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Porcine reproductive and respiratory syndrome virus (PRRSV) and transmissible gastroenteritis virus (TGEV) are two highly infectious and lethal viruses causing major economic losses to pig production. Here, we report generation of double-gene-knockout (DKO) pigs harboring edited knockout alleles for known receptor proteins CD163 and pAPN and show that DKO pigs are completely resistant to genotype 2 PRRSV and TGEV. We found no differences in meat-production or reproductive-performance traits between wild-type and DKO pigs, but detected increased iron in DKO muscle. Additional infection challenge experiments showed that DKO pigs exhibited decreased susceptibility to porcine deltacoronavirus (PDCoV), thus offering unprecedented in vivo evidence of pAPN as one of PDCoV receptors. Beyond showing that multiple gene edits can be combined in a livestock animal to achieve simultaneous resistance to two major viruses, our study introduces a valuable model for investigating infection mechanisms of porcine pathogenic viruses that exploit pAPN or CD163 for entry.

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Fifty-five samples (15.62%) collected from dogs and cats were identified as canine parvovirus (CPV) infection in Beijing during 2010–2013. The nucleotide identities and aa similarities were 98.2–100% and 97.7–100%, respectively, when compared with the reference isolates. Also, several synonymous and non-synonymous mutations were also recorded for the first time. New CPV-2a was dominant, accounting for 90.90% of the samples. Two of the 16 samples collected from cats were identified as new CPV-2a (12.5%), showing nucleotide identities of 100% with those from dogs. Twelve samples (15.78%) collected from completely immunized dogs were found to be new CPV-2a, which means CPV-2 vaccines may not provide sufficient protection for the epidemic strains.

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Bovine tuberculosis (bTB) is a worldwide zoonosis caused mainly by Mycobacterium bovis. The traditional diagnostic method used often is the tuberculin skin test, which uses bovine purified protein derivatives (PPD-B). However, it is difficult to maintain uniformity of PPD-B from batch to batch, and it shares common antigens with nonpathogenic environmental mycobacteria. To overcome these problems, M. bovis-specific antigens that showed good T cell stimulation, such as CFP-10, ESAT-6, Rv3615c, etc., have been used in the skin test, but there have been no large-scale clinical studies on these antigens. In this study, two combinations (CFP-10/ESAT-6/TB10.4 protein cocktail and CFP-10/ESAT-6/Rv3872/MPT63 protein cocktail) were developed and used as stimuli in the skin test. Cattle were double-blind tested to assess the efficiency of the protein cocktail-based skin tests. The results showed that the CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test can differentiate TB-infected cattle from Mycobacterium avium-infected ones and that it shows a high degree of agreement with the traditional tuberculin skin test ( ‫؍‬ 0.8536) and gamma interferon (IFN-␥) release assay ( ‫؍‬ 0.8154). Compared to the tuberculin skin test, the relative sensitivity and relative specificity of the CFP-10/ESAT-6/TB10.4-based skin test were 87% and 97%, respectively., The relative sensitivity and relative specificity of the CFP-10/ESAT-6/TB10.4-based skin test were 93% and 92%, respectively, on comparison with the IFN-␥ release assay. The correlation between the increases in skin thickness observed after the inoculation of stimuli was high ( (1, 2). M. bovis mainly infects cattle, causing bovine tuberculosis (bTB), and occasionally affects other species of mammals (3). M. bovis can also infect humans through the inhalation of aerosols or the ingestion of unpasteurized dairy products (4, 5). The eradication of bovine tuberculosis is based on reliable diagnostic methods.The traditional diagnostic method of identifying bTB is the tuberculin skin test, which is based on the detection of delayedtype hypersensitivity (DTH) induced by bovine purified protein derivative (PPD-B). The tuberculin skin test has been used worldwide and is economical and easily applied. However, PPD-B is a poorly defined mixture of proteins, lipids, and carbohydrates obtained from a virulent M. bovis culture, the uniformity of which is difficult to maintain from batch to batch (6). Moreover, the use of virulent M. bovis during the production of PPD-B may potentially harm workers. Importantly, shared antigenic components in PPD-B and nonpathogenic environmental mycobacteria can reduce the specificity of the tuberculin skin test (7,8). Although the gamma interferon (IFN-␥) release assay (IGRA) showed higher specificity than the single intradermal skin test, the high price of this kit limits its use in developing countries such as China (9, 10). To overcome the drawbacks of the traditional diagnostic methods, scientists have focused on screening new M. bovis-specific antige...

Bovine tuberculosis (bTB) is a chronic disease of cattle caused by Mycobacterium bovis. During early-stage infection, M. bovis-infected cattle shed mycobacteria through nasal secretions, which can be detected via nested-polymerase chain reaction (PCR) experiments. Little research has focused on immune responses in nested PCR-positive (bTB PCR-P) or nested PCR-negative (bTB PCR-N) M. bovis-infected cattle. Here, we investigated the transcriptomes of peripheral blood mononuclear cells (PBMCs), with or without stimulation by purified protein derivative of bovine tuberculin (PPD-B), among bTB PCR-P, bTB PCR-N, and healthy cattle using RNA-Seq. We also explored the potential value of PBMC transcripts as novel biomarkers for diagnosing bTB. Numerous differentially expressed genes were identified following pair-wise comparison of different groups, with or without PPD-B stimulation (adjusted p < 0.05). Compared with healthy cattle, bTB PCR-P, and bTB PCR-N cattle shared 5 significantly dysregulated biological pathways, including Cytokine-cytokine receptor interaction, NF-kappa B signaling pathway, Hematopoietic cell lineage, Osteoclast differentiation and HTLV-I infection. Notably, dysregulated biological pathways of bTB PCR-P and bTB PCR-N cattle were associated with cell death and phagocytosis, respectively. Lymphotoxin alpha and interleukin-8 could potentially differentiate M. bovis-infected and healthy cattle upon stimulation with PPD-B, with area-under-the-curve (AUC) values of 0.9991 and 0.9343, respectively. B cell lymphoma 2 and chitinase 3-like 1 might enable differentiation between bTB PCR-P and bTB PCR-N upon stimulation with PPD-B, with AUC values of 0.9100 and 0.8893, respectively. Thus, the PBMC transcriptome revealed the immune responses in M. bovis-infected cattle (bTB PCR-P and bTB PCR-N) and may provide a novel sight in bTB diagnosis.