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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Hemagglutinin (HA) cleavage is critical for virulence of influenza viruses. The amino acid residue at the P6 position of the HA cleavage site (HACS) has been shown to be most variable and to have a direct correlation with the cleavage efficiency and pathogenicity of H5N1 avian influenza viruses (AIVs) in mammals. Among these amino acid variants, serine has been associated with the highest virulence in mammals, and its detection may serve as an indicator for H5N1 AIVs with high pathogenicity and potential public risk. We developed a rapid detection method based on reverse-transcription (RT)-PCR and pyrosequencing to detect a mutation at the HACS that is associated with increased pathogenicity of H5N1 AIVs in mammals. Herein, we provide a specific, sensitive, and reliable method for rapid detection of one of the virulence determinants associated with increased pathogenicity of H5N1 AIVs in mammals. [ABSTRACT FROM AUTHOR]

The development history, etiology, epidemiology, clinical symptoms, pathological changes and diagnosis technology of porcine circovirus 3 were summarized, and countermeasures for prevention and control of the disease were put forward based on the prevalence status in China. [ABSTRACT FROM AUTHOR]

NF90 is a novel host antiviral factor that regulates PKR activation and stress granule formation in influenza A virus ( IAV)-infected cells, but the precise mechanisms by which it operates remain unclear. We identified NF90 as a novel interacting protein of IAV nonstructural protein 1 ( NS1). The interaction was dependent on the RNA-binding properties of NS1. NS1 associated with NF90 and PKR simultaneously; however, the interaction between NF90 and PKR was restricted by NS1. Knockdown of NF90 promoted inhibition of PKR phosphorylation induced by NS1, while coexpression of NF90 impeded reduction of PKR phosphorylation and stress granule formation triggered by NS1. In summary, NF90 exerts its antiviral activity by antagonizing the inhibitory role of NS1 on PKR phosphorylation. [ABSTRACT FROM AUTHOR]

To control the spread of tick-borne diseases, there is an urgent need to develop a reliable technique that can distinguish different species of ticks. DNA barcoding has been proved to be a powerful tool to identify species of arthropods, but this technique has not yet been developed for identifying ticks. Here, we screened and analyzed 1082 sequences of ticks from BOLD system and GenBank, consisting of 647 16S, 325 COI, and 110 18S. These sequences are reported in previous studies and considered to be correctly identified at the species level. Through the analyses of genetic divergences and neighbor-joining (NJ) phylogenetic relationships between the species of ticks, our results show that COI and 16S are reliable in discriminating species of ticks and the 18S could discriminate ticks at the genera level. New universal primers for 16S, 18S, and COI of ticks were designed and a DNA barcoding system for the Ixodida was developed. To assess the performance of this system, 57 specimens of ticks were collected within China. Our results show that DNA barcoding system could correctly identify the species of specimens in adult and subadult stages. This system would assist non-taxonomists to conveniently identify the species of Ixodida based on DNA sequences rather than morphological traits. However, there are still serious deficiencies in the information of 16S and COI of some species of ticks, and additional research is needed to resolve this problem. [ABSTRACT FROM AUTHOR]

Background: African swine fever virus (ASFV) is the etiologic agent of African swine fever (ASF), a disease of highly contagious and significant threat to pork production. At present, the sensitive detection methods are the keys to the disease control. Objective: Full-length p72 is produced by a eukaryotic system, and its monoclonal antibody (mAb) 34C10 is subsequently recovered. A blocking ELISA kit for detection of ASFV antibody is developed based on p72 trimers and 34C10. Methods: Full-length p72 is expressed and is used as an immunogen to prepare a panel of monoclonal antibodies. The mAb 34C10 is verified by immunofluorescence and tested by ELISAs with positive serums. The constant affinity of 34C10 is then confirmed. A blocking ELISA kit is further developed and is compared with two commercial kits. Results: The mAb 34C10 is specifically bound to p72 protein, and it exhibits a blocking effect to positive serum. The immunofluorescence assay experiment shows that 34C10 could bind to p72 expressed by baculoviruses, and the binding affinity of 34C10 is found to be as high as 1.85x1011 L/mol. The blocking ELISA kit shows high coincidence with a commercial ELISA kit. The sensitivity between these two kits is 97.6% (95%, CI: 90.65-99.58), and the specificity between them is 100% (95%, CI: 98.34-100). Conclusions: The blocking ELISA developed in this study may have great potential for diagnosis of ASF. The structure of the antigen p72 is found to be a key factor for the performance of the kit. Highlights: For the first time, the eukaryotic expressed full-length p72 protein is used to recover the monoclonal antibody, and it is coated as antigen during the development of the blocking ELISA kit. This study sheds new light on the development of the blocking ELISA kits, especially for the development of a diagnostic kit for the contagious virus with biosafety problems. [ABSTRACT FROM AUTHOR]

Abstract 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 ± 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. Highlights • A novel droplet digital PCR (ddPCR) targeting the cap gene was developed to detect PCV3. • The assay displayed good sensitivity, specificity, within experimental repeatability and across experimental reproducibility. • The assay is a robust diagnostic tool for sensitive detection and accurate quantitation of PCV3 DNA in clinical samples. [ABSTRACT FROM AUTHOR]

Molecular characterization of transgenic animals is crucial for the phenotype analysis, unintended effects prediction, commercial and economic requirements. In this study, a comprehensive molecular characterization of a transgenic pig, which expressed hCD46 gene, was verified using next-generation sequencing (NGS). One complete and one incomplete sequence of pBSCD46-neo and multiple backbone fragments were inserted into the host genome. The whole insertion sequence was 28,243 bp, and 12 bp sequence on the genome was deleted during the transgene. The cost of NGS is higher than other molecular methods, in order to reduce the cost and to make the sequencing strategy more economically effective, we carried out a computer simulation to identify the smallest amount of data that could meet the requirements of the molecular characterization analysis. 15 × depth of coverage was shown to be sufficient with good accuracy, while 3 × might be effective, 20 × was recommended. The obtained molecular characterization information would provide a stronger foundation for the safety evaluation of the GM animals. Meanwhile, the gene integrity of the porcine pathogen PERV was analyzed using the same procedure. No complete sequence of the PERV subtype was observed in the porcine genome, which was the same as the previous research. In consequence, NGS combined bioinformatics analysis is a reliable and accurate approach for the molecular characterization of GMOs, even for the safety evaluation. [ABSTRACT FROM AUTHOR]

The number of species and planting areas of genetically modified organisms (GMOs) has been rapidly developed during the past ten years. For the purpose of GMO inspection, quarantine and manipulation, we have now devised a high-throughput Bt-based GMOs screening method based on the liquid bead array. This novel method is based on the direct competitive recognition between biotinylated antibodies and beads-coupled antigens, searching for Bt presence in samples if it contains Bt Cry1 Aa, Bt Cry1 Ab, Bt Cry1 Ac, Bt Cry1 Ah, Bt Cry1 B, Bt Cry1 C, Bt Cry1 F, Bt Cry2 A, Bt Cry3 or Bt Cry9 C. Our method has a wide GMO species coverage so that more than 90% of the whole commercialized GMO species can be identified throughout the world. Under our optimization, specificity, sensitivity, repeatability and availability validation, the method shows a high specificity and 10–50 ng/mL sensitivity of quantification. We then assessed more than 1800 samples in the field and food market to prove capacity of our method in performing a high throughput screening work for GMO manipulation. Our method offers an applicant platform for further inspection and research on GMO plants. [ABSTRACT FROM AUTHOR]

Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that predominantly infects livestock such as cattle, sheep, and goats. Its nucleocapsid (N) protein is an ideal target antigen for SBV diagnosis. In this study, a stable BHK-21 cell line, BHK-21-EGFP-SBV-N, constitutively expressing the SBV N protein was obtained using a lentivector-mediated gene transfer system combined with puromycin selection. To facilitate the purification of recombinant SBV N protein, the coding sequence for a hexa-histidine tag was introduced into the C-terminus of the SBV N gene during construction of the recombinant lentivirus vector pLV-EGFP-SBV-N. The BHK-21-EGFP-SBV-N cell line was demonstrated to spontaneously emit strong enhanced green fluorescent protein (EGFP) signals that exhibited a discrete punctate distribution throughout the cytoplasm. SBV N mRNA and protein expression in this cell line were detected by real-time RT-PCR and western blot, respectively. The expressed recombinant SBV N protein carried an N-terminal EGFP tag, and was successfully purified using Ni–NTA agarose by means of its C-terminal His tag. The purified SBV N protein could be recognized by SBV antisera and an anti-SBV monoclonal antibody (mAb) 2C8 in an indirect enzyme-linked immunosorbent assay and western blot analyses. Indirect immunofluorescence assays further demonstrated that the stable cell line reacts with SBV antisera and mAb 2C8. These results suggest that the generated cell line has the potential to be used in the serological diagnosis of SBV. [ABSTRACT FROM AUTHOR]

Highlights: [•] Two soluble recombinant SBV N proteins were generated. [•] MAb clone 2C8 was successfully prepared using His-SBV-N as an immunogen. [•] MBP-SBV-N was used as a coating antigen to screen mAb-secreting hybridomas in ELISA assays. [•] The epitope recognized by 2C8 is located at amino acids 51–76 of the SBV N protein. [Copyright &y& Elsevier]

Abstract: Peste des petits ruminants virus (PPRV) is an important pathogen that seriously influences the productivity of small ruminants worldwide. Although PPRV is known to induce apoptosis in infected cells, the interaction between PPRV and permissive cells requires further elucidation. Here, we provide the first evidence that PPRV infection triggered autophagy in Vero cells based on the appearance of abundant double- and single-membrane vesicles, the accumulation of LC3 fluorescent puncta, the enhancement of LC3-I/-II conversion, and autophagic flux. We further demonstrated that induction of autophagy with rapamycin significantly increased PPRV progeny yield and nucleocapsid (N) protein expression, while inhibition of autophagy with siRNA targeting ATG7 resulted in diametrically opposite results. Our data indicate that PPRV exploits the autophagy machinery to facilitate its own replication in host cells, thus the production efficiency of live attenuated PPRV vaccines may be improved by targeting the autophagic pathway. [Copyright &y& Elsevier]

Abstract: Major caspid protein (MCP) is the major structural component of virus particles and revealed to be very responsible for classification of new tentative iridovirus isolates. In this paper, the complete sequences of MCP gene was firstly identified and characterized from soft-shelled turtle iridovirus (STIV). The MCP, classified as a late transcript by drug inhibition, encodes a protein of 463 aa with a predicted molecular weight of 50kDa. Indirect immunofluorescence (IIF) and virus neutralization assay were developed to determine the sensitivity and virus neutralizing activity of MCP-specific antiserum. Furthermore, the MCP temporal expression pattern during STIV infection in vitro was characterized by Western blot and RT-PCR assays. The results suggest that STIV could be classified as a member of genus Ranavirus in family Iridoviridae and has cell-type-specific programs of viral gene expression. [Copyright &y& Elsevier]

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© AOAC INTERNATIONAL 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© AOAC INTERNATIONAL 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: The zinc finger BED-type containing six knockout (ZBED6-KO) pigs were created to improve economic traits by increasing the expression of insulin-like growth factor 2. They were generated by CRISPR/CRISPR-associated protein 9 (Cas9) technology and a single-base deletion of ZBED6 was found. An efficient and rapid method was needed to detect this type of pig., Objective: This study aimed to develop a high-resolution melting (HRM) method to detect ZBED6-KO pigs., Methods: An unlabeled probe and two primers were designed to develop the HRM method. The limit of detection, specificity, and accuracy of the established method were tested by the constructed plasmid and DNA extracts of tissue specimens., Results: The limit of detection by the established method was 102 copies/µL. The HRM method with an unlabeled probe showed good specificity and high accuracy., Conclusions: The established HRM analysis with an unlabeled probe showed it to be a highly effective, rapid, and reliable method to distinguish ZBED6-KO pigs from wild-type pigs., Highlights: It is the first time that HRM analysis with an unlabeled probe has been used in the detection of genome editing pigs by the CRISPR/Cas9 technology., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.

Metabolites of serum and milk from genetically modified (GM) cows and contrast check (CK) cows were comparatively investigated. Serum and milk were collected from genetically modified (GM) cows and contrast check (CK) cows, and then, they were analyzed using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) and gas chromatography-mass spectrometry (GC-MS). Although the level of some blood biochemical indexes for GM cows was shifted up or down, they were generally in normal physiological condition. Serum samples from lactoferrin GM cows exhibited reduced levels of amino acids and elevated levels of indoleacetate, α-keto acids, long-chain fatty acids, etc. GM milk possessed elevated levels of pentose and amino sugar metabolites, including arabitol, xylulose, glucuronate, and N-acetylgalactosamine. Interestingly, some essential nutrients, such as certain unsaturated fatty acids (e.g., eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA)), and some necessary rare sugars were significantly upregulated. Compared to the CK group, a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was conducted based on the increased or decreased metabolites identified in the serum and milk samples of the GM group. The results showed that the GM cows were in healthy condition and their milk has improved benefits for customers. The milk from genetically modified cows was found to be a promising milk source for producing recombinant human lactoferrin (rhLF) for human beings.

The aim of this study was to investigate the occurrence and genomic characteristics of extended-spectrum-β-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 bla CTX-M-55 and bla CTX-M-14 genes, followed by bla CTX-M-15 and bla CTX-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 bla CTX-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 bla CTX-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-β-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., (Copyright © 2019 Feng et al.)

This study aimed to establish an event-specific multiplex PCR system using microsatellite markers and fluorescently labeled primers to detect six different genetically modified (GM) animal lines, including human lactoferrin GM cattle, human lysozyme GM cattle, human α-lactalbumin GM cattle, myostatin knockout pigs, phytase GM pigs, and ω-3 fatty acid desaturase gene GM pigs. Four different microsatellite loci for species identification, along with six GM animal-specific fragments, were selected as targets for primer design. The capillary gel electrophoresis results of multiplex PCR showed that the target fragments were amplified successfully. This high-throughput multiplex PCR detection system can be applied for the inspection and quarantine of GM animals.

Ticks host a wide range of zoonotic pathogens and are a significant source of diseases that affect humans and livestock. However, little is known about the pathogens associated with bat ticks. We have collected ectoparasites from bat carcasses over a seven year period. Nucleic acids (DNA and RNA) were extracted from 296 ticks removed from bats and the species designation was confirmed in all ticks as Argas (Carios) vespertilionis. A subset of these samples (n = 120) were tested for the presence of zoonotic pathogens by molecular methods. Babesia species, Rickettsia spp., within the spotted fever group (SFG), and Ehrlichia spp. were detected in ticks removed from 26 bats submitted from 14 counties across England. The prevalence of Rickettsia spp. was found to be highest in Pipistrellus pipistrellus from southern England. This study suggests that the tick species that host B. venatorum may include the genus Argas in addition to the genus Ixodes. As A. vespertilionis has been reported to feed on humans, detection of B. venatorum and SFG Rickettsia spp. could present a risk of disease transmission in England. No evidence for the presence of flaviviruses or Issyk-Kul virus (nairovirus) was found in these tick samples.

The present study aimed to investigate the serum metabolomic profiles in genetically modified cows carrying and expressing human lactalbumin α (LALBA) and non‑LALBA cows, and identify altered metabolic characteristics following the genetic modification. Serum biochemistry indexes were measured according to protocols recommended by International Federation of Clinical Chemistry. The metabolomic profiles were determined using the serum samples collected from LALBA (n=6) and non‑LALBA cows (n=6). Welch's two‑sample t‑test was used to identify the metabolites that significantly differed between the LALBA and non‑LALBA groups (fold‑change ≠ 1 and P<0.05), followed by random forest and pathway analysis. The serum biochemistry indexes of LALBA and non‑LALBA cows were within the normal ranges of healthy cows. A total of 273 metabolites were detected, among which 79 metabolites, including 46 increased and 33 decreased metabolites, differed significantly between the LALBA and non‑LALBA groups. Random forest analysis identified 30 potential key metabolites, including 14 elevated and 16 reduced metabolites. These metabolites were primarily involved in pathways concerning the metabolism of leucine, isoleucine, valine, tryptophan and lipids, such as myristate and eicosapentaenoate. However, the serum in LALBA cow had unique metabolomic signature compared with non‑LALBA cows. The accumulation of polyunsaturated fatty acids and amino acids, and the reduced levels of long chain saturated fatty acids in serum may benefit LALBA cows. However, further investigations are required to validate these benefits and the corresponding mechanisms.

Background: The 5' region of cytochrome oxidase I (COI) is the standard marker for DNA barcoding. However, COI has proved to be of limited use in identifying some species, and for some taxa, the coding sequence is not efficiently amplified by PCR. These deficiencies lead to uncertainty as to whether COI is the most suitable barcoding fragment for species identification of ticks., Methods: In this study, we directly compared the relative effectiveness of COI, 16S ribosomal DNA (rDNA), nuclear ribosomal internal transcribed spacer 2 (ITS2) and 12S rDNA for tick species identification. A total of 307 sequences from 84 specimens representing eight tick species were acquired by PCR. Besides the 1,834 published sequences of 189 tick species from GenBank and the Barcode of Life Database, 430 unpublished sequences representing 59 tick species were also successfully screened by Bayesian analyses. Thereafter, the performance of the four DNA markers to identify tick species was evaluated by identification success rates given by these markers using nearest neighbour (NN), BLASTn, liberal tree-based or liberal tree-based (+threshold) methods., Results: Genetic divergence analyses showed that the intra-specific divergence of each marker was much lower than the inter-specific divergence. Our results indicated that the rates of correct sequence identification for all four markers (COI, 16S rDNA, ITS2, 12S rDNA) were very high (> 96%) when using the NN methodology. We also found that COI was not significantly better than the other markers in terms of its rate of correct sequence identification. Overall, BLASTn and NN methods produced higher rates of correct species identification than that produced by the liberal tree-based methods (+threshold or otherwise)., Conclusions: As the standard DNA barcode, COI should be the first choice for tick species identification, while 16S rDNA, ITS2, and 12S rDNA could be used when COI does not produce reliable results. Besides, NN and BLASTn are efficient methods for species identification of ticks.

A ranavirus (RV) was isolated from Chinese giant salamanders (Andrias davidianus) in China in 2010 and provisionally designated Andrias davidianus ranavirus (ADRV). The complete genome sequence is 106,719 nucleotides long. Genomic sequence and phylogenetic analyses showed that ADRV has a high degree of conservation with other RVs.