Effect of pooled tracheal sample testing on the probability of Mycoplasma hyopneumoniae detection.

Tracheal pooling for Mycoplasma hyopneumoniae (M. hyopneumoniae) DNA detection allows for decreased diagnostic cost, one of the main constraints in surveillance programs. The objectives of this study were to estimate the sensitivity of pooled-sample testing for the detection of M. hyopneumoniae in tracheal samples and to develop probability of M. hyopneumoniae detection estimates for tracheal samples pooled by 3, 5, and 10. A total of 48 M. hyopneumoniae PCR-positive field samples were pooled 3-, 5-, and 10-times using field M. hyopneumoniae DNA-negative samples and tested in triplicate. The sensitivity was estimated at 0.96 (95% credible interval [Cred. Int.]: 0.93, 0.98) for pools of 3, 0.95 (95% Cred. Int: 0.92, 0.98) for pools of 5, and 0.93 (95% Cred. Int.: 0.89, 0.96) for pools of 10. All pool sizes resulted in PCR-positive if the individual tracheal sample Ct value was < 33. Additionally, there was no significant decrease in the probability of detecting at least one M. hyopneumoniae-infected pig given any pool size (3, 5, or 10) of tracheal swabs. Furthermore, this manuscript applies the probability of detection estimates to various real-life diagnostic testing scenarios. Combining increased total animals sampled with pooling can be a cost-effective tool to maximize the performance of M. hyopneumoniae surveillance programs.

Pigs lacking the SRCR5 domain of CD163 protein demonstrate heritable resistance to the PRRS virus and no changes in animal performance from birth to maturity.

Porcine reproductive and respiratory syndrome (PRRS) is one of the world's most persistent viral pig diseases, with a significant economic impact on the pig industry. PRRS affects pigs of all ages, causing late-term abortions and stillbirths in sows, respiratory disease in piglets, and increased susceptibility to secondary bacterial infection with a high mortality rate. PRRS disease is caused by a positive single-stranded RNA PRRS virus (PRRSV), which has a narrow host-cell tropism limited to monocyte-macrophage lineage cells. Several studies demonstrated that the removal of CD163 protein or, as a minimum, its scavenger receptor cysteine-rich domain 5 (SRCR5) precludes the viral genome release, conferring resistance to PRRSV in live animals. Today, very limited information exists about the impact of such edits on animal performance from birth to maturity in pigs. Using CRISPR-Cas9 with dual-guide RNAs and non-homologous end joining (NHEJ), first-generation (E0) pigs were produced with a deletion of exon 7 in the CD163 gene. The selected pigs were bred to produce the next three generations of pigs to establish multiple lines of pigs homozygous for the edited allele, thereby confirming that the CD163 gene with removed exon 7 was stable during multiple breeding cycles. The pigs were evaluated relative to non-edited pigs from birth to maturity, including any potential changes in meat composition and resistance to PRRSV. This study demonstrates that removing the SRCR5 domain from the CD163 protein confers resistance to PRRSV and, relative to unedited pigs, resulted in no detected differences in meat composition and no changes in the growth rate, health, and ability to farrow. Together, these results support the targeted use of gene editing in livestock animals to address significant diseases without adversely impacting the health and well-being of the animals or the food products derived from them.

Molecular characterization of Glaesserella parasuis strains circulating in North American swine production systems.

BACKGROUND: Glaesserella parasuis is the causative agent of Glässer's disease in pigs. Serotyping is the most common method used to type G. parasuis isolates. However, the high number of non-typables (NT) and low discriminatory power make serotyping problematic. In this study, 218 field clinical isolates and 15 G. parasuis reference strains were whole-genome sequenced (WGS). Multilocus sequence types (MLST), serotypes, core-genome phylogeny, antimicrobial resistance (AMR) genes, and putative virulence gene information was extracted. RESULTS: In silico WGS serotyping identified 11 of 15 serotypes. The most frequently detected serotypes were 7, 13, 4, and 2. MLST identified 72 sequence types (STs), of which 66 were novel. The most predominant ST was ST454. Core-genome phylogeny depicted 3 primary lineages (LI, LII, and LIII), with LIIIA sublineage isolates lacking all vtaA genes, based on the structure of the phylogenetic tree and the number of virulence genes. At least one group 1 vtaA virulence genes were observed in most isolates (97.2%), except for serotype 8 (ST299 and ST406), 15 (ST408 and ST552) and NT (ST448). A few group 1 vtaA genes were significantly associated with certain serotypes or STs. The putative virulence gene lsgB, was detected in 8.3% of the isolates which were predominantly of serotype 5/12. While most isolates carried the bcr, ksgA, and bacA genes, the following antimicrobial resistant genes were detected in lower frequency;  blaZ (6.9%), tetM (3.7%), spc (3.7%), tetB (2.8%), bla-ROB-1 (1.8%), ermA (1.8%), strA (1.4%), qnrB (0.5%), and aph3''Ia (0.5%).   CONCLUSION: This study showed the use of WGS to type G. parasuis isolates and can be considered an alternative to the more labor-intensive and traditional serotyping and standard MLST. Core-genome phylogeny provided the best strain discrimination. These findings will lead to a better understanding of the molecular epidemiology and virulence in G. parasuis that can be applied to the future development of diagnostic tools, autogenous vaccines, evaluation of antibiotic use, prevention, and disease control.

Case-control study on the association of porcine circovirus type 2 and other swine viral pathogens with postweaning multisystemic wasting syndrome.

A field-based case-control study was conducted to assess the strength of association of porcine circovirus type 2 (PCV2) and some major swine viruses with postweaning multisystemic wasting syndrome (PMWS). Cases were defined as individual pigs with a clinical history of progressive weight loss and histopathological lesions characteristic of PMWS. Controls were pigs without clinical signs and histopathological lesions typical of PMWS. A total of 31 cases and 56 controls was identified from diagnostic submissions. Serum and various tissues were collected from all animals and assayed for PCV, porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus, porcine enterovirus types 1-3, swine influenza virus, porcine respiratory coronavirus, transmissible gastroenteritis virus, porcine endogenous retrovirus, porcine lymphotropic herpesvirus type 1, and bovine viral diarrhea virus. The proportion of case and control pigs positive for each virus was determined and statistically compared for determining the strength of the association that each virus had with PMWS individually or in combinations. Porcine circovirus type 2 had the strongest association (OR = 9.3, P = 0.006) with PMWS among the viruses tested for. Risk for PWMS was much higher (OR = 31.2, P = 0.0009) if the animal was concurrently infected with PCV2 and PRRSV, suggesting that development of PMWS may be enhanced by cofactor(s). Because PCV2 was also found in 62.5% of the controls, PCV2 from 5 cases and 4 controls were selected and genetically compared. No significant genetic difference was observed between PCV2 from PMWS and control pigs.

Modified indirect porcine circovirus (PCV) type 2-based and recombinant capsid protein (ORF2)-based enzyme-linked immunosorbent assays for detection of antibodies to PCV.

Postweaning multisystemic wasting syndrome of swine associated with porcine circovirus (PCV) is a recently reported and economically important disease. Simple and reliable diagnostic methods are needed for detecting antibodies to PCV type 2 (PCV2) for monitoring of PCV infection. Here, we report the development of two modified indirect enzyme-linked immunosorbent assays (ELISAs): a PCV2 ELISA based on cell-culture-propagated PCV2 and an ORF2 ELISA based on recombinant major capsid protein. PCV2 and ORF2 ELISA detected antibodies to PCV2 and the capsid protein, respectively, in sera from pigs experimentally infected with PCV2 as early as 14 and 21 days postinoculation (dpi). The kinetics of the antibody response to PCV2 and the major capsid protein were similar. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs for both assays were less than 30%. To validate the assays, PCV2 and ORF2 ELISAs were performed with 783 serum samples of young and adult pigs collected from different herds in the Midwestern United States and compared with an indirect immunofluorescent assay (IIF). Six out of 60 samples collected from nursery and growing pigs in 1987 were positive by both ELISA and IIF. Compared with IIF, the diagnostic sensitivity, specificity, and accuracy of PCV2 and ORF2 ELISAs were similar (>90%). The tests showed no cross-reactivity with antibodies to porcine parvovirus and porcine reproductive and respiratory syndrome virus. There was good agreement between the two ELISAs and between the ELISAs and IIF. The availability of the two ELISAs should accelerate our understanding of the host immune response to PCV2 and facilitate the development of prevention and control strategies by elucidating the ecology of PCV2 within swine populations.

Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein.

Porcine circovirus 2 (PCV2), a single-stranded DNA virus associated with post-weaning multisystemic wasting syndrome of swine, has two potential open reading frames, ORF1 and ORF2, greater than 600 nucleotides in length. ORF1 is predicted to encode a replication-associated protein (Rep) essential for replication of viral DNA, while ORF2 contains a conserved basic amino acid sequence at the N terminus resembling that of the major structural protein of chicken anaemia virus. Thus far, the structural protein(s) of PCV2 have not been identified. In this study, a viral structural protein of 30 kDa was identified in purified PCV2 particles. ORF2 of PCV2 was cloned into a baculovirus expression vector and the gene product was expressed in insect cells. The expressed ORF2 gene product had a molecular mass of 30 kDa, similar to that detected in purified virus particles. The recombinant ORF2 protein self-assembled to form capsid-like particles when viewed by electron microscopy. Antibodies against the ORF2 protein were detected in samples of sera obtained from pigs as early as 3 weeks after experimental infection with PCV2. These results show that the major structural protein of PCV2 is encoded by ORF2 and has a molecular mass of 30 kDa.