Characterization and comparative analysis of transcriptional profiles of porcine colostrum and mature milk at different parities.

BACKGROUND: Porcine milk is a complex fluid, containing a myriad of immunological, biochemical, and cellular components, made to satisfy the nutritional requirements of the neonate. Whole milk contains many different cell types, including mammary epithelial cells, neutrophils, macrophages, and lymphocytes, as well nanoparticles, such as milk exosomes. To-date, only a limited number of livestock transcriptomic studies have reported sequencing of milk. Moreover, those studies focused only on sequencing somatic cells as a proxy for the mammary gland with the goal of investigating differences in the lactation process. Recent studies have indicated that RNA originating from multiple cell types present in milk can withstand harsh environments, such as the digestive system, and transmit regulatory molecules from maternal to neonate. Transcriptomic profiling of porcine whole milk, which is reflective of the combined cell populations, could help elucidate these mechanisms. To this end, total RNA from colostrum and mature milk samples were sequenced from 65 sows at differing parities. A stringent bioinformatic pipeline was used to identify and characterize 70,841 transcripts. RESULTS: The 70,841 identified transcripts included 42,733 previously annotated transcripts and 28,108 novel transcripts. Differential gene expression analysis was conducted using a generalized linear model coupled with the Lancaster method for P-value aggregation across transcripts. In total, 1667 differentially expressed genes (DEG) were identified for the milk type main effect, and 33 DEG were identified for the milk type x parity interaction. Several gene ontology (GO) terms related to immune response were significant for the milk type main effect, supporting the well-known fact that immunoglobulins and immune cells are transferred to the neonate via colostrum. CONCLUSIONS: This is the first study to perform global transcriptome analysis from whole milk samples in sows from different parities. Our results provide important information and insight into synthesis of milk proteins and innate immunity and potential targets for future improvement of swine lactation and piglet development.

In-Feed Tylosin Phosphate Administration to Feedlot Cattle Minimally Affects Antimicrobial Resistance.

ABSTRACT: The macrolide class antimicrobial tylosin (trade name Tylan) is approved by the U.S. Food and Drug Administration for continuous inclusion in feed for liver abscess prevention. To address concerns that this antimicrobial application may threaten human health, a population of feedlot steers was split into a control treatment (n = 42) and a tylosin treatment (n = 42). Feed rations were identical except for the inclusion of tylosin at 60 to 90 mg per head per day. Fecal swab (n = 335), pen surface material (n = 256), feed (n = 56), and water trough (n = 32) samples were obtained over four sample occasions: November (1 day before the start of tylosin inclusion in feed), January (80 days of tylosin in feed), April (167 days), and June (253 days). These samples were cultured for Escherichia coli, tetracycline-resistant E. coli, third-generation cephalosporin-resistant E. coli, Enterococcus, tetracycline-resistant Enterococcus, and erythromycin-resistant Enterococcus. Metagenomic DNA was isolated from each June fecal swab and pen surface material sample. Metagenomic DNA samples were pooled by pen for 14 fecal and 14 pen surface material samples. Quantitative PCR was employed to assess the abundances of the following 10 antimicrobial resistance genes: aac(6')-Ie-aph(2″)-Ia, aadA1, blaCMY-2, blaCTX-M, blaKPC-2, erm(B), mecA, tet(A), tet(B), and tet(M). Nasal swab samples (n = 335) were obtained from each steer during each sample period and cultured for the presence of Staphylococcus aureus and methicillin-resistant S. aureus. Of these measurements, only January and June mean fecal swab erythromycin-resistant Enterococcus colony counts for tylosin-treated cattle were significantly higher (P ≤ 0.05) than the range of mean values for control treatments. These results suggest that in-feed tylosin through the end of finishing has a narrow and minimal antimicrobial resistance impact.

Timing of transcriptomic and proteomic changes in the bovine placentome after parturition.

Proper post-partum reproductive performance is important for reproductive efficiency in beef cows, and dystocia decreases post-partum fertility. Crossbred beef cows (n = 1676) were evaluated for lifetime performance based on degree of dystocia at presentation of the first calf. Cows that experienced moderate or severe dystocia produced fewer calves during their productive life (P < 0.01). The exact mechanism is unclear, but may be due to the contributions of dystocia to abnormal placental separation. Proteolytic activity is hypothesized to contribute to placental separation in ruminants; however, when ovine placentomes were collected following caesarian section, no proteolytic activity was detected. We hypothesized that stage 2 of parturition was necessary to stimulate proteolysis and initiate placental separation. Serial placentome collections were performed on mature cows (n = 21 initiated; 7 with complete sampling) at hourly intervals for the first 2 h after expulsion of the calf. An intact piece of each placentome was fixed for histological evaluation, and a separate piece of caruncular and cotyledonary tissue from each placentome was frozen for transcriptomic and proteolytic analysis. A full set of placentomes was collected from only 7 of 21 cows at 0, 1, and 2 h, and all cows had expelled fetal membranes by 6 h. Histological, transcriptomic and proteolytic analysis was performed on placentomes from cows from which three placentomes were collected (n = 7). The microscopic distance between maternal and fetal tissues increased at 1 h (P = 0.01). Relative transcript abundance of matrix metalloprotease 14 (MMP14) tended to increase with time (P = 0.06). The relative transcript abundance of plasminogen activator urokinase-type (PLAU) was greater in caruncles than cotyledons (P = 0.01), and tended (P = 0.10) to increase in the caruncle between 0 and 2 h while remaining unchanged in the cotyledon over the same span of time. Greater PLAU and plasminogen activator tissue-type (PLAT) proteolytic activity was detected by zymography in the caruncle than the cotyledon immediately post-partum (P < 0.01). From these findings we conclude that 1) dystocia during the first parity decreases lifetime productivity in beef cattle, 2) the PA system is present at both the transcript and protein level in the bovine plactentome during parturition and 3) proteolytic activity is localized to the caruncular aspect of the placentome.

Development and model testing of antemortem screening methodology to predict required drug withholds in heifers.

A simple, cow-side test for the presence of drug residues in live animal fluids would provide useful information for tissue drug residue avoidance programs. This work describes adaptation and evaluation of rapid screening tests to detect drug residues in serum and urine. Medicated heifers had urine, serum, and tissue biopsy samples taken while on drug treatment. Samples were tested by rapid methods and high-performance liquid chromatography (HPLC). The adapted microbial inhibition method, kidney inhibition swab test, was useful in detecting sulfadimethoxine in serum, and its response correlated with the prescribed withdrawal time for the drug, 5 to 6 days posttreatment. The lateral flow screening method for flunixin and beta-lactams, adapted for urine, was useful in predicting flunixin in liver detected by HPLC, 96 h posttreatment. The same adapted methods were not useful to detect ceftiofur in serum or urine due to a lack of sensitivity at the levels of interest. These antemortem screening test studies demonstrated that the method selected, and the sampling matrix chosen (urine or serum), will depend on the drug used and should be based on animal treatment history if available. The live animal tests demonstrated the potential for verification that an individual animal is free of drug residues before sale for human consumption.

A defect in dystrophin causes a novel porcine stress syndrome.

BACKGROUND: Losses of slaughter-weight pigs due to transport stress are both welfare and economic concerns to pork producers. Historically, the HAL-1843 mutation in ryanodine receptor 1 was considered responsible for most of the losses; however, DNA testing has effectively eliminated this mutation from commercial herds. We identified two sibling barrows in the USMARC swine herd that died from apparent symptoms of a stress syndrome after transport at 12 weeks of age. The symptoms included open-mouth breathing, skin discoloration, vocalization and loss of mobility. RESULTS: We repeated the original mating along with sire-daughter matings to produce additional offspring. At 8 weeks of age, heart rate and electrocardiographs (ECG) were monitored during isoflurane anesthesia challenge (3% for 3 min). Four males from the original sire-dam mating and two males from a sire-daughter mating died after one minute of anesthesia. Animals from additional litters were identified as having a stress response, sometimes resulting in death, during regular processing and weighing. Affected animals had elevated plasma creatine phosphokinase (CPK) levels before and immediately after isoflurane challenge and cardiac arrhythmias. A pedigree containing 250 pigs, including 49 affected animals, was genotyped with the Illumina PorcineSNP60 Beadchip and only one chromosomal region, SSCX at 25.1-27.7 Mb over the dystrophin gene (DMD), was significantly associated with the syndrome. An arginine to tryptophan (R1958W) polymorphism in exon 41 of DMD was the most significant marker associated with stress susceptibility. Immunoblots of affected heart and skeletal muscle showed a dramatic reduction of dystrophin protein and histopathology of affected hearts indicated muscle fiber degeneration. CONCLUSIONS: A novel stress syndrome was characterized in pigs and the causative genetic factor most likely resides within DMD that results in less dystrophin protein and cardiac abnormalities that can lead to death under stressful conditions. The identification of predictive markers will allow us to determine the prevalence of this disease in commercial swine populations. This defect also provides a unique biomedical model for human cardiomyopathy associated with muscular dystrophy that may be superior to those available because of the similarities in anatomy and physiology and allow advances in gene therapies for human disease.