Detection of Mycoplasma hyopneumoniae viability using a PCR-based assay.

Mycoplasma hyopneumoniae detection in clinical specimens is accomplished by PCR targeting bacterial DNA. However, the high stability of DNA and the lack of relationship between bacterial viability and DNA detection by PCR can lead to diagnostic interpretation issues. Bacterial messenger RNA is rapidly degraded after cell death, and consequently, assays targeting mRNA detection can be used for the exclusive detection of viable bacterial cells. Therefore, this study aimed at developing a PCR-based assay for the detection of M. hyopneumoniae mRNA and at validating its applicability to differentiate viable from inert bacteria. Development of the RNA-based PCR encompassed studies to determine its analytical sensitivity, specificity, and repeatability, as well as its diagnostic accuracy. Comparisons between DNA and mRNA detection for the same target gene were performed to evaluate the ability of the RNA-based PCR to detect exclusively viable M. hyopneumoniae after bacterial inactivation using various methods. The RNA-based PCR was also compared to the DNA-based PCR as a tool to monitor the growth of M. hyopneumoniae in vitro. Under the conditions of this study, the developed RNA-based PCR assay detected only viable or very recently inactivated M. hyopneumoniae, while the DNA-based PCR consistently detected cells irrespective of their viability status. Changes in growth activity over time were only observable via RNA-based PCR. This viability PCR assay could be directly applied to evaluate the clearance of M. hyopneumoniae or to determine the viability of the bacterium at late stages of eradication 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.

The effect of timing of Improvest administration on growth performance and carcass characteristics in gilts.

Improvest (IMP; Zoetis Inc., Parsippany, NJ) has been approved by the U.S. Food and Drug Administration for use in gilts. Improvest is administered twice: the first dose should be administered no earlier than 9 wk of age and the second dose (D2) at least 4 wk after the first dose. The aim of this study was to determine how the timing of IMP before harvest affects growth performance and carcass characteristics in gilts. A total of 1,632 gilts were allocated to four groups (12 pens/treatment; 34 gilts/pen): 1) a control group did not receive IMP; 2) T-early gilts received IMP on day 7 (day 0 = 10 wk postweaning), and D2 on day 40 (i.e., 35 d prior to first removal for harvest); 3) T-medium gilts received IMP on day 21 and D2 on day 56 (i.e., 19 d prior to first removal for harvest); 4) T-late gilts received IMP on day 35 and D2 on day 70 (i.e., 5 d before first removal for harvest). Pigs were selected for harvest by visual observation on days 75, 89, 103, and 117: 1) the heaviest 7 gilts/pen for each treatment on day 75; 2) the heaviest 10 gilts/pen of each treatment at day 89; 3) the heaviest 10 gilts/pen of each treatment on day 103; and 4) the remaining 7 gilts/pen on day 117. Weights and feed disappearance were recorded every 2 wk and during harvest dates to calculate average daily gain (ADG), average daily feed intake (ADFI), and feed efficiency (Gain:Feed; G:F). Generalized linear mixed models of SAS were used to analyze all variables. The increase in ADFI over Control gilts was observed 15 d post D2 and continued through 77 d post D2, with advantages in ADG occurring between 15 and 35 d post D2. Control and IMP treated gilts had similar G:F 15 to 33 d post D2. The overall ADG and ADFI from day 0 to market, final live weights, and hot carcass weights were significantly greater (P ≤ 0.05) in IMP gilts compared to Control. When G:F based on live weight was averaged across all groups (i.e., from day 0 to market), T-early had the lowest (P ≤ 0.05) G:F compared to Control, T-medium, and T-late gilts, which did not differ. Carcasses from IMP gilts had increased (P < 0.01) backfat, but similar (P = 0.5) Longissimus muscle depth, compared to Control. Within a cohort of similar aged gilts finishing during the summer, this study indicates that the trajectory of growth is enhanced within a similar window post D2 of IMP. Gilts treated with IMP had heavier carcasses with increased backfat and similar Longissimus muscle depth.

Vaccination decreases the risk of influenza A virus reassortment but not genetic variation in pigs.

Although vaccination is broadly used in North American swine breeding herds, managing swine influenza is challenging primarily due to the continuous evolution of influenza A virus (IAV) and the ability of the virus to transmit among vaccinated pigs. Studies that have simultaneously assessed the impact of vaccination on the emergence of IAV reassortment and genetic variation in pigs are limited. Here, we directly sequenced 28 bronchoalveolar lavage fluid (BALF) samples collected from vaccinated and unvaccinated pigs co-infected with H1N1 and H3N2 IAV strains, and characterized 202 individual viral plaques recovered from 13 BALF samples. We identified 54 reassortant viruses that were grouped in 17 single and 16 mixed genotypes. Notably, we found that prime-boost vaccinated pigs had less reassortant viruses than nonvaccinated pigs, likely due to a reduction in the number of days pigs were co-infected with both challenge viruses. However, direct sequencing from BALF samples revealed limited impact of vaccination on viral variant frequency, evolutionary rates, and nucleotide diversity in any IAV coding regions. Overall, our results highlight the value of IAV vaccination not only at limiting virus replication in pigs but also at protecting public health by restricting the generation of novel reassortants with zoonotic and/or pandemic potential.

Swine influenza A viruses cause severe illness among pigs and financial losses on pig farms worldwide. These viruses can also infect humans and have caused deadly human pandemics in the past. Influenza A viruses are dangerous because viruses can be transferred between humans, birds and pigs. These co-infections can allow the viruses to swap genetic material. Viral genetic exchanges can result in new virus strains that are more dangerous or that can infect other types of animals more easily. Farmers vaccinate their pigs to control the swine influenza A virus. The vaccines are regularly updated to match circulating virus strains. But the virus evolves rapidly to escape vaccine-induced immunity, and infections are common even in vaccinated pigs. Learning about how vaccination affects the evolution of influenza A viruses in pigs could help scientists prevent outbreaks on pig farms and avoid spillover pandemics in humans. Li et al. show that influenza A viruses are less likely to swap genetic material in vaccinated and boosted pigs than in unvaccinated animals. In the experiments, Li et al. collected swine influenza A samples from the lungs of pigs that had received different vaccination protocols. Next, Li et al. used next-generation sequencing to identify new mutations in the virus or genetic swaps among different strains. In pigs infected with both the H1N1 and H3N2 strains of influenza, the two viruses began trading genes within a week. But less genetic mixing occurred in vaccinated and boosted pigs because they spent less time infected with both viruses than in unvaccinated pigs. The vaccination status of the pig did not have much effect on how many new mutations occurred in the viruses. The experiments show that vaccinating and boosting pigs against influenza A viruses may protect against genetic swapping among influenza viruses. If future studies on pig farms confirm the results, the information gleaned from the study could help scientists improve farm vaccine protocols to further reduce influenza risks to animals and people.

Effect of antibiotic treatment on Mycoplasma hyopneumoniae detection and infectious potential.

Mycoplasma hyopneumoniae (M. hyopneumoniae) causes significant economic losses in the swine industry. Antibiotics with activity against Mycoplasma spp. are employed for disease mitigation and pathogen elimination. However, veterinarians are often challenged with the detection of M. hyopneumoniae by PCR after antibiotic treatment, thus raising the question whether the bacterium is still infectious. The objective of this study was to evaluate the effect of tulathromycin treatment on M. hyopneumoniae detection and infectious potential during the acute and chronic phases of infection. For each infection phase, one age-matched naïve gilt was placed in contact with one M. hyopneumoniae infected gilt that was either treated with tulathromycin, treated and vaccinated, or non-treated, for 14 days. Four replicates per treatment group were performed for each infection phase. A numerical reduction in relative bacterial load was observed in acutely treated gilts compared to non-treated gilts. The rate at which naïve gilts became infected with M. hyopneumoniae was numerically reduced when co-housed with treated, acutely infected gilts compared to those housed with non-treated, infected gilts. During the chronic infection phase, M. hyopneumoniae was detected by PCR in more than 50 % of treated infected gilts and persisted for up to three months post-treatment. Transmission was not detected in all treatment groups however, the possibility that the pathogen was infectious could not be completely ruled out. Further research focused on assessing M. hyopneumoniae detection and viability post-treatment is necessary to guide control and elimination efforts.

Water intake of pigs consuming tiamulin during the nursery phase.

Mass medication to manage population health can be achieved by providing therapeutics in the drinking water. Young nursery pigs are highly sensitive to the flavor and smell of water. Medications that reduce water palatability often lead to an interruption in water and feed intake. With the availability of several generic water-soluble antimicrobials for pigs, questions have arisen about their palatability compared with the original product. In this study, we compared the intake of water containing tiamulin hydrogen fumarate from two different manufacturers with the intake of unmedicated water. The hypothesis was that the intake of tiamulin-containing water would be similar to unmedicated water. Water intake was monitored upon entry into the nursery and just prior to leaving the nursery. Also, average daily gain (ADG) and feed efficiency (FE) were determined. A total of 300 pigs were individually weighed (4.2-10.9 kg; avg = 6.8 kg) for randomization to pen (n = 30 pens). The experiment had two time points: 1) early nursery (periods 1-3) and 2) late nursery (period 4). Pens were randomly assigned to a sequence (period 1-3) in a crossover experimental design containing three 10-d periods, with 5 d for the resetting of baseline where unmedicated water was provided followed by 5 d on tiamulin source addition [i.e., TriamuloxTM (Zoetis, Parsippany, NJ); Denagard (Elanco Animal Health, Greenfield, IN)] or unmedicated water. After period 3 was concluded, all pens were given unmedicated water (via nipple waterers) and the number of pigs per pen was reduced to six pigs to maintain adequate space per pig. Ten days prior to pigs leaving the nursery, a fourth period was performed. After a 5-d water baseline was achieved, pens were treated with either unmedicated water or Triamulox- or Denagard-containing water. Pigs had ad libitum access to water and feed. During the testing periods, daily water intake was measured by a cup water system in each pen. Feed intake was measured every 5 d. There was no effect of treatment on initial body weights or weights at the beginning or end of each period (P ≥ 0.51). Therefore, there was no effect of treatment on ADG (P ≥ 0.23). Water intake (P ≥ 0.16) and FE (P ≥ 0.35) were not affected by treatment. Water consumption was similar among all treatments in each of the four periods. There appears to be no aversion to water intake when tiamulin hydrogen fumarate is added to the drinking water.

Exploring heterologous prime-boost vaccination approaches to enhance influenza control in pigs.

Influenza A viruses evolve rapidly to escape host immunity. In swine, this viral evolution has resulted in the emergence of multiple H1 and H3 influenza A virus (IAV) lineages in the United States (US) pig populations. The heterologous prime-boost vaccination strategy is a promising way to deal with diverse IAV infection in multiple animal models. However, whether or not this vaccination strategy is applicable to US swine to impart immunity against infection from North American strains of IAV is still unknown. We performed a vaccination-challenge study to evaluate the protective efficacy of using multivalent inactivated vaccine and/or a live attenuated IAV vaccine (LAIV) in pigs following multiple prime-boost vaccination protocols against a simultaneous H1N1 and H3N2 IAV infection. Our data show that pigs in the heterologous prime-boost vaccination group had more favorable outcomes consistent with a better response against virus challenge than non-vaccinated pigs. Additionally, delivering a multivalent heterologous inactivated vaccine boost to pigs following a single LAIV administration was also beneficial. We concluded the heterologous prime boost vaccination strategy may potentiate responses to suboptimal immunogens and holds the potential applicability to control IAV in the North American swine industry. However, more studies are needed to validate the application of this vaccination approach under field conditions.

A randomized controlled trial to evaluate performance of pigs raised in antibiotic-free or conventional production systems following challenge with porcine reproductive and respiratory syndrome virus.

The trial objective was to compare the performance and animal health parameters of pigs raised according to one of 3 antibiotic (AB) protocols: standard AB medication consisting of mass treatment on days 4 and 21 and judicious AB therapy given therapeutically thereafter as group medication in water and feed or by individual injection (group T1, N = 702); modified AB medication identical to group T1 but with mass treatment only on day 4 and without subsequent therapeutic feed medication (group T2, N = 675); or an antibiotic-free (ABF) regimen (group T3, N = 702). All pigs were vaccinated with a modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine 3 days after weaning. Using a seeder pig model to mimic endemic field infection dynamics, pigs were contact-challenged with virulent PRRSV lineage 1 strain 174 four weeks after vaccination. At finishing, average daily gain (ADG) and mean feed conversion ratio (FCR) were significantly better (p ≤ 0.05) for the T1 and T2 groups compared to the T3 group. There were no significant differences in post-weaning ADG and FCR between the T1 and T2 groups. Mortality and removals significantly favored (p ≤0.05) the T1 and T2 groups (20.94% and 24.89%, respectively) versus the T3 group (57.98%). Net revenue per pig was $105.43, $98.79, and $33.81 for the T1, T2 and T3 groups, respectively. Under the conditions of this study, these results indicate that in a PRRSV-endemic setting involving bacterial co-infections, an ABF production strategy may leave pigs at considerable risk of exposure to severe clinical disease and that judicious use of antibiotics can significantly improve animal health.

Serologic profiling of Haemophilus parasuis-vaccinated sows and their litters using a novel oligopeptide permease A enzyme-linked immunosorbent assay reveals unexpected patterns of serological response and maternal antibody transfer.

Haemophilus parasuis is an economically important swine pathogen with 15 recognized serovars. An enzyme-linked immunosorbent assay (ELISA) was developed that detects serum antibodies to the oligopeptide permease A (OppA) polypeptide membrane protein present in the reference strains for 13 of the H. parasuis serovars. Using the OppA-ELISA, H. parasuis serologic profiles were assessed on 2 swine farms, with seroconversion defined as an OppA-ELISA sample-to-positive (S/P) ratio ≥0.5. Ten gilts from each farm were vaccinated for H. parasuis using either a live avirulent culture vaccine (farm 1) or an inactivated autogenous vaccine (farm 2). Seroconversion occurred in 100% of farm 1 gilts and 90% of farm 2 gilts, with a mean S/P ratio (MSPR) of 3.36 and 1.43, respectively. The OppA-ELISA MSPRs were determined for 2 piglets, 1 male and 1 female, randomly selected from 10 first-parity (P1), 10 second-parity (P2), and 10 third-parity (P3) litters farrowed by respective vaccinated gilts on each farm. On both farms, postfarrowing MSPRs and rate of seropositivity were highest in P1 versus P2 and P3 dams. Parity 1 piglets had higher MSPRs and rates of seropositivity versus later parities, with the difference being significant (P < 0.05) on farm 2. Polymerase chain reaction analysis of nasal swabs indicated that 100% of farm 1 piglets and 47-84%, depending on parity, of farm 2 piglets were H. parasuis-colonized at weaning. The results indicated that H. parasuis vaccination of gilts will not maintain serologic responses in the OppA-ELISA over their reproductive lifetimes, and that maternally derived antibodies do not prevent H. parasuis colonization of piglets.

Gene expression profiling in the lungs of pigs with different susceptibilities to Glässer's disease.

BACKGROUND: Haemophilus parasuis is the causative agent of Glässer's disease in pigs. Currently, little is known about the molecular mechanisms that contribute to disease susceptibility. This study used a porcine oligonucleotide microarray to identify genes that were differentially expressed (DE) in the lungs of colostrum-deprived animals previously characterized as being either 'Fully Resistant' (FR) or 'Susceptible' to infection by H. parasuis in a bacterial challenge experiment. RESULTS: Gene expression profiles of 'FR' and 'Susceptible' animals were obtained by the identification of genes that were differentially expressed between each of these groups and mock-inoculated 'Control' animals. At 24 hours post-inoculation, a total of 21 and 58 DE genes were identified in 'FR' and 'Susceptible' animals respectively. At 72 hours, the numbers of genes were 20 and 347 respectively. 'FR' animals at 24 hours exhibited an increased expression of genes encoding extracellular matrix and TGF-beta signalling components, possibly indicative of tissue repair following the successful early resolution of infection. The gene expression profile of 'FR' animals at 72 hours supported the hypothesis that higher levels of antibacterial activity were responsible for the 'FR' phenotype, possibly due to an increase in natural immunoglobulin A and decrease in signalling by the immunoregulatory transcription factor peroxisome proliferator-activated receptor gamma (PPAR-gamma). The expression profile of 'Susceptible' animals at both time-points was characterized by an imbalance in signalling between pro and anti-inflammatory cytokines and an increased expression of genes involved in biological processes associated with inflammation. These include the pro-inflammatory cytokine genes resistin (RETN) and interleukin 1-beta (IL1B). At 72 hours, a reduction in the expression of genes involved in antigen presentation by both MHC class I and II molecules was observed, which could have contributed to the inability of 'Susceptible' animals to clear infection. CONCLUSIONS: This study is the first to have identified discrete sets of DE genes in pigs of differing susceptibility to H. parasuis infection. Consequently, several candidate genes and pathways for disease resistance or susceptibility phenotypes have been identified. In addition, the findings have shed light on the molecular pathology associated with Glässer's disease.

Differences in susceptibility to Haemophilus parasuis infection in pigs.

In animal breeding programs, deoxyribonucleic acid (DNA) markers can be used to identify sires that are less susceptible to disease. These DNA markers are typically discovered in populations that display differences in susceptibility. To find those differences, it was hypothesized that sires influence their offspring responses to infection with H. parasuis. To identify differences in susceptibility, colostrum-deprived pigs derived from 6 sires were inoculated with a virulent strain of H. parasuis serovar 5. Pigs were infected at 21-d of age and euthanized 1, 2, or 3 days post-infection. Rectal temperatures, bacterial detection, clinical signs, and lesions were measured by comparing disease susceptibility in the offspring from each sire. The effect of the sire on the severity of disease in the offspring was statistically analyzed using to a 2-way ANOVA with sire and test day as fixed effects. Significant differences among sires were found for lesions, rectal temperatures from days 0-1 and 0-2 (P < 0.05) and marginal effects for clinical signs (P = 0.08). On average, the offspring of sire H94 was the most susceptible to challenge. Responses to infection were categorized to determine the clinical responses and analyzed by Chi square. Overall, 10% of all pigs infected were fully resistant to H. parasuis infection. Boar H94 didn't produce any fully resistant offspring. Differences in susceptibility to H. parasuis were observed, and the results support the hypothesis that sires influence their offspring's response to infection. Tissues from this population could be used to identify DNA markers for genetic selection of sires that produce offspring more resistant to H. parasuis infection.