Generation of a Commercial-Scale Founder Population of Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs Using CRISPR-Cas.

Disease resistance genes in livestock provide health benefits to animals and opportunities for farmers to meet the growing demand for affordable, high-quality protein. Previously, researchers used gene editing to modify the porcine CD163 gene and demonstrated resistance to a harmful virus that causes porcine reproductive and respiratory syndrome (PRRS). To maximize potential benefits, this disease resistance trait needs to be present in commercially relevant breeding populations for multiplication and distribution of pigs. Toward this goal, a first-of-its-kind, scaled gene editing program was established to introduce a single modified CD163 allele into four genetically diverse, elite porcine lines. This effort produced healthy pigs that resisted PRRS virus infection as determined by macrophage and animal challenges. This founder population will be used for additional disease and trait testing, multiplication, and commercial distribution upon regulatory approval. Applying CRISPR-Cas to eliminate a viral disease represents a major step toward improving animal health.

Genetically modified pigs lacking CD163 PSTII-domain-coding exon 13 are completely resistant to PRRSV infection.

CD163 expressed on cell surface of porcine alveolar macrophages (PAMs) serves as a cellular entry receptor for porcine reproductive and respiratory syndrome virus (PRRSV). The extracellular portion of CD163 contains nine scavenger receptor cysteine-rich (SRCR) and two proline-serine-threonine (PST) domains. Genomic editing of pigs to remove the entire CD163 or just the SRCR5 domain confers resistance to infection with both PRRSV-1 and PRRSV-2 viruses. By performing a mutational analysis of CD163, previous in vitro infection experiments showed resistance to PRRSV infection following deletion of exon 13 which encodes the first 12 amino acids of the 16 amino acid PSTII domain. These findings predicted that removal of exon 13 can be used as a strategy to produce gene-edited pigs fully resistant to PRRSV infection. In this study, to determine whether the deletion of exon 13 is sufficient to confer resistance of pigs to PRRSV infection, we produced pigs possessing a defined CD163 exon 13 deletion (ΔExon13 pigs) and evaluated their susceptibility to viral infection. Wild type (WT) and CD163 modified pigs, placed in the same room, were infected with PRRSV-2. The modified pigs remained PCR and serologically negative for PRRSV throughout the study; whereas the WT pigs supported PRRSV infection and showed PRRSV related pathology. Importantly, our data also suggested that removal of exon 13 did not affect the main physiological function associated with CD163 in vivo. These results demonstrate that a modification of CD163 through a precise deletion of exon 13 provides a strategy for protection against PRRSV infection.

Amoxicillin and amoxicillin-clavulanate resistance in urinary Escherichia coli antibiograms of cats and dogs from the Midwestern United States.

BACKGROUND: Antibiograms are stewardship tools that provide antimicrobial resistance data for regional bacterial isolates to guide treatment of infections. OBJECTIVES: To develop regional antibiograms of urinary Escherichia coli isolates from cats and dogs. ANIMALS: Escherichia coli isolates cultured from feline (N = 143) and canine (640) urine from 2013 to 2017, from Kansas State University (N = 335) and private practice (N = 448) patients in the Midwestern United States. METHODS: Retrospective review of urine culture and susceptibility results. Antibiograms were created for 10 commonly used antimicrobial agents using Clinical and Laboratory Standards Institutes guidelines. RESULTS: No isolates from cats were susceptible to amoxicillin-clavulanate (susceptibility [S] ≤ 0.25/0.12) or amoxicillin (S ≤ 0.25); isolates from dogs had low susceptibility to amoxicillin 53% (S ≤ 8). Conversely, isolates from dogs had high susceptibility to amoxicillin-clavulanate 92% (S ≤ 8/4), despite equal 90th percentile minimum inhibitory concentrations (8 µg/mL) for feline and canine populations. Resistance to other antimicrobials was uncommon (≤7% for isolates from cats, ≤14% for isolates from dogs). CONCLUSIONS AND CLINICAL IMPORTANCE: The disparity in susceptibility for amoxicillin and amoxicillin-clavulanate between isolates from cats and dogs likely reflects higher breakpoints for urinary tract infections (UTIs) in dogs. Urine concentration data for these antimicrobials in cats might support a UTI-specific breakpoint for cats and increase potential therapeutic options for managing UTIs in cats with first-line antimicrobials. Decreased susceptibility among isolates from dogs to amoxicillin (53%) compared to amoxicillin-clavulanate (92%) might support amoxicillin-clavulanate as a better empirical choice for UTIs in dogs in this geographical region.