Simultaneous Detection of Porcine Respiratory Coronavirus, Porcine Reproductive and Respiratory Syndrome Virus, Swine Influenza Virus, and Pseudorabies Virus via Quadruplex One-Step RT-qPCR.

Porcine respiratory coronavirus (PRCoV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), and pseudorabies virus (PRV) are significant viruses causing respiratory diseases in pigs. Sick pigs exhibit similar clinical symptoms such as fever, cough, runny nose, and dyspnea, making it very difficult to accurately differentially diagnose these diseases on site. In this study, a quadruplex one-step reverse-transcription real-time quantitative PCR (RT-qPCR) for the detection of PRCoV, PRRSV, SIV, and PRV was established. The assay showed strong specificity, high sensitivity, and good repeatability. It could detect only PRCoV, PRRSV, SIV, and PRV, without cross-reactions with TGEV, PEDV, PRoV, ASFV, FMDV, PCV2, PDCoV, and CSFV. The limits of detection (LODs) for PRCoV, PRRSV, SIV, and PRV were 129.594, 133.205, 139.791, and 136.600 copies/reaction, respectively. The intra-assay and inter-assay coefficients of variation (CVs) ranged from 0.29% to 1.89%. The established quadruplex RT-qPCR was used to test 4909 clinical specimens, which were collected in Guangxi Province, China, from July 2022 to September 2023. PRCoV, PRRSV, SIV, and PRV showed positivity rates of 1.36%, 10.17%, 4.87%, and 0.84%, respectively. In addition, the previously reported RT-qPCR was also used to test these specimens, and the agreement between these methods was higher than 99.43%. The established quadruplex RT-qPCR can accurately detect these four porcine respiratory viruses simultaneously, providing an accurate and reliable detection technique for clinical diagnosis.

A triplex crystal digital PCR for the detection of genotypes I and II African swine fever virus.

African swine fever (ASF) is a highly contagious and lethal viral disease that causes severe hemorrhagic fever in pigs. It keeps spreading around the world, posing a severe socioeconomic risk and endangering biodiversity and domestic food security. ASF first outbroke in China in 2018, and has spread to most provinces nationwide. Genotypes I and II ASF virus (ASFV) as the etiological pathogens have been found in China. In this study, three pairs of specific primers and probes targeting the ASFV B646L gene, F1055L gene, and E183L gene were designed to detect universal, genotype I, and genotype II strains, respectively. A triplex crystal digital PCR (cdPCR) was established on the basis of optimizing various reaction conditions. The assay demonstrated remarkably sensitive with low limits of detection (LODs) of 5.120, 4.218, 4.588 copies/reaction for B646L, F1055L, and E183L gene, respectively; excellent repeatability with 1.24-2.01% intra-assay coefficients of variation (CVs) and 1.32-2.53% inter-assay CVs; good specificity for only detection of genotypes I and II ASFV, without cross-reactivity with PCV2, PRV, SIV, PRRSV, PEDV, FMDV, and CSFV. The triplex cdPCR was used to test 1,275 clinical samples from Guangxi province of China, and the positivity rates were 5.05, 3.22, and 1.02% for genotype I, genotype II, and co-infection of genotypes I and II, respectively. These 1,275 clinical samples were also detected using a reported reference triplex real-time quantitative PCR (qPCR), and the agreements of detection results between these two methods were more than 98.98%. In conclusion, the developed triplex cdPCR could be used as a rapid, sensitive, and accurate method to detect and differentiate genotypes I and II strains of ASFV.

Development of a triplex real-time quantitative PCR for detection and differentiation of genotypes I and II African swine fever virus.

African swine fever virus (ASFV) was first identified in 1921 and is extensively prevalent around the world nowadays, which has a significant negative impact on the swine industry. In China, genotype II ASFV was first discovered in 2018, and has spread quickly to different provinces in a very short time; genotype I ASFV was first found in 2020, and has been reported in several provinces since then. To establish an accurate method for detection and differentiation of genotypes I and II ASFV, three primers and probes were designed targeting the ASFV B646L gene for different genotypes, the F1055L gene for genotype I, and the E183L gene for genotype II, and a triplex real-time quantitative PCR (qPCR) for differential detection of genotypes I and II ASFV was developed after optimizing the reaction conditions. The assay showed high sensitivity, and the limits of detection (LOD) of the B646L, F1055L, and E183L genes were 399.647 copies/reaction, 374.409 copies/reaction, and 355.083 copies/reaction, respectively; the coefficients of variation (CVs) of the intra-assay and the inter-assay were 0.22-1.88% and 0.16-1.68%, respectively, showing that this method had good repeatability; the assay could detect only ASFV, without cross-reactivity with other swine viruses including PRRSV, PEDV, PDCoV, CSFV, PRV, and PCV2, showing excellent specificity of this method. A total of 3,519 clinical samples from Guangxi province, southern China, were tested by the developed assay, and 8.16% (287/3,519) samples were found to be positive for ASFV, of which 0.17% (6/3,519) samples were positive for genotype I, 7.19% (253/3,519) samples for genotype II, and 0.80% (28/3,519) samples for genotypes I and II. At the same time, these clinical samples were also tested by a previously reported multiplex qPCR, and the agreement between these two methods was more than 99.94%. In summary, the developed triplex qPCR provided a fast, specific and accurate method for detection and differentiation of genotypes I and II ASFV.

Triplex Crystal Digital PCR for the Detection and Differentiation of the Wild-Type Strain and the MGF505-2R and I177L Gene-Deleted Strain of African Swine Fever Virus.

African swine fever (ASF) is a severe and highly contagious viral disease that affects domestic pigs and wild boars, characterized by a high fever and internal bleeding. The disease is caused by African swine fever virus (ASFV), which is prevalent worldwide and has led to significant economic losses in the global pig industry. In this study, three pairs of specific primers and TaqMan probes were designed for the ASFV B646L, MGF505-2R and I177L genes. After optimizing the reaction conditions of the annealing temperature, primer concentration and probe concentration, triplex crystal digital PCR (cdPCR) and triplex real-time quantitative PCR (qPCR) were developed for the detection and differentiation of the wild-type ASFV strain and the MGF505-2R and/or I177L gene-deleted ASFV strains. The results indicate that both triplex cdPCR and triplex qPCR were highly specific, sensitive and repeatable. The assays could detect only the B646L, MGF505-2R and I177L genes, without cross-reaction with other swine viruses (i.e., PRRSV, CSFV, PCV2, PCV3, PEDV, PDCoV and PRV). The limit of detection (LOD) of triplex cdPCR was 12 copies/reaction, and the LOD of triplex qPCR was 500 copies/reaction. The intra-assay and inter-assay coefficients of variation (CVs) for repeatability and reproducibility were less than 2.7% for triplex cdPCR and less than 1.8% for triplex qPCR. A total of 1510 clinical tissue samples were tested with both methods, and the positivity rates of ASFV were 14.17% (214/1510) with triplex cdPCR and 12.98% (196/1510) with triplex qPCR, with a coincidence rate of 98.81% between the two methods. The positivity rate for the MGF505-2R gene-deleted ASFV strains was 0.33% (5/1510), and no I177L gene-deleted ASFV strain was found. The results indicate that triplex cdPCR and triplex qPCR developed in this study can provide rapid, sensitive and accurate methods for the detection and differentiation of the ASFV B646L, MGF505-2R and I177L genes.

A relatively high zoonotic trematode prevalence in Orientogalba ollula and the developmental characteristics of isolated trematodes by experimental infection in the animal model.

BACKGROUND: Food-borne parasitic diseases decrease food safety and threaten public health. The snail species is an intermediate host for numerous human parasitic trematodes. Orientogalba ollula has been reported as intermediate hosts of many zoonotic trematodes. Here, we investigated the prevalence of zoonotic trematodes within O. ollula in Guangxi, China, and assessed their zoonotic potential. METHODS: Snails were collected from 54 sites in 9 cities throughout Guangxi. The snail and trematode larvae species were determined by combining morphological characteristics and molecular markers. The trematodes prevalence and constituent ratio were calculated and compared among different habitat environments. Phylogenetic trees of the trematode species were constructed using the neighbor-joining method with nuclear internal transcribed spacer 2 (ITS2) sequences. The developmental cycles of the isolated trematodes were examined by experimental infection in ducks. The developmental characteristics of Echinostoma revolutum was recorded by dissecting infected ducklings from 1-day post infection (dpi) to 10 dpi. RESULTS: The overall prevalence of trematode larvae was 22.1% (1818/8238) in O. ollula from 11 sample sites. Morphological together with molecular identification, showed that E. revolutum, Australapatemon sp., Hypoderaeum conoideum, Pharyngostomum cordatum, and Echinostoma sp. parasitized O. ollula, with the highest infection rate of E. revolutum (13.0%). However, no Fasciola larvae were detected. The trematodes prevalence and constituent ratio varied in two sub-biotypes (P < 0.01). A neighbor-joining tree analysis of ITS2 sequences resulted in distinct monophyletic clades supported by sequences from isolated larvae with high bootstrap values. Ducklings exposed to O. ollula infected with Echinostoma sp., E. revolutum, and H. conoideum larvae were successfully infected. The animal model for Echinostoma revolutum was successfully established. E. revolutum matured from larvae to adult at 10 dpi in the intestine of the duck, and the developmental characteristics of E. revolutum were characterized by the maturation of the reproductive and digestive organs at 6-8 dpi. CONCLUSIONS: This study revealed a high prevalence of zoonotic trematodes in O. ollula from Guangxi, China. Existing trematodes infection in animals and human clinical cases, coupled with the wide geographical distribution of O. ollula, necessitate further evaluations of the potential risk of spillover of zoonotic infection from animal to human and vice versa.

Whole-genome analyses of APEC carrying mcr-1 in some coastal areas of China from 2019 to 2020.

OBJECTIVES: Polymyxin is considered as one of the 'last lines of defense' for the treatment of multidrug resistant bacteria. Increased use of polymyxin during recent years poses a risk to public health. The purpose of this study was to investigate the carrying situation of the mcr-1 drug-resistance gene in waterfowl in some coastal areas of China from 2019 to 2020. METHODS: Fifty-seven isolated avian pathogenic Escherichia coli strains were selected from 493 APEC isolates for whole-genome sequencing. The 24 mcr-1-positive APEC strains were tested for conjugation and genome-wide analysis, including sequence type (ST) analysis, serotype analysis, and drug-resistance gene analysis. Numerous mcr-1-positive E. coli were downloaded from the National Center for Biotechnology Information (NCBI) for comparative genomic analysis. RESULTS: Antimicrobial susceptibility test results showed that 57 APEC isolates were highly resistant to gentamicin, cefotaxime, and ofloxacin, and 24 mcr-1-positive APEC isolates were resistant to polymyxin. Fourteen isolates of mcr-1-positive APEC plasmids were successfully conjugated to EC600. Both ST156 and ST10 were found in high proportions in human and avian sources through genome-wide analysis; it is worth noting that these two isolates of APEC were detected to contain the blaNDM-1 and blaNDM-4 genes, respectively. CONCLUSION: In this study, the epidemiological investigation of the mcr-1 gene was carried out on APEC in some coastal areas of China from 2019 to 2020, and our results have enriched the data on the transmission of APEC isolates carrying the mcr-1 gene in waterfowl.

The Development of a Multiplex Real-Time Quantitative PCR Assay for the Differential Detection of the Wild-Type Strain and the MGF505-2R, EP402R and I177L Gene-Deleted Strain of the African Swine Fever Virus.

African swine fever virus (ASFV) causes African swine fever (ASF), a devastating hemorrhagic disease of domestic pigs and wild boars. Currently, the MGF505R, EP402R (CD2v) and I177L gene-deleted ASFV strains were confirmed to be the ideal vaccine candidate strains. To develop an assay for differentiating the wild-type and gene-deleted ASFV strains, four pairs of specific primers and TaqMan probes targeting the ASFV B646L (p72), I177L, MGF505-2R and EP402R (CD2v) genes were designed. A multiplex real-time qPCR assay for the differential detection of the wild-type and gene-deleted ASFV strains was developed after optimizing the reaction conditions, including the annealing temperature, primer concentration and probe concentration. The results showed that the multiplex real-time qPCR assay can specifically test the ASFV B646L (p72), I177L, MGF505-2R and EP402R (CD2v) genes with a limit of detection (LOD) of 32.1 copies/µL for the B646L (p72) gene, and 3.21 copies/µL for the I177L, MGF505-2R and EP402R (CD2v) genes. However, the assay cannot test for the classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), porcine circovirus type 2 (PCV2), PCV3 and pseudorabies virus (PRV). The assay demonstrated good repeatability and reproducibility with coefficients of variation (CV) less than 1.56% for both the intra- and inter-assay. The assay was used to test 4239 clinical samples, and the results showed that 12.60% (534/4239) samples were positive for ASFV, of which 10 samples lacked the EP402R gene, 6 samples lacked the MGF505-2R gene and 14 samples lacked the EP402R and MGF505-2R genes. The results indicated that the multiplex real-time qPCR developed in this study can provide a rapid, sensitive and specific diagnostic tool for the differential detection of the ASFV B646L, I177L, MGF505-2R and EP402R genes.

O145 may be emerging as a predominant serogroup of Avian pathogenic Escherichia coli (APEC) in China.

Among the numerous serotypes of Avian pathogenic Escherichia coli (APEC), O1, O2 and O78 have long been considered the predominant serogroups. O145, a pivotal serogroup in non-O157 Shiga toxin-producing Escherichia coli, has never been considered an important serogroup among APEC. The prevalence of APEC O145 was determined from the results of molecular serogrouping based on 42 sequenced isolates from Jiangsu and Guangxi Provinces in China. After realizing the potential importance of O145, 224 APEC isolates isolated from Jiangsu, Guangxi, Anhui, Shandong, Henan, Yunnan and Fujian provinces were screened using PCR amplification. The results showed that the proportion of O145 detected was 37.9 % (85/224), which was higher than those of the three traditional APEC serogroups. The virulence evaluation experiment showed that this serogroup may have stronger pathogenicity. Here, we report for the first time that O145 may be emerging as a predominant serogroup of APEC in China. The possible reasons for its prevalence and oversight were analyzed through genomic analysis. Furthermore, pangenome analysis with STEC O145 was performed to assess the potential threat to humans. The discovery of the ubiquity of O145 may not be coincidental, which may also account for the failure of vaccines that target the three major serogroups. Therefore, this newly predominant serogroup should be paid more attention and the focus should not be limited to the so-called three major APEC serogroups.

[Ultrastructural observation on nymphal Armillifer sp. by scanning electron microscopy and phylogenetic analysis based on 18S rRNA].

OBJECTIVE: To observe the ultrastructure of nymphal Armillifer sp. isolated from Macaca fascicularis by using scanning electron microscope (SEM), and analyze the phylogenetic relationships based on 18S rRNA gene sequences. METHODS: The parasite samples stored in 70% alcohol were fixed by glutaraldehyde and osmium peroxide. Ultrastructural characters of those samples were observed under SEM. Amplification and sequencing of the 18S rRNA gene were performed following the extraction of total genome DNA. Sequence analysis was performed based on multiple alignment using ClustalX1.83, while phylogenetic analysis was made by Neighbor-Joining method using MEGA4.0. RESULTS: The nymphs were in cylindrical shape, the body slightly claviform tapering to posterior end. Abdominal annuli were gradually widened from anterior to posterior parts, the 12th-13th abdominal annuli of which were similar in width. The annuli ranged closer in the front half body, whereas in the latter part there were certain gaps between them. The circular-shaped mouth located in the middle of head ventrally. Folds were seen in inner margin of the mouth with a pair of curved hooks on both sides above it which practically disposed in a straight line. Two pairs of large sensory papillae were observed symmetrically over the last thoracic annulus of cephalothoraxs lying below the outer hook, and the first abdominal annulus was near the median ventral line. The number of abdominal annuli was 29, not including 2 incomplete terminal annuli. Rounded sensory papillae were fully distributed on the body surface, except the dorsal side of head and the ventral part of the terminal annulus. Agglomerate-like anus opening was observed at the end of ventral abdominal annuli and distinctly sub-terminal. These morphological features demonstrated that the nymphs were highly similar with that of Armillifer moniliformis Diesing, 1835. A fragment of 18SrRNA gene (1 836 bp) sequences was obtained by PCR combined with sequencing, and was registered to the GeneBank database with an accession number HM048870. The phylogenetic tree indicated that A. moniliformis, A.agkistrodon and A.armillatus were at the same clade with a bootstrap value at 95%, and A. moniliformis and A. agkistrodon were solo at a clade with a bootstrap value of 75%. CONCLUSION: The nymphs isolated from Macaca fascicularis are identified as A. moniliformis temporarily.