Molecular Detection and Genotyping of Toxoplasma gondii in Pigs for Human Consumption in Hunan Province, China.

Toxoplasma gondii is one of the most important sources of foodborne diseases. In this study, the molecular prevalence and genotypes of T. gondii were investigated in pigs in Hunan province, China. A total of 339 brain tissue samples of pigs were collected from April 2015 to December 2017 in Hunan province and were used to detect the T. gondii B1 gene. Of these, 34 (10%; 95% confidence interval: 8.7-12.6) samples were tested positive for the T. gondii B1 gene. Positive samples were genotyped at 10 genetic markers (SAG1, SAG2 [5' + 3' SAG2, alter. SAG2], SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) using polymerase chain reaction-restriction fragment length polymorphism technology. Moreover, one sample was identified as genotype ToxoDB#10 (Type I), and another sample was suspected to be unusual genotype ToxoDB#61 that has never been reported in China. This study showed that T. gondii is prevalent in pigs in Hunan province, posing a food safety threat to the public health in the investigated areas. Our result has implications for better understanding the genetic diversity of T. gondii infections in animals in China.

Identification and characterization of multiple porcine astrovirus genotypes in Hunan province, China.

Astroviruses (AstVs) can infect a variety of hosts, including mammalian and avian species, and are commonly associated with enteric infections. Recently, mammalian AstVs have been linked to extra-intestinal manifestations, including neurologic disorders in humans, cattle and minks, demonstrating zoonotic potential. So far, five porcine AstV (PAstV) genotypes have been identified, with PAstV1, PAstV2, PAstV3 and PAstV5 implicated in cross-species transmission. Our knowledge about PAstV epidemiology in China is still limited. In this study, two duplex differential RT-PCR assays were developed to investigate the distribution and prevalence of PAstV1, PAstV2, PAstV4 and PAstV5. Two hundred eighteen samples were collected from 33 farms and pigs with known diarrhea status in nine regions of Hunan province in China. Specifically, 126 small intestines, 51 fecal swabs, 20 lungs, 19 spleens and two kidneys were obtained. PAstVs were detected in all nine regions and in 81.8% (27/33) of the pig farms investigated. The overall prevalence of PAstV was 46.3% (101/218), with PAstV5 as the predominant type, with a positive rate of 24.8% (54/218). The prevalence of PAstV4, PAstV1 and PAstV2 was 16.1% (35/218), 14.7% (32/218) and 10.1% (22/218), respectively. Besides being present in intestines and fecal swabs, PAstV RNA was also detected in lungs, spleens and kidneys. Sequencing revealed a high level of genetic divergence within each genotype, and a higher positive rate of PAstV5 was associated with pigs with diarrhea compared to pigs without diarrhea. This study revealed for the first time that PAstV4 is circulating in China, and that PAstV5 is the dominant genotype in pig herds in Hunan province in China.

Seroprevalence of Toxoplasma gondii infection in sows in Hunan province, China.

Toxoplasma gondii infections are prevalent in animals and humans worldwide. Although the prevalence of T. gondii has been reported in many animals in China, little is known of T. gondii infection in sows. Antibodies to T. gondii in sows in Hunan province, subtropical China, were examined using indirect hemagglutination test (IHAT). Overall, 31.3% (373/1191) of the examined sows were seropositive for T. gondii. Among 11 representative regions of Hunan province, the seroprevalence ranged from 14.8% to 45.1%. In addition, the T. gondii seroprevalence was higher in summer (37.4%) and autumn (34.9%) than in spring (24.6%) and winter (23.9%). Regarding different antibody titers, the seroprevalence ranged from 1.8% (titer ≥ 1: 1024) to 17.4% (titer = 1:64). The findings of the present investigation revealed the high seroprevalence of T. gondii in sows in Hunan province, China, which poses a potential risk for T. gondii infection in humans and animals in this province. Therefore, effective measures should be taken to prevent and control toxoplasmosis of pigs in this province. This is the first report of the comprehensive survey of T. gondii seroprevalence in sows in Hunan Province, subtropical China.

High seroprevalence of Chlamydia infection in sows in Hunan province, subtropical China.

Chlamydia spp. are Gram-negative obligate intracellular bacteria, which are responsible for significant public health problems in humans and have major economic impact on animals. In the present study, the seroprevalence of Chlamydia infection in sows in Hunan province, subtropical China, was examined using indirect hemagglutination assay (IHA). Antibodies to Chlamydia were detected in 747 of 1,191 (62.7%, 95% CI 60-65.5) serum samples (IHA titer ≥ 1:16). The Chlamydia seroprevalence ranged from 35% (95% CI 25.7-44.4) to 77.1% (95% CI 69.1-85.2) among different regions in Hunan province, and the differences were statistically significant (P < 0.01). In addition, the seroprevalence of Chlamydia infection in sows was higher in summer (75.7%, 95% CI 71.3-80) and spring (63.2%, 95% CI 57.5-68.8) than in autumn (56.9%, 95% CI 51.5-62.3) and winter (48.6%, 95% CI 42-55.3), and the differences were statistically significant (P < 0.01). The results of the present investigation indicated the high seroprevalence of Chlamydia infection in sows in Hunan province, subtropical China, which poses a potential risk for human infection with Chlamydia in this province. This is the first report of Chlamydia seroprevalence in sows over the last two decades in Hunan province, subtropical China.

Seroprevalence of porcine cytomegalovirus and sapovirus infection in pigs in Hunan province, China.

The seroprevalence of porcine cytomegalovirus (PCMV) and sapovirus (SaV) infections in pigs was investigated in Hunan province, China, between May 2005 and October 2010. A total of 500 pig serum samples collected from 10 representative administrative regions in Hunan province were evaluated for antibodies against PCMV and SaV using enzyme-linked immunosorbent assay (ELISA). The overall seroprevalence of porcine cytomegalovirus and sapovirus in pigs was 96.40% (482/500) and 63.40% (317/500), and the seropositivity of 10 herds we surveyed varied, ranging from 94.74% to 98.48% and 56.36% to 72.50%, respectively. The highest prevalence was found in breeding sows (96.67% for PCMV and 83.33% for SaVs). The results of the present survey indicated that infections with porcine cytomegalovirus and sapovirus are highly prevalent in pigs in Hunan province, China.

RT-PCR test for detecting porcine sapovirus in weanling piglets in Hunan Province, China.

The prevalence of porcine sapovirus infection in weanling pigs was investigated in Hunan Province, China, between August 2006 and October 2007. A total of 153 diarrheic fecal samples from ten intensive pig farms from ten representative administrative regions in Hunan province were examined for porcine sapoviruses using RT-PCR. Twenty-two of 153 (14.37 %) samples were found to contain porcine sapoviruses. Phylogenetic analysis showed that all the porcine sapovirus isolates in Hunan Province belonged to the porcine sapovirus genogroup III. The results of the present investigation have implications for the control of porcine sapovirus infection in pigs in Hunan Province, China.

First report of Neospora caninum infection in pigs in China.

Neospora caninum is a protozoan parasite which can infect many mammals and birds with a worldwide distribution. However, no molecular data are available about the occurrence of N. caninum in pigs. In this study, the serological and molecular prevalence of N. caninum infection in farmed pigs were investigated in Hunan province, China, between January 2017 and December 2018. A total of 1,500 serum samples collected from 10 herds in Hunan province were evaluated using a competitive-inhibition enzyme-linked immunoassay assay (cELISA). The overall seroprevalence of N. caninum in the examined pigs was 1.9%. The seroprevalence of N. caninum ranged from 0.3% to 4.6% among different regions in Hunan province of China (p < .05). DNA was extracted from brain samples, and the Nc-5 gene and ITS-1 region were amplified and then sequenced. Three (0.5%) of the examined 600 brain tissues were found to contain N. caninum DNA. Our phylogenetic analyses indicated that N. caninum samples were classified into two distinct groups. Although the prevalence is low within the pig groups investigated, our results revealed the emergence of N. caninum infection in pigs in China. The finding of the present study provides molecular evidence that the pigs are the natural intermediate host of N. caninum and may have major epidemiological importance.

Determination of antibody induction by highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) vaccine: A comparison of two ELISA kits.

Two commercial porcine reproductive and respiratory syndrome virus (PRRSV) antibody ELISA kits (IDEXX and LSI) are currently in extensive use. To determine which kit is more suitable for the evaluation of HP-PRRSV vaccine efficacy, the two kits were used to test 546 serum samples. The agreement between the results was unsatisfactory, with a kappa statistic of 0.681 and a linear correlation coefficient of 0.665. In tests of samples from experimentally vaccinated and PRRSV-negative herds, IDEXX-ELISA identified antibody-positive conversion earlier and showed a higher specificity compared to LSI-ELISA. The serological profile obtained by neutralization testing was closer to that obtained by IDEXX-ELISA than by LSI-ELISA in the late immunization period. The findings reveal that IDEXX-ELISA is the more suitable for the evaluation of antibody response to HP-PRRSV vaccine and for guiding vaccination strategies.

Seroprevalence and Risk Factors of Toxoplasma gondii Infection in Growth Stages of Pigs in Hunan Province, Subtropical China.

Toxoplasma gondii infection is prevalent in humans and animals worldwide. In the present study, a total of 612 serum samples were examined using indirect hemagglutination test (IHAT) for T. gondii infection. Antibodies to T. gondii were detected in 49 (8.0%, confidence interval [95% CI]: 5.9-10.2) serum samples (IHA titer ≥1:16). The T. gondii seroprevalence ranged from 4.4% (95% CI: 0-10.5) to 14.3% (95% CI: 0-40.2) among different regions in Hunan province of subtropical China. The highest seroprevalence was found in breeding sows (18.8%). The T. gondii seroprevalence was higher in winter (18.3%, 95% CI: 8.5-28.1) and spring (10.9%, 95% CI: 5.7-16.1) than in summer (6.4%, 95% CI: 2.8-10.1) and autumn (4.9%, 95% CI: 2.2-7.7), and the differences were statistically significant excepting summer. In addition, developmental stage and season were identified as risk factors for T. gondii infection. Our findings revealed the seroprevalence of T. gondii in growth stages of pigs in the Hunan province of subtropical China, indicating that it may cause public health and economic problems. To our knowledge, this is the first report of the comprehensive survey of T. gondii seroprevalence in pigs in the Hunan province of subtropical China.

Construction of an HRP-streptavidin bound antigen and its application in an ELISA for porcine circovirus 2 antibodies.

A fusion protein SBP-Cap∆41, consisting of Cap∆41 (without 41 amino acids at the N-terminus) protein of porcine circovirus 2 (PCV2) and a streptavidin binding peptide (SBP), was constructed. This fusion protein binds to HRP-labeled streptavidin (HRP-SA) through high affinity between SBP and SA, forming an HRP-streptavidin bound antigen (Hsb-Ag) with both immunoreactivity and enzymatic activity, which can be used in a double-antigen sandwich ELISA for detection of PCV2 antibodies. Comparison of the characteristics of the HSb-Cap∆41 and chemical conjugates of the recombinant Cap∆41 protein showed that the HSb-Cap∆41 based double-antigen sandwich ELISA (HBDS-ELISA) had higher specificity and sensitivity. Use of the HBDS-ELISA detected PCV2-IgG in 9 injected pigs as early as 10 days p.i., 3 days earlier than both a double-antigen sandwich ELISA (DS-ELISA) based on a chemically conjugated antigen, and a commercial indirect ELISA kit.

Identification of Bacterial Surface Antigens by Screening Peptide Phage Libraries Using Whole Bacteria Cell-Purified Antisera.

Bacterial surface proteins can be good vaccine candidates. In the present study, we used polyclonal antibodies purified with intact Erysipelothrix rhusiopthiae to screen phage-displayed random dodecapeptide and loop-constrained heptapeptide libraries, which led to the identification of mimotopes. Homology search of the mimotope sequences against E. rhusiopthiae-encoded ORF sequences revealed 14 new antigens that may localize on the surface of E. rhusiopthiae. When these putative surface proteins were used to immunize mice, 9/11 antigens induced protective immunity. Thus, we have demonstrated that a combination of using the whole bacterial cells to purify antibodies and using the phage-displayed peptide libraries to determine the antigen specificities of the antibodies can lead to the discovery of novel bacterial surface antigens. This can be a general approach for identifying surface antigens for other bacterial species.

Small-sized circular genomes similar to genome of porcine circovirus 2.

Circular genomes smaller than and similar to the genome of porcine circovirus 2 were obtained from pig tissues along with the full-length genome of porcine circovirus 2. The 922-, 839-, and 617-nucleotide-long genomes exhibit high homology to the rep gene plus the origin of replication sequence of porcine circovirus 2.

Complete genome sequences of classical Swine Fever virus isolates belonging to a new subgenotype, 2.1c, from hunan province, china.

Two isolates of a new classical swine fever virus (CSFV) subgenotype, 2.1c (HNLY-2011 and HNSD-2012), were recently isolated from pigs in Hunan Province, China. The most significant difference in the amino acid sequences of the polyproteins from subgenotypes 2.1a and 2.1b is an SPA → TPV amino acid substitution at positions 777 and 779 in the E2 protein.

Epitope screening of the PCV2 Cap protein by use of a random peptide-displayed library and polyclonal antibody.

Capsid protein (Cap), the only structural protein of porcine circovirus 2 (PCV2), is involved in the host protective response and is a target for vaccine development. To find a rapid and easy way to fully map the antigenic epitopes of Cap, purified Cap-specific polyclonal antibodies were used to screen a random heptapeptide phage display library. After three rounds of screening, twenty phage clones that had binding activity to Cap-specific antibodies (tested by phage ELISA) were sequenced. When the inserted amino acid sequences were aligned with the Cap protein sequence, eight core regions in Cap ((50)SRTFGYT(56), (62)VRTPSW(67), (68)AVDMMR(73), (79)FLPPGG(84), (86)SNPRSVPF(93), (102)KVEFWP(107), (119)GSSXXXLDDN(128) and (229)PPLNP(233)) were identified, three of which ((50)SRTFGYT(56), (86)SNPRSVPF(93) and (102)KVEFWP(107)) for the first time. Nine phagetopes representing the eight regions were chosen to immunize Kunming mice. All except minotopes (50)SRTFGYT(56) and (229)PPLNP(233) induced antibodies against PCV2 when injected into Kunming mice.

Phylogenetic analysis using E2 gene of classical swine fever virus reveals a new subgenotype in China.

Outbreaks of classical swine fever (CSF) have caused serious economic consequences in China. Phylogenetic analysis based on full-length E2 gene sequences showed that five classical swine fever virus (CSFV) isolates collected from Hunan province in 2011 and 2012, together with seven other isolates from neighboring provinces, Guangdong (5) and Guangxi (2), could be classified as a new subgenotype 2.1c, which may have been endemic in the south of China for at least fourteen years. Subgenotype 2.1c isolates share 90.2-94.9% and 89.9-93.8% nucleotide sequence similarity separately with those of subgenotype 2.1a and 2.1b in E2 gene, which are lower than the nucleotide identities between subgenotype 2.1a and 2.1b (91.1-95.7%). Further analysis based on a partial E2 gene sequence (216 nt) indicated that subgenotype 2.1c isolates are also circulating in Thailand. Alignment of E2 amino acid sequences showed that subgenotype 2.1c isolates exhibit a SPA → TPV substitution at positions 777 and 779 compared with subgenotypes 2.1a and 2.1b.

Identification of new defective interfering RNA species associated with porcine reproductive and respiratory syndrome virus infection.

In the present study, seven new defective interfering (DI) RNA species of porcine reproductive and respiratory syndrome virus (PRRSV) were identified. RT-PCR, Northern blot and sequence analyses indicated that these DI RNA specie have deletions of 8513-9176 nucleotides located between Nsp1/Nsp2 and Nsp10. Compared with the previous DI RNAs of PRRSV reported, they have three distinct characteristics: much smaller deletion sizes; different nucleotide repeats (2-12nt) used in the junction sites and in-frame deletions. The results further suggested that the similarity-assisted RNA recombination may be the main cause of generation of DI RNAs in PRRSV and probably in other arteriviruses.