rate of piglets was 28.6%, less than 70%. This low percentage imply that the e cacy of vaccination in piglets could be affected by the presence of MDA.

Conclusion
This study has determined the serological statistics of CSFV and PRRSV antibodies from immunized and unvaccinated pigs in Hunan province of China from 2017 to 2019. Continuous monitoring of these viral antibodies among pigs in China is hereby recommended as well as improved immune procedure with intensi ed awareness campaign.

Study locations and design
Hunan province is located in the south-central region of China, a farming zone known for high pig production. The sampling area is consisted of 8 herds and the herds were located in the north eastern (Changsha and Yueyang), northern (Changde and Yiyang), southern eastern (Chenzhou), and southern (Hengyang, Shaoyang, Yongzhou) parts of Hunan province (Fig. 1). The number of pigs to be sampled per herd was based on the availability of pigs and willingness of the farmers to allow for their pigs to be sampled. Furthermore, Face-to-face interviews were carried out with farmers (> 18 years old) who were mostly involved in swine breeding and management, and lled the registration form covered information of samples, such as the growth stage, age, and the immunity condition of pigs (Additional le 1).
For detecting CSFV antibodies, a total of 1449 blood samples from pigs corresponding to nonvaccination (n = 129) and vaccination (n = 1320) of CSFV, different size herds sampled during years 2017-2019 were used (Fig. 1). Also, totally 1571 swine blood samples relevant to non-vaccination or vaccination of PRRSV, from the same herds were collected for testing PRRSV antibodies (Fig. 1).
Three millilitres of blood sample was collected from each pig by using a 5 ml syringe and corresponding needle. The blood was then gently transferred into a 5 ml sterile tube, labeled appropriately and standing overnight. Then, the blood was centrifuged at 3000 × g for 10 min to separate sera from clotted blood. The sera were then decanted into an appropriately labeled 2 ml plastic serum storage tube, and stored at -20 ℃ until used.

Laboratory analysis
Two commercial kits: IDEXX PRRS X3 Ab Test (indirect for PRRS) and IDEXX CSFV Ab Test (E2 competitive for CSF) from IDEXX Laboratories, Inc. (Westbrook, ME, USA) were used to test for the presences of PRRSV and CSFV antibodies in sera, respectively. The manufacturer's instructions were strictly adhered to and results were read and interpreted using an ELISA reader at a wavelength