Ruminant-specific retrotransposons shape regulatory evolution of bovine immunity.

Cattle are an important livestock species, and mapping the genomic architecture of agriculturally relevant traits such as disease susceptibility is a major challenge in the bovine research community. Lineage-specific transposable elements (TEs) are increasingly recognized to contribute to gene regulatory evolution and variation, but this possibility has been largely unexplored in ruminant genomes. We conducted epigenomic profiling of the type II interferon (IFN) response in bovine cells and found thousands of ruminant-specific TEs including MER41_BT and Bov-A2 elements predicted to act as IFN-inducible enhancer elements. CRISPR knockout experiments in bovine cells established that critical immune factors including IFNAR2 and IL2RB are transcriptionally regulated by TE-derived enhancers. Finally, population genomic analysis of 38 individuals revealed that a subset of polymorphic TE insertions may function as enhancers in modern cattle. Our study reveals that lineage-specific TEs have shaped the evolution of ruminant IFN responses and potentially continue to contribute to immune gene regulatory differences across modern breeds and individuals. Together with previous work in human cells, our findings demonstrate that lineage-specific TEs have been independently co-opted to regulate IFN-inducible gene expression in multiple species, supporting TE co-option as a recurrent mechanism driving the evolution of IFN-inducible transcriptional networks.

Impact of Four Ovine TMEM154 Haplotypes on Ewes during Multiyear Lentivirus Exposure.

Polypeptide variation encoded by the ovine transmembrane protein 154 gene (TMEM154) is associated with susceptibility to ovine lentivirus, the causative agent of Ovine Progressive Pneumonia (OPP) and Visna/Maedi. Our aim was to compare the four most prevalent TMEM154 haplotypes on the incidence of infection and ewe productivity during natural multiyear virus exposure. Prospective cohort studies were designed to test gene action and estimate effects of TMEM154 haplotypes encoding distinctive variant residues: K35 ("1"), I70 ("2"), ancestral ("3"), and A4del/M44 ("4"). Exposure consisted of co-mingling infected ewes at a rate greater than 30% with serological status evaluated every four months. For ewes with one or two copies of the highly susceptible haplotypes "2" and "3", the infection prevalence steadily increased to nearly 100% at 55 months. Haplotypes "2" and "3" were equally susceptible and dominant to haplotype "1". A difference was not detected (p < 0.53) in the magnitude of effect with haplotype combinations of "1" and "4". The ewe infection prevalence with "1,1"; "1,4"; and "4,4" was 10% to 40% at 55 months. The latter suggested that two copies of the K35 amino acid substitution ("1") were as effective as a homozygous TMEM154 "knockout" with the frame-shift deletion mutation ("4") in reducing infection susceptibility. When considering ewe reproductive performance, a difference was not detected when comparing haplotypes "2", and "3" to each other, or "1" and "4" to each other. Our study indicated that ewes with two copies of the severely truncated versions of TMEM154 ("4,4") had normal lamb productivity. Without complete understanding of the natural function of TMEM154 our recommendations to producers interested in using TMEM154 selection to reduce their flock's genetic predisposition to OPP are encouraged to increase the frequency of TMEM154 haplotype K35 ("1") since it encodes a full-length protein with minimal difference to the ancestral polypeptide.

Differences between predicted outer membrane proteins of genotype 1 and 2 Mannheimia haemolytica.

BACKGROUND: Mannheimia haemolytica strains isolated from North American cattle have been classified into two genotypes (1 and 2). Although members of both genotypes have been isolated from the upper and lower respiratory tracts of cattle with or without bovine respiratory disease (BRD), genotype 2 strains are much more frequently isolated from diseased lungs than genotype 1 strains. The mechanisms behind the increased association of genotype 2 M. haemolytica with BRD are not fully understood. To address that, and to search for interventions against genotype 2 M. haemolytica, complete, closed chromosome assemblies for 35 genotype 1 and 34 genotype 2 strains were generated and compared. Searches were conducted for the pan genome, core genes shared between the genotypes, and for genes specific to either genotype. Additionally, genes encoding outer membrane proteins (OMPs) specific to genotype 2 M. haemolytica were identified, and the diversity of their protein isoforms was characterized with predominantly unassembled, short-read genomic sequences for up to 1075 additional strains. RESULTS: The pan genome of the 69 sequenced M. haemolytica strains consisted of 3111 genes, of which 1880 comprised a shared core between the genotypes. A core of 112 and 179 genes or gene variants were specific to genotype 1 and 2, respectively. Seven genes encoding predicted OMPs; a peptidase S6, a ligand-gated channel, an autotransporter outer membrane beta-barrel domain-containing protein (AOMB-BD-CP), a porin, and three different trimeric autotransporter adhesins were specific to genotype 2 as their genotype 1 homologs were either pseudogenes, or not detected. The AOMB-BD-CP gene, however, appeared to be truncated across all examined genotype 2 strains and to likely encode dysfunctional protein. Homologous gene sequences from additional M. haemolytica strains confirmed the specificity of the remaining six genotype 2 OMP genes and revealed they encoded low isoform diversity at the population level. CONCLUSION: Genotype 2 M. haemolytica possess genes encoding conserved OMPs not found intact in more commensally prone genotype 1 strains. Some of the genotype 2 specific genes identified in this study are likely to have important biological roles in the pathogenicity of genotype 2 M. haemolytica, which is the primary bacterial cause of BRD.

Genomic signatures of Mannheimia haemolytica that associate with the lungs of cattle with respiratory disease, an integrative conjugative element, and antibiotic resistance genes.

BACKGROUND: Mannheimia haemolytica typically resides in cattle as a commensal member of the upper respiratory tract microbiome. However, some strains can invade their lungs and cause respiratory disease and death, including those with multi-drug resistance. A nucleotide polymorphism typing system was developed for M. haemolytica from the genome sequences of 1133 North American isolates, and used to identify genetic differences between isolates from the lungs and upper respiratory tract of cattle with and without clinical signs of respiratory disease. RESULTS: A total of 26,081 nucleotide polymorphisms were characterized after quality control filtering of 48,403 putative polymorphisms. Phylogenetic analyses of nucleotide polymorphism genotypes split M. haemolytica into two major genotypes (1 and 2) that each were further divided into multiple subtypes. Multiple polymorphisms were identified with alleles that tagged genotypes 1 or 2, and their respective subtypes. Only genotype 2 M. haemolytica associated with the lungs of diseased cattle and the sequence of a particular integrative and conjugative element (ICE). Additionally, isolates belonging to one subtype of genotype 2 (2b), had the majority of antibiotic resistance genes detected in this study, which were assorted into seven combinations that ranged from 1 to 12 resistance genes. CONCLUSIONS: Typing of diverse M. haemolytica by nucleotide polymorphism genotypes successfully identified associations with diseased cattle lungs, ICE sequence, and antibiotic resistance genes. Management of cattle by their carriage of M. haemolytica could be an effective intervention strategy to reduce the prevalence of respiratory disease and supplemental needs for antibiotic treatments in North American herds.

Large genomic differences between Moraxella bovoculi isolates acquired from the eyes of cattle with infectious bovine keratoconjunctivitis versus the deep nasopharynx of asymptomatic cattle.

Moraxella bovoculi is a recently described bacterium that is associated with infectious bovine keratoconjunctivitis (IBK) or "pinkeye" in cattle. In this study, closed circularized genomes were generated for seven M. bovoculi isolates: three that originated from the eyes of clinical IBK bovine cases and four from the deep nasopharynx of asymptomatic cattle. Isolates that originated from the eyes of IBK cases profoundly differed from those that originated from the nasopharynx of asymptomatic cattle in genome structure, gene content and polymorphism diversity and consequently placed into two distinct phylogenetic groups. These results suggest that there are genetically distinct strains of M. bovoculi that may not associate with IBK.

Genetic subgroup of small ruminant lentiviruses that infects sheep homozygous for TMEM154 frameshift deletion mutation A4Δ53.

Small ruminant lentivirus (SRLV) infections of sheep are influenced by genetics on both the host and pathogen sides. Genetic variation in the ovine transmembrane 154 (TMEM154) gene associates with infection susceptibility, and distinct SRLV genetic subgroups infect sheep in association with their TMEM154 diplotypes. In this study, a novel SRLV subgroup was identified that naturally infected sheep with various TMEM154 diplotypes, including those homozygous for a rare frameshift mutation (A4 delta53), which is predicted to abolish TMEM154 protein function. Thus, these SRLVs may infect sheep that lack functional TMEM154, and may not be restricted by TMEM154 diplotypes in establishing infections.

Reduced lentivirus susceptibility in sheep with TMEM154 mutations.

Visna/Maedi, or ovine progressive pneumonia (OPP) as it is known in the United States, is an incurable slow-acting disease of sheep caused by persistent lentivirus infection. This disease affects multiple tissues, including those of the respiratory and central nervous systems. Our aim was to identify ovine genetic risk factors for lentivirus infection. Sixty-nine matched pairs of infected cases and uninfected controls were identified among 736 naturally exposed sheep older than five years of age. These pairs were used in a genome-wide association study with 50,614 markers. A single SNP was identified in the ovine transmembrane protein (TMEM154) that exceeded genome-wide significance (unadjusted p-value 3×10(-9)). Sanger sequencing of the ovine TMEM154 coding region identified six missense and two frameshift deletion mutations in the predicted signal peptide and extracellular domain. Two TMEM154 haplotypes encoding glutamate (E) at position 35 were associated with infection while a third haplotype with lysine (K) at position 35 was not. Haplotypes encoding full-length E35 isoforms were analyzed together as genetic risk factors in a multi-breed, matched case-control design, with 61 pairs of 4-year-old ewes. The odds of infection for ewes with one copy of a full-length TMEM154 E35 allele were 28 times greater than the odds for those without (p-value<0.0001, 95% CI 5-1,100). In a combined analysis of nine cohorts with 2,705 sheep from Nebraska, Idaho, and Iowa, the relative risk of infection was 2.85 times greater for sheep with a full-length TMEM154 E35 allele (p-value<0.0001, 95% CI 2.36-3.43). Although rare, some sheep were homozygous for TMEM154 deletion mutations and remained uninfected despite a lifetime of significant exposure. Together, these findings indicate that TMEM154 may play a central role in ovine lentivirus infection and removing sheep with the most susceptible genotypes may help eradicate OPP and protect flocks from reinfection.

Small ruminant lentivirus genetic subgroups associate with sheep TMEM154 genotypes.

Small ruminant lentiviruses (SRLVs) are prevalent in North American sheep and a major cause of production losses for the U.S. sheep industry. Sheep susceptibility to SRLV infection is influenced by genetic variation within the ovine transmembrane 154 gene (TMEM154). Animals with either of two distinct TMEM154 haplotypes that both encode glutamate at position 35 of the protein (E35) are at greater risk of SRLV infection than those homozygous with a lysine (K35) haplotype. Prior to this study, it was unknown if TMEM154 associations with infection are influenced by SRLV genetic subgroups. Accordingly, our goals were to characterize SRLVs naturally infecting sheep from a diverse U.S. Midwestern flock and test them for associations with TMEM154 E35K genotypes. Two regions of the SRLV genome were targeted for proviral amplification, cloning, sequence analysis, and association testing with TMEM154 E35K genotypes: gag and the transmembrane region of env. Independent analyses of gag and env sequences showed that they clustered in two subgroups (1 and 2), they were distinct from SRLV subtypes originating from Europe, and that subgroup 1 associated with hemizygous and homozygous TMEM154 K35 genotypes and subgroup 2 with hemi- and homozygous E35 genotypes (gag p<0.001, env p=0.01). These results indicate that SRLVs in the U.S. have adapted to infect sheep with specific TMEM154 E35K genotypes. Consequently, both host and SRLV genotypes affect the relative risk of SRLV infection in sheep.

The Effect of Probiotics in a Milk Replacer on Leukocyte Differential Counts, Phenotype, and Function in Neonatal Dairy Calves.

Probiotics have been investigated for many health benefits; however, few studies have been performed to determine the effects of oral probiotics on peripheral blood and respiratory immune cells in cattle. Our objectives were to determine changes in health and growth status, differential blood cell counts and function, and blood and lung cell function using flow cytometry and PCR in dairy calves fed a milk replacer with (PRO, N = 10) or without (CON, N = 10) the addition of probiotics to the milk replacer and dry rations from birth to weaning. Performance and clinical scores were not different between the treatment groups. Treatment-by-day interactions for peripheral blood leukocyte populations differed in cell number and percentages. A greater percentage of leukocytes expressed the cell surface markers CD3, CD4, CD8, CD11b, and CD205 on d 21 in CON animals. Lung lavages were performed on five animals from each treatment group on d 52. There were no differences between treatment groups for the expression of cytokines and Toll-Like Receptors as measured using Polymerase Chain Reaction, possibly due to the small sample size. Oral probiotics appear to affect peripheral blood immune cells and function. Their effect on overall calf health remains to be determined.

Probiotics in milk replacer affect the microbiome of the lung in neonatal dairy calves.

Introduction: Probiotics have been investigated for their many health benefits and impact on the microbiota of the gut. Recent data have also supported a gut-lung axis regarding the bacterial populations (microbiomes) of the two locations; however, little research has been performed to determine the effects of oral probiotics on the microbiome of the bovine respiratory tract. We hypothesized that probiotic treatment would result in changes in the lung microbiome as measured in lung lavage fluid. Our overall goal was to characterize bacterial populations in the lungs of calves fed probiotics in milk replacer and dry rations from birth to weaning. Methods: A group of 20 dairy calves was split into two treatment groups: probiotic (TRT; N = 10, milk replacer +5 g/d probiotics; Bovamine Dairy, Chr. Hansen, Inc., Milwaukee, WI) and control (CON; N = 10, milk replacer only). On day 0, birth weight was obtained, and calves were provided colostrum as per the dairy SOP. On day 2, probiotics were added to the milk replacer of the treated group and then included in their dry ration. Lung lavages were performed on day 52 on five random calves selected from each treatment group. DNA was extracted from lavage fluid, and 16S ribosomal RNA (rRNA) gene hypervariable regions 1-3 were amplified by PCR and sequenced using next-generation sequencing (Illumina MiSeq) for the identification of the bacterial taxa present. Taxa were classified into both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs). Results: Overall, the evaluation of these samples revealed that the bacterial genera identified in the lung lavage samples of probiotic-fed calves as compared to the control calves were significantly different based on the OTU dataset (p < 0.05) and approached significance for the ASV dataset (p < 0.06). Additionally, when comparing the diversity of taxa in lung lavage samples to nasal and tonsil samples, taxa diversity of lung samples was significantly lower (p < 0.05). Discussion: In conclusion, analysis of the respiratory microbiome in lung lavage samples after probiotic treatment provides insight into the distribution of bacterial populations in response to oral probiotics and demonstrates that oral probiotics affect more than the gut microbiome.

Effect of TMEM154 E35K variant (haplotypes 1 and 3) on the incidence of ovine lentivirus infection and ewe productivity during lifetime exposure.

Ovine progressive pneumonia virus (OPPV) is a small ruminant lentivirus that is widespread throughout U.S. sheep flocks. Infections with OPPV are lifelong and effects are multi-systemic with significant implications for animal well-being and productivity. A protein isoform with lysine at position 35 (K35, haplotype "1") encoded by the ovine transmembrane protein 154 (TMEM154) gene has been associated with reduced susceptibility to infection when two copies are present (i.e., diplotype "1,1"). Conversely, the ancestral protein isoform with glutamate at position 35 (E35, haplotype "3") is associated with high susceptibility to infection when at least one copy is present. The beneficial effect of TMEM154 K35 alleles on ewe productivity has not been previously measured in controlled challenge experiments and was a major objective of this study. Ewes with TMEM154 diplotypes "1,1"; "1,3"; and "3,3" (n = 31, 47, and 30, respectively) were born and reared by OPPV-infected dams and managed under continual natural exposure to OPPV. Ewes were tested for serological status at 4-mo intervals for up to 5.5 yr. The incidence of infection in ewes with diplotype "1,1" was 6.5% to 9.7% and significantly lower (P < 0.001) than ewes with diplotype "1,3" (60.5 to 97.3%) or "3,3" (64.0 to 91.4%). Furthermore, the incidence among ewes with diplotype "1,1" did not increase from 10 to 67 mo of age (P > 0.99), whereas the incidence among diplotype "1,3" and "3,3" ewes increased steadily until reaching an asymptote at approximately 52 mo of age. Total number and weight of lamb weaned per ewe exposed through 5.5 yr from ewes with diplotype "1,1" far exceeded (P ≤ 0.05) those with diplotypes "1,3" and "3,3" by, on average, 2.1 lambs and 40 kg, respectively. The present study confirmed that TMEM154 diplotype "1,1" animals have reduced incidence of OPPV infection and, correspondingly, improved productivity. In flocks with a high frequency of TMEM154 haplotype "3," selection for haplotype "1" appears to be a cost-effective approach to mitigate the impact of this economically important disease.

Evaluating Accuracy of DNA Pool Construction Based on White Blood Cell Counts.

Pooling individual samples prior to DNA extraction can mitigate the cost of DNA extraction and genotyping; however, these methods need to accurately generate equal representation of individuals within pools. The objective of this study was to determine accuracy of pool construction of blood samples based on white blood cell counts compared to two common DNA quantification methods. Fifty individual bovine blood samples were collected, and then pooled with all individuals represented in each pool. Pools were constructed with the target of equal representation of each individual animal based on number of white blood cells, spectrophotometric readings, spectrofluorometric readings, and whole blood volume with 9 pools per method and a total of 36 pools. Pools and individual samples that comprised the pools were genotyped using a commercially available genotyping array. ASReml was used to estimate variance components for individual animal contribution to pools. The correlation between animal contributions between two pools was estimated using bivariate analysis with starting values set to the result of a univariate analysis. Adonis test on distance matrix from the animal correlation showed clustering with method, and higher correlations between methods than within (P < 1 × 10-6). White blood cell count was predictive of sample representation when compared to pooling based on DNA concentration. Therefore, constructing pools using white blood cell counts prior to DNA extraction may reduce cost associated with DNA extraction and genotyping and improve representation of individuals in a pool.

In Vitro Infection Dynamics of Japanese Encephalitis Virus in Established Porcine Cell Lines.

Japanese encephalitis virus (JEV) is a zoonotic mosquito-borne pathogen that regularly causes severe neurological disease in humans in Southeast Asia and the Western Pacific region. Pigs are one of the main amplifying hosts of JEV and play a central role in the virus transmission cycle. The objective of this study was to identify in vitro cell systems to investigate early effects of JEV infection including viral replication and host cell death. Here, we demonstrate the susceptibility of several porcine cell lines to the attenuated genotype III JEV strain SA14-14-2. Monolayers of porcine nasal turbinate (PT-K75), kidney (SK-RST), testis (ST), and monocyte-derived macrophage (CΔ2+) cells were infected with SA14-14-2 for up to five days at a multiplicity of infection (MOI) of 0.1. The hamster kidney cell line BHK-21, previously shown to be susceptible to SA14-14-2, was used as a positive control. Culture supernatants and cells were collected between 0 and 120 h post infection (hpi), and monolayers were observed for cytopathic effect (CPE) using brightfield microscopy. The number of infectious virus particles was quantified by plaque assay and cell viability was determined using trypan blue staining. An indirect immunofluorescence assay was used to detect the presence of JEV NS1 antigens in cells infected at 1 MOI. All four porcine cell lines demonstrated susceptibility to SA14-14-2 and produced infectious virus by 12 hpi. Virus titers peaked at 48 hpi in CΔ2+, BHK-21, and SK-RST cells, at 72 hpi in PT-K75, and at 120 hpi in ST cells. CPE was visible in infected CΔ2+ and BHK-21 cells, but not the other three cell lines. The proportion of viable cells, as measured by trypan blue exclusion, declined after 24 hpi in BHK-21 and 48 hpi in CΔ2+ cells, but did not substantially decline in SK-RST, PT-K75 or ST cells. At 48 hpi, JEV NS1 was detected in all infected cell lines by fluorescence microscopy. These findings demonstrate several porcine cell lines which have the potential to serve as useful research tools for investigating JEV infection dynamics and host cell mechanisms in a natural amplifying host species, such as pigs, in vitro.

Hematology parameters as potential indicators of feed efficiency in pigs.

The identification of an inexpensive, indirect measure of feed efficiency in swine could be a useful tool to help identify animals with improved phenotypes to supplement expensive phenotypes including individual feed intakes. The purpose of this study was to determine whether hematology parameters in pigs at the beginning and end of a feed efficiency study, or changes in those values over the study, were associated with average daily gain (ADG), average daily feed intake (ADFI), or gain-to-feed (G:F). Whole blood samples were taken at days 0 and 42 from pigs (n = 178) that were monitored for individual feed intakes and body weight gain during a 6-week study. Blood samples were analyzed for blood cell parameters including white blood cell (WBC), neutrophil, lymphocyte, monocyte, eosinophil and basophil counts, red blood cell (RBC) counts, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC), platelet count, and mean platelet volume (MPV). Feed efficiency parameters were predicted using an ANOVA model including fixed effects of farrowing group and pen (sex constant) and individual hematology parameters at day 0, day 42 or their change as covariates. At day 0, platelet count was positively associated with ADFI (P < 0.05) and negatively associated with G:F (P < 0.1), and lymphocyte count was positively associated with ADFI (P < 0.05). At day 42, neutrophil, RBC counts, hemoglobin and hematocrit were associated with ADFI (P < 10-3). Over the course of the study, changes in RBC measurements including RBC, hemoglobin, MCV, MCH, and MCHC (P < 10-4) which may improve oxygen carrying capacity, were associated with ADG and ADFI. The change in hematocrit over the course of the study was the only parameter that was associated with all three measures of feed efficiency (P < 0.05). Changes in RBC parameters, especially hematocrit, may be useful measurements to supplement feed efficiency phenotypes in swine.

Cytokine and Haptoglobin Profiles From Shipping Through Sickness and Recovery in Metaphylaxis- or Un-Treated Cattle.

Fifty-six head of cattle, 28 animals with bovine respiratory disease complex (BRDC), and 28 healthy animals that were matched by treatment, sale barn of origin, day, and interactions among these variables, were identified from a population of 180 animals (60 each purchased at three sale barns located in Missouri, Tennessee, and Kentucky) enrolled in a study comparing animals receiving metaphylaxis to saline-treated controls. Cattle were transported to a feedlot in KS and assigned to treatment group. Blood samples were collected at Day 0 (at sale barn), Day 1, Day 9, and Day 28 (at KS feedlot), and transported to the US Meat Animal Research Center in Clay Center, NE where plasma was harvested and stored at -80°C until assayed for the cytokines IFN-γ, IL-1ß, IL-6, and TNF-α, and the acute stress protein haptoglobin (HPT). Our objectives were to determine if cytokine and haptoglobin profiles differed between control and metaphylaxis treatment groups over time, and if profiles differed between animals presenting with BRDC and those that remained healthy. There was no difference between the treated animals and their non-treated counterparts for any of the analytes measured. Sale barn of origin tended to affect TNF-α concentration. Differences for all analytes changed over days, and on specific days was associated with state of origin and treatment. The Treatment by Day by Case interaction was significant for HPT. The analyte most associated with BRDC was HPT on D9, possibly indicating that many of the cattle were not exposed to respiratory pathogens prior to entering the feedlot.

Detection of bovine inflammatory cytokines IL-1ß, IL-6, and TNF-α with a multiplex electrochemiluminescent assay platform.

Commercially available bovine-specific assays are limited in number, and multiplex assays for this species are rare. Our objective was to develop a multiplex assay for the bovine inflammatory cytokines IL-1ß, IL-6, and TNF-α using the Meso Scale Discovery U-PLEX platform. "Do-It-Yourself" ELISA kits that contained polyclonal antibodies, both unlabeled and biotinylated, and the specific recombinant bovine cytokine standard, were purchased for each of these three cytokines. The biotinylated antibodies were coupled to linkers that bind to specific locations within each well of the U-PLEX plate. Unique linkers were used for each of the cytokines. The unlabeled antibodies were conjugated with electrochemiluminescent labels to serve as detection antibodies. Each cytokine assay was optimized individually prior to performing an optimization on the multiplex assay containing reagents for all three cytokines. To calculate cytokine concentrations, standard curves were developed using the recombinant cytokines and were run concurrently on each plate. Standard curves for IL-1ß and TNF-α were run at concentrations ranging from 0 to 50,000 pg/mL, and for IL-6 from 0 to 10,000 pg/mL. The average lowest level of detection concentration measured by the standard curves were 5.3 pg/mL, 0.92 pg/mL, and 22.34 pg/mL for IL-1ß, IL-6, and TNF-α respectively, as determined by data from seven plates containing bovine plasma samples from a combination of healthy and diseased cattle. The U-PLEX platform was a viable means to develop custom analyte- and species-specific multiplex assays using privately developed or purchased sets of commercially available reagents.

Distribution of Shiga-toxigenic Escherichia coli O157 in the gastrointestinal tract of naturally O157-shedding cattle at necropsy.

Shiga-toxigenic Escherichia coli (STEC) O157 occurrence was determined along the entire gastrointestinal tract (GIT) of each of four naturally shedding cattle and at three sites in 61 slaughter cattle. STEC O157 was distributed along the entire GIT, though interanimal distribution was variable. Neither feces nor rectoanal-junction samples accurately predicted the STEC O157-negative status of any particular animal.

Variation in the response of bovine alveolar lavage cells to diverse species of probiotic bacteria.

OBJECTIVE: Probiotics are fed to improve enteric health, and they may also affect respiratory immunity through their exposure to the upper respiratory tract upon ingestion. However, their effect on the respiratory system is not known. Our aim was to determine how probiotics affect functions and markers of bronchoalveolar lung lavage cells (BAL) isolated from lungs of calves at slaughter. RESULTS: Treatments consisted of ten probiotic species and one control treatment. Probiotics and BAL were incubated 1:1 for 2 h at 37 °C and 5% CO2. The cell surface markers measured included CD14, CD205, and CD18, and E. coli bioparticles were used to measure phagocytosis and oxidative burst. Differences were considered significant at P ≤ 0.05 and were noted for percent cells fluorescing and mean fluorescence intensity for CD14 and CD205. Additionally, oxidative burst was different as measured by both percentage of cells fluorescing and mean fluorescence intensity, and phagocytosis differed among species as measured by mean fluorescence intensity. Overall, probiotic species differed in their ability to suppress or increase leukocyte function showing that probiotic bacteria differentially modulate BAL.

The regulatory actions of retinoic acid on M2 polarization of porcine macrophages.

We previously demonstrated that the most bioactive vitamin A metabolite, all-trans retinoic acid (ATRA), increased T helper 2-associated responses induced in pigs by infection with the parasitic nematode Ascaris suum We also showed that ATRA potentiated the mRNA expression of several IL-4 induced chemokines (chemokine (CC motif) ligand 11 [(CCL11), CCL17, CCL22 and CCL26] associated with alternative activation (M2a) in porcine macrophages in vitro. Herein, several mechanisms whereby ATRA affects IL-4 signaling are profiled using large-scale real time PCR and RNA-Seq analysis. Twenty-three genes associated with M2a markers in other species were independently upregulated by both IL-4 and ATRA, including the adenosine receptor A2B (ADORA2B), cysteinyl leukotriene receptor 2 (CYSLTR2) and the vitamin D receptor (VDR). ATRA synergistically enhanced IL-4 up-regulation of Hepatitis A virus cellular receptor 2 (HAVCR2) and transglutaminase 2 (TGM2) and further repressed IL-4 down-regulated CD163 and Cytochrome b-245, beta polypeptide (CYBB) mRNA. Macrophages treated with ATRA exhibited a dose-dependent reduction in phagocytosis of opsonized Staphylococcus aureus. In addition, the combination of IL-4 and ATRA up-regulated the anti-inflammatory protein, IL-1R antagonist (IL1RN) and TGM2. These data indicate that ATRA induces a state of partial alternative activation in porcine macrophages, and amplifies certain aspects of M2a activation induced by IL-4. Given the prevalence of allergic and parasitic diseases worldwide and the close similarities in the porcine and human immune responses, these findings have important implications for the nutritional regulation of allergic inflammation at mucosal surfaces.

Effect of grinding and long-term storage on the toxicity of white snakeroot (Ageratina altissima) in goats.

White snakeroot (Ageratina altissima) contains the putative toxin tremetone and can produce a disease called "trembles" or "milk sickness". However the toxicity of tremetone has not been demonstrated in vivo. It has been reported that the plant is less toxic after drying and grinding. The objectives of these studies were to determine: 1) the toxic effect of grinding white snakeroot 4 months prior to dosing and, 2) the toxic effect of storing white snakeroot at ambient temperature for 5 years. Dried white snakeroot, ground 1 day, 1 month, and 4 months prior to dosing, was orally gavaged to goats at 2% of their body weight for up to 28 days or until they were minimally poisoned (minimal muscular weakness and increased serum creatine kinase (CK) activities). All four goats dosed with white snakeroot that had been ground 4 months previously and stored at room temperature were poisoned, became exercise intolerant, and had increased serum CK activities (>5600 U/ L). White snakeroot stored for 5 years was toxic as 3 of 5 dosed goats developed clinical disease within only 6 days of dosing even though approximately 80% of the tremetone in the plant had disappeared during the 5-year storage period. The results from this study demonstrate that previous grinding and extended storage did not significantly alter white snakeroot toxicity. The results also indicate that tremetone concentration is not the singular indicator of toxicity and that other white snakeroot toxins or toxic tremetone degradation products remain in dried, stored white snakeroot.