Identification of a broadly neutralizing epitope within Gc protein of Akabane virus using newly prepared neutralizing monoclonal antibodies.

Akabane virus (AKAV) is characterized by abortion, stillbirth, premature birth, and congenital deformities in livestock and is widely distributed throughout Australia, Southeast Asia, East Asia, the Middle East, and Africa. Gc protein is the major neutralizing target of AKAV and is often considered as an immunogen to prepare neutralizing antibodies. In this study, we prepared and characterized three monoclonal antibodies (mAbs), 4D1, 4E6, and 4F12, against the Gc protein of AKAV (TJ2016 strain). Western blot (WB) and indirect immunofluorescence assay (IFA) analysis proved that the mAbs can react with both the truncated recombinant AKAV Gc protein and the natural Gc protein produced in the AKAV-infected cells. Further research demonstrated that these mAbs possess neutralizing activity. We next defined a neutralizing epitope 1134SVQSFDGKL1142 by screening a panel of overlapping peptides spanning the truncated Gc protein (aa991∼1232) using the generated neutralizing mAbs. Bioinformatic analysis shows that the neutralizing epitope is highly conserved across different genotypes of AKAV. The newly produced neutralizing mAbs and the identified neutralizing epitope in this study enrich the antigenic epitope information of the AKAV Gc protein and could have potential applications in the development of antigen and antibody detection systems that are specific to AKAV.

Generation of Stable Cell Lines Expressing Akabane Virus N Protein and Insight into Its Function in Viral Replication.

Akabane virus (AKAV) is a world wide epidemic arbovirus belonging to the Bunyavirales order that predominantly infects livestock and causes severe congenital malformations. The nucleocapsid (N) protein of AKAV possesses multiple important functions in the virus life cycle, and it is an ideal choice for AKAV detection. In this study, we successfully constructed two stable BHK-21 cell lines (C8H2 and F7E5) that constitutively express the AKAV N protein using a lentivirus system combined with puromycin selection. RT-PCR analysis confirmed that the AKAV N gene was integrated into the BHK-21 cell genome and consistently transcribed. Indirect immunofluorescence (IFA) and Western blot (WB) assays proved that both C8H2 and F7E5 cells could react with the AKAV N protein mAb specifically, indicating potential applications in AKAV detection. Furthermore, we analyzed the growth kinetics of AKAV in the C8H2 and F7E5 cell lines and observed temporary inhibition of viral replication at 12, 24 and 36 h postinfection (hpi) compared to BHK-21 cells. Subsequent investigations suggested that the reduced viral replication was linked to the down-regulation of the viral mRNAs (Gc and RdRp). In summary, we have established materials for detecting AKAV and gained new insights into the function of the AKAV N protein.

Characterization and Double-Antibody Sandwich ELISA Application of a Monoclonal Antibody Against Akabane Virus Nucleocapsid Protein.

BACKGROUND: Akabane virus (AKAV) is a Culicoides-borne Orthobunyavirus that is teratogenic to the fetus of cattle and small ruminant species. OBJECTIVE: This study aimed to develop an effective diagnostic assay for the detection of AKAV using produced monoclonal antibody (mAb). METHOD: First, the mAb against N protein of AKAV was produced and characterized by Western blot (WB) and indirect immunofluorescence (IFA) assays. Then, the linear epitope of AKAV N protein against the mAb was identified and the mAb was applied to establish a double-antibody sandwich ELISA (DAS-ELISA). RESULTS: One AKAV N-reactive monoclonal mAb was generated and designated as 2D3. WB and IFA assays indicated that 2D3 could react with both recombinant N protein and AKAV isolate TJ2016. The linear epitope recognized by mAb 2D3 was located at amino acids 168-182 of AKAV N protein. The DAS-ELISA established on based mAb 2D3 was able to detect both the purified AKAV N protein (with a detection limit of 6.25 ng/mL) and AKAV-infected cell culture supernatant (with a detection limit of 250 TCID50/mL). CONCLUSIONS: Taken together, we successfully prepared a mAb 2D3 against AKAV N protein and identified its corresponding linear epitope, and then established a DAS-ELISA for the detection of AKAV antigen. HIGHLIGHTS: A produced mAb against AKAV N protein was used to define a linear epitope of AKAV and establish a DAS-ELISA for AKAV antigen detection.

A Novel, Reverse Transcription, Droplet Digital PCR Assay for the Combined, Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus 2 with Swine Acute Diarrhea Syndrome Coronavirus.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread over the world since its emergence. Although the dominant route of SARS-CoV-2 infection is respiratory, a number of studies revealed infection risk from contaminated surfaces and products, including porcine-derived food and other products. The SARS-CoV-2 outbreak has been severely threatening public health, and disrupting porcine products trade and the pig industry. Swine acute diarrhea syndrome coronavirus (SADS-CoV), which was responsible for large-scale, fatal disease in piglets, emerged in 2017 and has caused enormous economic losses in the pig industry. Currently, reverse transcription real-time PCR (RT-rPCR) is the gold standard method for SARS-CoV-2 diagnosis and is most commonly used for SADS-CoV detection. However, inaccurate detection of the SARS-CoV-2 infection obtained by RT-rPCR is increasingly reported, especially in specimens with low viral load. OBJECTIVE: This study aimed to develop an accurate reverse transcription droplet digital PCR (RT-ddPCR) assay for the detection of SARS-CoV-2 and SADS-CoV simultaneously. METHODS: Two pairs of primers and one double-quenched probe targeting the RNA-dependent RNA polymerase (RDRP) region of the open reading frame 1ab (ORF1ab) gene of SARS-CoV-2 and the corresponding ORF1ab region of SADS-CoV were designed to develop the RT-ddPCR assay. The sensitivity, specificity, repeatability, and reproducibility were tested using complementary RNAs (cRNAs) and clinical specimens. RESULTS: The detection limits of RT-ddPCR were 1.48 ± 0.18 and 1.38 ± 0.17 copies in a 20 µL reaction for SARS-CoV-2 and SADS-CoV cRNAs, respectively (n = 8), showing approximately 4- and 10-fold greater sensitivity than the RT-rPCR assay. This assay also exhibited good specificity, repeatability, and reproducibility. CONCLUSION: The established RT-ddPCR assay was shown to be a highly effective, accurate, and reliable method for the sensitive detection of SARS-CoV-2 and SADS-CoV. HIGHLIGHTS: This RT-ddPCR assay could be used to detect both SARS-CoV-2 and SADS-CoV in a sample with one double-quenched probe, and is also the first reported RT-ddPCR assay for SADS-CoV detection.

Characterization and reverse genetic establishment of cattle derived Akabane virus in China.

BACKGROUND: Akabane virus (AKAV) is an important insect-borne virus which is widely distributed throughout the world except the Europe and is considered as a great threat to herbivore health. RESULTS: An AKAV strain defined as TJ2016 was firstly isolated from the bovine sera in China in 2016. Sequence analysis of the S and M segments suggested that the isolated AKAV strain was closely related to the AKAV strains JaGAr39 and JaLAB39, which belonged to AKAV genogroup II. To further study the pathogenic mechanism of AKAV, the full-length cDNA clone of TJ2016 S, M, and L segment was constructed separately into the TVT7R plasmid at the downsteam of T7 promoter and named as TVT7R-S, TVT7R-M, and TVT7R-L, respectively. The above three plasmids were further transfected into the BSR-T7/5 cells simultaneously with a ratio of 1:1:1 to produce the rescued virus AKAV. Compared with the parental wild type AKAV (wtAKAV), the rescued virus (rAKAV) was proved to be with similar cytopathic effects (CPE), plaque sizes and growth kinetics in BHK-21 cells. CONCLUSION: We successfully isolated a AKAV strain TJ2016 from the sera of cattle and established a reverse genetic platform for AKAV genome manipulation. The established reverse genetic system is also a powerful tool for further research on AKAV pathogenesis and even vaccine studies.

A new landscape of rabbit gut microbiota shaped by the infection of precocious parasites of Eimeria intestinalis.

Rabbit intestinal coccidiosis is caused by one or several Eimeria species, which cause intestinal damage and secondary bacterial infection. However, the impact of Eimeria infection on gut microbiota is much unknown. To evaluate the influence, we detected the feces flora of SPF rabbits infected with the 1 × 104 oocysts of E. intestinalis wild type (WT) and a precocious line (EIP8), a highly pathogenic species, by 16S rRNA sequencing. The microbiota of newly weaned rabbits post vaccination with low doses of EIP8 oocysts was also detected. In SPF rabbits, while Ruminococcaceae, Lachnospiraceae, and Bacteroidaceae were dominant families in all groups, EIP8 infection induced less changes in beta-diversity. In EIP8-infected rabbits, the intestinal flora whose abundance changed post infection accounted for less than 5.23 % of the entire flora. In comparison, it accounted for 27.18 % in WT group on d14 PI, while it was more than 20 % in diclazuril control group on d7 or d10 PI. The amount of fecal IgA and the abundance of IgA-production-related bacteria were similar in either EIP8 or WT infected rabbits. In the newly weaned rabbits, vaccination with EIP8 provided sufficient protection against challenge with WT parasites, as the body weight gain of vaccinated rabbits was similar to that of untreated animals, as well as more than 80 % reduction of oocyst output was detected when compared with unimmunized and challenged animals. Moreover, the vaccination had no significant impact on rabbit microbiota. Together, our findings suggested that the precocious line of E. intestinalis, compared with WT, induced a new fecal microbiota biodiversity in rabbits.

Application of an AlphaLISA method for rapid sensitive detection of African swine fever virus in porcine serum.

Infection with African swine fever virus (ASFV) causes an acute and highly lethal hemorrhagic disease that has been responsible for huge economic losses in China. To exactly detect the antigen of ASFV, we established a rapid, no-wash, one-step sandwich-type immunoassay based on the amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) using two monoclonal antibodies (mAbs) M-5 and M-6 against ASFV p72. ASFV p72 in samples was captured by biotinylated mAb M-5 connected to the donor bead surface via streptavidin and "sandwiched" by mAb M-6 which was coated onto the acceptor bead. Efficacy and sensitivity trials revealed that the AlphaLISA could detect ≥0.78 ng/ml of purified p72 and with a linear range of 0.78-100 ng/ml. The AlphaLISA was specific for ASFV and did not cross-react with other common pathogenic porcine viruses. Compared with RealPCR ASFV DNA test and ASFV antigen detection kit, the sensitivity of the AlphaLISA evaluated in 60 porcine serum samples was 93% and 100%, respectively. The specificity was 100% and 91.7%, respectively. This study presents a good laboratory diagnostic tool for sensitive and efficient detection of ASFV in porcine serum. KEY POINTS: • MAbs M-5 and M-6 recognized various epitopes of ASFV p72. • The established ASFV p72 AlphaLISA showed well specificity, high sensitivity, and satisfied correlation coefficient.

Multiplex Real-Time PCR Method for Simultaneous Detection and Differentiation of Goat pox Virus, Sheeppox Virus, and Lumpy Skin Disease Virus.

BACKGROUND: The diseases caused by the Capripoxvirus species have very similar symptoms and are difficult to distinguish clinically. According to a recent report, Capripoxvirus are not strictly host specific. OBJECTIVE: This study aimed to identify the viruses from ovine (include sheep and goat) or bovine, which will assist in selecting the appropriate vaccine and correct measures to control diseases. METHOD: Universal primers for all Capripoxvirus and specific probes for lumpy skin disease virus, sheeppox virus, and goatpox virus were designed and analyzed to identify the viruses from ovine (including sheep and goats) or bovine species. The parameters of the system, such as the annealing temperatures and the quantities of primers and probes used, were optimized. The sensitivity, specificity, and reproducibility were tested. RESULTS: Each probe showed a specific fluorescent signal, with no cross reaction with other pathogens that cause symptoms similar to those of the poxviruses. The LOD was 102 copies of the target genome DNA. The 557 local clinical samples and samples from Ethiopia were successfully detected and the results were consistent with a restriction fragment length polymorphism PCR analysis of the P32 and RPO30 genes and gene sequencing. CONCLUSIONS: This optimized real-time PCR detection system has good diagnostic sensitivity and specificity and can be used for the rapid and effective differential diagnosis of these diseases in goats, sheep, and cattle. HIGHLIGHTS: It is a rapid detection method to distinguish the viruses from ovine (include sheep and goat) or bovine.

Development of a Colloidal Gold Immunochromatographic Strip for the Rapid Detection of Channel Catfish Virus.

BACKGROUND: Channel catfish virus disease (CCVD) has resulted in great economic losses and has restricted the development of fisheries. There is therefore, a need for rapid and efficient diagnostic methods to control the spread of CCVD. OBJECTIVE: A colloidal gold immunochromatographic strip has been developed for the detection of CCVD. METHODS: In this study, a colloidal gold immunochromatographic strip for channel catfish virus (CCV) detection was developed using the monoclonal antibody 8B6 conjugated with colloidal gold as the detector antibody. A rabbit anti-CCV antibody was used as the capture complex at the test line, and a goat anti-mouse IgG antibody was used as the capture antibody at the control line. The strip was characterized in its specificity, sensitivity, and stability. In addition, an infection experiment was performed to test the applicability of the test strip. RESULT: The strip was able to detect concentrations of the virus (104 tissue culture infective dose (TCID50)/mL) and showed analytical specificity when tested against other viral pathogens. The strips were still usable after 30 days of storage at 60°C. It was possible to detect CCV experimentally in infected fish within 10-15 min of using the strip. CONCLUSIONS: The strip can be used as a rapid and convenient tool for on-site diagnosis to control outbreaks and the spread of CCVD. HIGHLIGHTS: The immunochromatographic strip was the first to be developed and applied for the detection of CCVD.

A quantum dot fluorescent microsphere based immunochromatographic strip for detection of brucellosis.

BACKGROUND: Brucellosis is a serious zoonosis disease that frequently causes significant economic loss in animal husbandry and threatens human health. Therefore, we established a rapid, accurate, simple and sensitive fluorescent immunochromatographic strip test (ICST) based on quantum dots (QDs) for detection the antibodies of Brucella infection animals serum. RESULTS: The test strips were successfully prepared by quantum dot fluorescent microspheres (QDFM) as tracers, which were covalently coupled to an outer membrane protein of Brucella OMP22. The outer membrane protein OMP28 and monoclonal antibodies of OMP22 were separately dispensed onto a nitrocellulose membrane as test and quality control lines, respectively. The critical threshold for determining negative or positive through the ratio of the fluorescent signal of the test line and the control line (HT / HC) is 0.0492. The repeatability was excellent with an overall average CV of 8.78%. Under optimum conditions, the limit of detection was 1.05 ng/mL (1:512 dilution). With regard to the detection of brucellosis in 150 clinical samples, the total coincidence rate of ICST and Rose Bengal plate test (RBPT) was 97.3%, the coincidence rate of positive samples was 98.8%, the coincidence rate of negative samples was 95.3%, the sensitivity of RBPT is 1:32, and no cross reaction with the sera of other related diseases was observed. CONCLUSION: In our present study, the QDFM has promising application for on-site screening of brucellosis owing to its high detection speed, high sensitivity, high specificity and low cost.

Genetically modified adipose-derived stem cells with matrix metalloproteinase 3 promote scarless cutaneous repair.

Adipose-derived stem cells (ASCs) possess strong regenerative potencies and have been used to improve wound healing in animal models and clinical studies. However, the use of ASCs on scarless wound healing is not satisfactory. Matrix metalloproteinase 3 (MMP-3) is involved in extracellular matrix (ECM) remolding and scar formation. We aimed to investigate the effect of ASCs stable expressing MMP-3 (ASCs-MMP-3) on wound healing and scarring. A cutaneous wound healing animal model was used to assess the effect of ASCs and ASCs-MMP-3 on wound healing and scar formation. The target protein levels in the wound tissues were determined by western blot assay. Our results demonstrated that ASCs alone promoted wound healing but had a negligible effect on reducing scarring. ASCs-MMP-3 not only possessed the ability of ASCs to speed up wound healing, but also incorporated the capability of MMP-3 to reduce scaring. Overexpressing of MMP-3 decreased the collagen I, transforming growth factor (TGF)-ß1, and α-smooth muscle actin (α-SMA) levels and enhanced collagen III and TGF-ß3 levels which contributed to reducing scar formation. Our studies suggested that ASCs-MMP-3 is a potential candidate for developing effective therapeutic strategies for scarless wound healing.

Identification and Molecular Analysis of Ixodid Ticks (Acari: Ixodidae) Infesting Domestic Animals and Tick-Borne Pathogens at the Tarim Basin of Southern Xinjiang, China.

Livestock husbandry is vital to economy of the Tarim Basin, Xinjiang Autonomous Region, China. However, there have been few surveys of the distribution of ixodid ticks (Acari: Ixodidae) and tick-borne pathogens affecting domestic animals at these locations. In this study, 3,916 adult ixodid ticks infesting domestic animals were collected from 23 sampling sites during 2012-2016. Ticks were identified to species based on morphology, and the identification was confirmed based on mitochondrial 16S and 12S rRNA sequences. Ten tick species belonging to 4 genera were identified, including Rhipicephalus turanicus, Hyalomma anatolicum, Rh. bursa, H. asiaticum asiaticum, and Rh. sanguineus. DNA sequences of Rickettsia spp. (spotted fever group) and Anaplasma spp. were detected in these ticks. Phylogenetic analyses revealed possible existence of undescribed Babesia spp. and Borrelia spp. This study illustrates potential threat to domestic animals and humans from tick-borne pathogens.

Development of polyclonal-antibody-coated immunomagnetic beads for separation and detection of koi herpesvirus in large-volume samples.

To separate and concentrate koi herpesvirus (KHV) from large-volume samples, a separation method based on immunomagnetic beads (IMBs) coated with polyclonal antibody directed against KHV was developed. After treatment with IMBs, viral DNA was extracted from samples and used as a template for quantitative PCR (qPCR). The results showed that the concentration of the template DNA extracted from the virus that had been separated using IMBs was 9.65-fold higher than that from virus not treated with IMBs. The detection limit of the IMBs/qPCR method was found to be at least 10 times lower than that of qPCR alone.

Comparative evaluation of two commercial ELISA kits for detection of antibodies against Akabane virus in cattle serum.

BACKGROUND: Akabane disease (AD), a barrier to international trade for endemic areas with far economic impact on the countries, is caused by Akabane virus (AKAV). Commercial enzyme-linked immunosorbent assay (ELISA) is a commonly used diagnostic technique for AKAV infection, including the IDEXX and IDVET ELISA kits. However, the comparative evaluation of the IDEXX and IDVET ELISA kits has not been published. The object of this study was to evaluate the test performance of the two commercial ELISA kits in detecting serum anti-AKAV antibodies in cattle. RESULTS: With virus neutralization test (VNT) as the "relative gold standard", the diagnostic sensitivity (DSe) was 80.39% (123/153) and 93.46% (143/153) for the IDEXX and IDVET ELISA kit, when suspect samples were included. The diagnostic specificity (DSp) for the IDEXX and IDVET ELISA kit was 93.48% (502/537) and 82.31% (442/537), respectively. CONCLUSION: Both of the tested ELISA kits could be applied to detect antibodies against AKAV in cattle serum. The IDVET ELISA kit had a higher DSe. The IDEXX ELISA kit possessed the higher DSp. These results have important implications if the kits are used to screen herds or individual cattle in surveillance programs, or at border crossings for import-export inspection and quarantine.

Emergence and Comparative Genomics Analysis of Extended-Spectrum-ß-Lactamase-Producing Escherichia coli Carrying mcr-1 in Fennec Fox Imported from Sudan to China.

The aim of this study was to investigate the occurrence and genomic characteristics of extended-spectrum-ß-lactamase-producing Escherichia coli (ESBL-EC) in fennec fox imported from Sudan to China. We screened 88 fecal samples from fennec fox for ESBL-EC, using cefotaxime- and meropenem-supplemented selective medium. Antimicrobial susceptibility testing was performed by the agar dilution method except for colistin and tigecycline; for colistin and tigecycline, testing was conducted by the broth microdilution method. ESBL-EC bacteria were sequenced, and their genomes were characterized. Plasmid conjugation, S1 nuclease pulsed-field gel electrophoresis (PFGE), and Southern blotting were performed for a MCR-1-producing isolate. The genetic environment of mcr-1 and ESBL genes was also investigated. A total of 29 ESBL-EC bacteria were isolated from 88 fennec fox (32.9%), while no carbapenemase producers were found. The most prevalent genotypes were the blaCTX-M-55 and blaCTX-M-14 genes, followed by blaCTX-M-15 and blaCTX-M-64 We detected nine sequence types among 29 ESBL-EC. Furthermore, the mcr-1 gene was detected in isolate EcFF273. Conjugation analysis confirmed that the mcr-1 gene was transferable. S1 PFGE, Southern blotting, and whole-genome sequencing revealed that mcr-1 and blaCTX-M-64 were both located on a 65-kb IncI2 plasmid. This study reports for the first time the occurrence of ESBL-EC in fennec fox. The high prevalence of ESBL producers and the occurrence of MCR-1 producer in fennec fox imported into China from Sudan are unexpected. In addition, it clearly demonstrated that commensal E. coli strains can be reservoirs of blaCTX-M and mcr-1, potentially contributing to the dissemination and transfer of such genes to pathogenic bacteria among fennec fox. Our results support the implication of fennec fox as a biological vector for ESBL-producing members of the Enterobacteriaceae family.IMPORTANCE The extended-spectrum-ß-lactamase (ESBL)-producing members of the Enterobacteriaceae family are a global concern for both animal and human health. There is some information indicating a high prevalence of ESBL producers in food animals. Moreover, there have been an increasing number of reports on ESBL-producing strains resistant to the last-resort antibiotic colistin with the global dissemination of the plasmid-mediated mcr-1 gene, which is believed to have originated in animal breeding. However, little is known regarding the burden of ESBL-producing Enterobacteriaceae on wild animals. No data were available on the prevalence of antimicrobial resistance (AMR) among wild animals imported into China. This is the first study to investigate the microbiological and genomics surveillance investigation of ESBL colonization among fennec fox (Vulpes zerda) imported from Sudan to China, and we uncovered a high prevalence of ESBL-EC. Furthermore, the underlying mechanism of colistin resistance in an isolate that harbored mcr-1 was also investigated. Results of characterization and analysis of 29 ESBL-producing E. coli may have important implications on our understanding of the transmission dynamics of these bacteria. We emphasize the importance of improved multisectoral surveillance for colistin-resistant E. coli in this region.

Multiplex detection of six swine viruses on an integrated centrifugal disk using loop-mediated isothermal amplification.

Advances in molecular testing and microfluidic technologies have opened new avenues for rapid detection of animal viruses. We used a centrifugal microfluidic disk (CMFD) to detect 6 important swine viruses, including foot-and-mouth disease virus, classical swine fever virus, porcine reproductive and respiratory swine virus-North American genotype, porcine circovirus 2, pseudorabies virus, and porcine parvovirus. Through integrating the loop-mediated isothermal amplification (LAMP) method and microfluidic chip technology, the CMFD could be successfully performed at 62℃ in 60 min. The detection limit of the CMFD was 3.2 × 102 copies per reaction, close to the sensitivity of tube-type LAMP turbidity methods (1 × 102 copies per reaction). In addition, the CMFD was highly specific in detecting the targeted viruses with no cross-reaction with other viruses, including porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine rotavirus. The coincidence rate of CMFD and conventional PCR was ~94%; the CMFD was more sensitive than conventional PCR for detecting mixed viral infections. The positive detection rate of 6 viruses in clinical samples by CMFD was 44.0% (102 of 232), whereas PCR was 40.1% (93 of 232). Thirty-six clinical samples were determined to be coinfected with 2 or more viruses. CMFD can be used for rapid, sensitive, and accurate detection of 6 swine viruses, offering a reliable assay for monitoring these pathogens, especially for detecting viruses in widespread mixed-infection clinical samples.

Stable transfection of Eimeria necatrix through nucleofection of second generation merozoites.

Eimeria spp., the causative agents of coccidiosis, are the most common protozoan pathogens of chickens. Infection with these parasites can result in poor development or death of animals leading to a devastating economic impact on poultry production. The establishment of transfection protocols for genetic manipulation of Eimeria species and stable expression of genes would help advance the biology of these parasites as well as establish these organisms as novel vaccine delivery vehicles. Here, we report the selection of the first stable transgenic E. necatrix population, EnHA1, consitutively expressing the EYFP reporter following transfection of the 2nd generation merozoites with a linear DNA fragment harboring the EYFP reporter gene, the HA1 gene from the avian influenza virus H9N2 and the TgDHFR-TS selectable marker, which confers resistance to pyrimethamine. Transfected merozoites were inoculated into chickens via the cloacal route, and feces from 18 h to 72 h post inoculation were collected and subjected to subsequent serial passages, FACS sorting and pyrimethamine selection. A gradual increase in the number of EYFP-expressing sporulated oocysts was noticed with more than 90% EYFP + oocysts obtained after five passages. Immunofluorescence assay confirmed successful expression of the HA1 antigen in the EnHA1 population. The ability to genetically manipulate E. necatrix merozoites and express heterologous genes in this parasite will pave the way for possible use of this organism as a vaccine-delivery vehicle.

Establishment of a sensitive TaqMan-based real-time PCR assay for porcine circovirus type 3 and its application in retrospective quarantine of imported boars to China.

Porcine circovirus type 3 (PCV3) is a novel pathogen first identified in the United States in 2016. As there is a high possibility that no clinical signs of infection are observed in the host, an accurate and sensitive method is needed for quarantine on numerous live pigs especially for international pig trade. In this study, a TaqMan-based real-time PCR assay specifically for PCV3 was established without cross-reactions with other non-targeted pig viruses. The sensitivity of the current approach is about 1.5 × 101  copies µL-1 plasmid DNA while the sensitivity of the conventional PCR is about 1.5 × 102  copies µL-1 plasmid DNA. Further, this assay was applied in the retrospective quarantine on serum samples of 601 commercial live boars imported to China from the United States, France and the United Kingdom from 2011 to 2017. The results revealed that PCV3 could be detected positive in the commercial boars imported from the United States and the above-mentioned western European countries and phylogenetic study also revealed that viral isolates were grouped with some isolates from Korea and the United States. Our study suggested that PCV3 may be prevalent globally since 2011.

Development of a droplet digital PCR assay for sensitive detection of porcine circovirus 3.

Porcine circovirus 3 (PCV3), a newly emerged circovirus, is associated with porcine dermatitis and nephropathy syndrome, reproductive failure and multi-systemic inflammation disease, and is widely distributed in pig populations worldwide. Therefore, developing specific diagnostic assays will be important for controlling this emerging pathogen. In this study, we developed a novel droplet digital PCR (ddPCR) assay targeting the PCV3 cap gene to improve the sensitivity of PCV3 detection. The established assay is highly specific to PCV3, and does not cross react with other important swine pathogens. The assay's detection limit was 1.68 ±â€¯0.29 copies of PCV3 DNA per reaction (n = 8), an approximately 10-fold greater sensitivity than that of our previously developed quantitative real-time PCR (qPCR) assay for the same virus. The ddPCR assay results were highly reproducible, with intra- and inter-assay coefficient of variation values of <9.0%. Of the 239 archived pig tissue and serum samples, 42 tested positive for PCV3 by the ddPCR assay. Among the 42 positive samples, 31 tested positive by the qPCR assay. Notably, PCV3 was detected in the serum samples collected from commercially imported healthy boars from the US, France and the UK during 2011-2017. The overall agreement between the two assays was 95.39% (228/239). Furthermore, the linear regression analysis showed that the ddPCR and the qPCR results were significantly correlated with an R2 value of 0.9945. Collectively, these results indicate that the ddPCR assay is a robust diagnostic tool for sensitive detection of PCV3, even in samples with low viral loads.

Development of a novel reverse transcription droplet digital PCR assay for the sensitive detection of Senecavirus A.

In pigs, Senecavirus A (SVA) causes a vesicular disease that is clinically indistinguishable from foot-and-mouth disease, vesicular stomatitis and swine vesicular disease. Sensitive and specific detection of SVA is critical for controlling this emerging disease. In this study, a novel reverse transcription droplet digital PCR (RT-ddPCR) assay, targeting the conserved viral polymerase 3D gene, was established for the detection of SVA. This assay exhibited good linearity, repeatability and reproducibility, and maintained linearity at extremely low concentrations of SVA nucleic acid templates. The detection limit of RT-ddPCR was 1.53 ± 0.22 copies of SVA RNA per reaction (n = 8), and the assay showed approximately 10-fold greater sensitivity than a reverse transcription real-time PCR (RT-rPCR) assay. Moreover, specificity analysis showed that the RT-ddPCR for SVA had no cross-reactivity with other important swine pathogens. In clinical diagnosis of 134 pig serum and tissue samples, 26 and 21 samples were identified as positive by RT-ddPCR and RT-rPCR, respectively. The overall agreement between the two assays was 96.27% (129/134). Further linear regression analysis showed a significant correlation between the RT-ddPCR and RT-rPCR assays with an R2 value of 0.9761. Our results indicate that the RT-ddPCR assay is a robust diagnostic tool for the sensitive detection of SVA, even in samples with a low viral load.