Publications by authors named "Carol G Chitko-McKown"

35 Publications

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

Vet Immunol Immunopathol 2021 Jul 25;237:110274. Epub 2021 May 25.

College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA.

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-1β, 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-1β 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-1β, 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.
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http://dx.doi.org/10.1016/j.vetimm.2021.110274DOI Listing
July 2021

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

Front Vet Sci 2021 19;8:611927. Epub 2021 Mar 19.

College of Veterinary Medicine, Kansas State University, Manhattan, KS, United States.

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-1β, 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.
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http://dx.doi.org/10.3389/fvets.2021.611927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8017278PMC
March 2021

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

Front Genet 2021 5;12:635846. Epub 2021 Feb 5.

Department of Animal Science, South Dakota State University, Brookings, SD, United States.

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 ( < 1 × 10). 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.
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http://dx.doi.org/10.3389/fgene.2021.635846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7893106PMC
February 2021

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

BMC Microbiol 2020 08 12;20(1):250. Epub 2020 Aug 12.

United States Department of Agriculture, Genetics, Breeding, and Animal Health Research Unit, Agricultural Research Service, U.S. Meat Animal Research Center, Clay Center, NE, USA.

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.
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http://dx.doi.org/10.1186/s12866-020-01932-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424683PMC
August 2020

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

BMC Res Notes 2020 Mar 17;13(1):159. Epub 2020 Mar 17.

Chr. Hansen, Inc., Animal Health & Nutrition, Milwaukee, WI, 53214, USA.

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% CO. 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.
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http://dx.doi.org/10.1186/s13104-020-4921-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077026PMC
March 2020

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

Dev Comp Immunol 2019 09 8;98:20-33. Epub 2019 Apr 8.

U.S. Department of Agriculture - Agriculture Research Service, Beltsville Human Nutrition Research Center, Diet, Genomics and Immunology Laboratory, Beltsville, MD, 20705, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.dci.2019.03.020DOI Listing
September 2019

A bovine CD18 signal peptide variant with increased binding activity to leukotoxin.

F1000Res 2018 28;7:1985. Epub 2018 Dec 28.

USDA, US Meat Animal Research Center (USMARC), Clay Center, Nebraska, 68933, USA.

is the major bacterial infectious agent of bovine respiratory disease complex and causes severe morbidity and mortality during lung infections. secretes a protein leukotoxin (Lkt) that binds to the CD18 receptor on leukocytes, initiates lysis, induces inflammation, and causes acute fibrinous bronchopneumonia. Lkt binds the 22-amino acid CD18 signal peptide domain, which remains uncleaved in ruminant species. Our aim was to identify missense variation in the bovine CD18 signal peptide and measure the effects on Lkt binding. Missense variants in the integrin beta 2 gene ( ) encoding CD18 were identified by whole genome sequencing of 96 cattle from 19 breeds, and targeted Sanger sequencing of 1238 cattle from 46 breeds. The ability of different CD18 signal peptide variants to bind Lkt was evaluated by preincubating the toxin with synthetic peptides and applying the mixture to susceptible bovine cell cultures in cytotoxicity-blocking assays. We identified 14 missense variants encoded on 15 predicted haplotypes, including a rare signal peptide variant with a cysteine at position 5 (C ) instead of arginine (R ). Preincubating Lkt with synthetic signal peptides with C blocked cytotoxicity significantly better than those with R . The most potent synthetic peptide (C PQLLLLAGLLA) had 30-fold more binding activity compared to that with R . The results suggest that missense variants in the CD18 signal peptide affect Lkt binding, and animals carrying the C allele may be more susceptible to the effects of Lkt. The results also identify a potent class of non-antibiotic Lkt inhibitors that could potentially protect cattle from cytotoxic effects during acute lung infections.
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http://dx.doi.org/10.12688/f1000research.17187.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6406179PMC
September 2019

Complete blood count data and leukocyte expression of cytokine genes and cytokine receptor genes associated with bovine respiratory disease in calves.

BMC Res Notes 2018 Nov 3;11(1):786. Epub 2018 Nov 3.

USDA, ARS, U.S. Meat Animal Research Center, P.O. Box 166, Clay Center, NE, 68933, USA.

Objective: The purpose of this study was to evaluate potential relationships between cytokine gene expression, complete blood counts (CBC) and animals that were sick or would become sick. The CBC and the transcript abundance of cytokines and their receptors expressed in leukocytes were measured from calves at two early timepoints, and again after diagnosis with bovine respiratory disease (BRD).

Results: Blood was collected from calves at pre-conditioning (n = 796) and weaning (n = 791) for CBC. Blood counts were also measured for the calves with BRD (n = 13), and asymptomatic calves (n = 75) after weaning. The CBC were compared for these animals at 3 time points. At diagnosis, neutrophils were higher and basophils lower in sick animals (P < 0.05). To further characterize BRD responses, transcript abundance of 84 cytokine genes were evaluated in 5 calves with BRD and 9 asymptomatic animals at all time points. There was more data for CBC than transcript abundance; hence, animal and temporary environmental correlations between CBC and transcript abundance were exploited to improve the power of the transcript abundance data. Expression of CCL16, CXCR1, CCR1 was increased in BRD positive animals compared to controls (P-corrected < 0.1). Cytokine expression data may help to provide insight into an animal's health.
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http://dx.doi.org/10.1186/s13104-018-3900-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6215650PMC
November 2018

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

Res Vet Sci 2018 Jun 14;118:419-422. Epub 2018 Apr 14.

U.S. Meat Animal Research Center, U.S. Department of Agriculture, Clay Center, NE 68933, United States.

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.
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http://dx.doi.org/10.1016/j.rvsc.2018.04.006DOI Listing
June 2018

Phenotyping and susceptibility of established porcine cells lines to African Swine Fever Virus infection and viral production.

Sci Rep 2017 09 4;7(1):10369. Epub 2017 Sep 4.

Virology Department, Centro Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.

African swine fever virus (ASFV) is a highly pathogenic, double-stranded DNA virus with a marked tropism for cells of the monocyte-macrophage lineage, affecting swine species and provoking severe economic losses and health threats. In the present study, four established porcine cell lines, IPAM-WT, IPAM-CD163, C∆2+ and WSL, were compared to porcine alveolar macrophage (PAM) in terms of surface marker phenotype, susceptibility to ASFV infection and virus production. The virulent ASFV Armenia/07, E70 or the naturally attenuated NHV/P68 strains were used as viral models. Cells expressed only low levels of specific receptors linked to the monocyte/macrophage lineage, with low levels of infection overall, with the exception of WSL, which showed more efficient production of strain NHV/P68 but not of strains E70 and Armenia/07.
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http://dx.doi.org/10.1038/s41598-017-09948-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5583235PMC
September 2017

Evaluation of the effect of serum antibody abundance against bovine coronavirus on bovine coronavirus shedding and risk of respiratory tract disease in beef calves from birth through the first five weeks in a feedlot.

Am J Vet Res 2017 Sep;78(9):1065-1076

OBJECTIVE To evaluate the effect of serum antibody abundance against bovine coronavirus (BCV) on BCV shedding and risk of bovine respiratory disease (BRD) in beef calves from birth through the first 5 weeks in a feedlot. ANIMALS 890 natural-service crossbred beef calves from 4 research herds. PROCEDURES Serial blood samples for measurement of serum anti-BCV antibody abundance by an ELISA and nasal swab specimens for detection of BCV and other viral and bacterial BRD pathogens by real-time PCR methods were collected from all calves or subsets of calves at predetermined times from birth through the first 5 weeks after feedlot entry. Test results were compared among herds, over time, and between calves that did and did not develop BRD. The associations of various herd and calf factors with test results were also evaluated. RESULTS At the calf level, serum anti-BCV antibody abundance was not associated with BCV shedding, but BCV shedding was positively associated with BRD incidence before and after weaning. The mean serum anti-BCV antibody abundance at weaning for a group of calves was inversely related with the subsequent incidence of BRD in that group; however, the serum anti-BCV antibody abundance at weaning for individual calves was not predictive of which calves would develop BRD after feedlot entry. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that serum anti-BCV antibody abundance as determined with ELISA were not associated with BCV shedding or risk of BRD in individual beef calves from birth through the first 5 weeks after feedlot entry.
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http://dx.doi.org/10.2460/ajvr.78.9.1065DOI Listing
September 2017

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

Theriogenology 2017 Sep 27;100:1-7. Epub 2017 May 27.

USDA(1), ARS, U.S. Meat Animal Research Center, P.O. Box 166, Clay Center, NE 68933, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.theriogenology.2017.05.020DOI Listing
September 2017

Comparison of the diagnostic performance of bacterial culture of nasopharyngeal swab and bronchoalveolar lavage fluid samples obtained from calves with bovine respiratory disease.

Am J Vet Res 2017 Mar;78(3):350-358

OBJECTIVE To compare predictive values, extent of agreement, and gamithromycin susceptibility between bacterial culture results of nasopharyngeal swab (NPS) and bronchoalveolar lavage fluid (BALF) samples obtained from calves with bovine respiratory disease (BRD). ANIMALS 28 beef calves with clinical BRD. PROCEDURES Pooled bilateral NPS samples and BALF samples were obtained for bacterial culture from calves immediately before and at various times during the 5 days after gamithromycin (6 mg/kg, SC, once) administration. For each culture-positive sample, up to 12 Mannheimia haemolytica, 6 Pasteurella multocida, and 6 Histophilus somni colonies underwent gamithromycin susceptibility testing. Whole-genome sequencing was performed on all M haemolytica isolates. For paired NPS and BALF samples collected 5 days after gamithromycin administration, the positive and negative predictive values for culture results of NPS samples relative to those of BALF samples and the extent of agreement between the sampling methods were determined. RESULTS Positive and negative predictive values of NPS samples were 67% and 100% for M haemolytica, 75% and 100% for P multocida, and 100% and 96% for H somni. Extent of agreement between results for NPS and BALF samples was substantial for M haemolytica (κ, 0.71) and H somni (κ, 0.78) and almost perfect for P multocida (κ, 0.81). Gamithromycin susceptibility varied within the same sample and between paired NPS and BALF samples. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated culture results of NPS and BALF samples from calves with BRD should be interpreted cautiously considering disease prevalence within the population, sample collection relative to antimicrobial administration, and limitations of diagnostic testing methods.
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http://dx.doi.org/10.2460/ajvr.78.3.350DOI Listing
March 2017

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

BMC Genomics 2016 11 29;17(1):982. Epub 2016 Nov 29.

United States Department of Agriculture, Agricultural Research Service, U.S. Meat Animal Research Center, Clay Center, NE, USA.

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.
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http://dx.doi.org/10.1186/s12864-016-3316-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127058PMC
November 2016

Resolving Bovine viral diarrhea virus subtypes from persistently infected U.S. beef calves with complete genome sequence.

J Vet Diagn Invest 2016 Sep 7;28(5):519-28. Epub 2016 Jul 7.

U.S. Meat Animal Research Center, Clay Center, NE (Workman, Heaton, Harhay, Smith, Chitko-McKown)Great Plains Veterinary Educational Center, School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Clay Center, NE (Grotelueschen)Cattle Empire LLC, Satanta, KS (Sjeklocha)Nebraska Veterinary Diagnostic Center, School of Veterinary Medicine and Biomedical Sciences (Brodersen), University of Nebraska-Lincoln, Lincoln, NEDepartment of Animal Science (Petersen), University of Nebraska-Lincoln, Lincoln, NE.

Bovine viral diarrhea virus (BVDV) is classified into 2 genotypes, BVDV-1 and BVDV-2, each of which contains distinct subtypes with genetic and antigenic variation. To effectively control BVDV by vaccination, it is important to know which subtypes of the virus are circulating and how their prevalence is changing over time. Accordingly, the purpose of our study was to estimate the current prevalence and diversity of BVDV subtypes from persistently infected (PI) beef calves in the central United States. Phylogenetic analysis of the 5'-UTR (5' untranslated region) for 119 virus strains revealed that a majority (82%) belonged to genotype 1b, and the remaining strains were distributed between genotypes 1a (9%) and 2 (8%); however, BVDV-2 subtypes could not be confidently resolved. Therefore, to better define the variability of U.S. BVDV isolates and further investigate the division of BVDV-2 isolates into subtypes, complete genome sequences were obtained for these isolates as well as representatives of BVDV-1a and -1b. Phylogenetic analyses of the complete coding sequence provided more conclusive genetic classification and revealed that U.S. BVDV-2 isolates belong to at least 3 distinct genetic groups that are statistically supported by both complete and individual coding gene analyses. These results show that a more complex set of BVDV-2 subtypes has been circulating in this region than was previously thought.
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http://dx.doi.org/10.1177/1040638716654943DOI Listing
September 2016

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

Vet Res 2016 Feb 13;47:31. Epub 2016 Feb 13.

United States Department of Agriculture (USDA), Agricultural Research Service (ARS), U.S. Meat Animal Research Center (USMARC), State Spur 18D, Clay Center, NE, 68933, USA.

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.
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http://dx.doi.org/10.1186/s13567-016-0316-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4752781PMC
February 2016

Complete Closed Genome Sequences of a Mannheimia haemolytica Serotype A1 Leukotoxin Deletion Mutant and Its Wild-Type Parent Strain.

Genome Announc 2015 May 7;3(3). Epub 2015 May 7.

Meat Animal Research Center (USMARC), Clay Center, USDA, ARS, Clay Center, Nebraska, USA.

Mannheimia haemolytica is a bacterial pathogen that secretes leukotoxin (LktA) which binds to leukocyte membranes via CD18, causing bacterial pneumonia in ruminants. We report the complete closed genome sequences of a leukotoxin mutant and its parent strain that are frequently used in respiratory disease studies.
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http://dx.doi.org/10.1128/genomeA.00417-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4424311PMC
May 2015

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

Vet Res 2015 Mar 5;46:22. Epub 2015 Mar 5.

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.
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http://dx.doi.org/10.1186/s13567-015-0162-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4349320PMC
March 2015

SNPs for parentage testing and traceability in globally diverse breeds of sheep.

PLoS One 2014 16;9(4):e94851. Epub 2014 Apr 16.

U.S. Meat Animal Research Center (USMARC), Clay Center, Nebraska, United States of America.

DNA-based parentage determination accelerates genetic improvement in sheep by increasing pedigree accuracy. Single nucleotide polymorphism (SNP) markers can be used for determining parentage and to provide unique molecular identifiers for tracing sheep products to their source. However, the utility of a particular "parentage SNP" varies by breed depending on its minor allele frequency (MAF) and its sequence context. Our aims were to identify parentage SNPs with exceptional qualities for use in globally diverse breeds and to develop a subset for use in North American sheep. Starting with genotypes from 2,915 sheep and 74 breed groups provided by the International Sheep Genomics Consortium (ISGC), we analyzed 47,693 autosomal SNPs by multiple criteria and selected 163 with desirable properties for parentage testing. On average, each of the 163 SNPs was highly informative (MAF≥0.3) in 48±5 breed groups. Nearby polymorphisms that could otherwise confound genetic testing were identified by whole genome and Sanger sequencing of 166 sheep from 54 breed groups. A genetic test with 109 of the 163 parentage SNPs was developed for matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The scoring rates and accuracies for these 109 SNPs were greater than 99% in a panel of North American sheep. In a blinded set of 96 families (sire, dam, and non-identical twin lambs), each parent of every lamb was identified without using the other parent's genotype. In 74 ISGC breed groups, the median estimates for probability of a coincidental match between two animals (PI), and the fraction of potential adults excluded from parentage (PE) were 1.1×10(-39) and 0.999987, respectively, for the 109 SNPs combined. The availability of a well-characterized set of 163 parentage SNPs facilitates the development of high-throughput genetic technologies for implementing accurate and economical parentage testing and traceability in many of the world's sheep breeds.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0094851PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3989260PMC
February 2015

Complete Closed Genome Sequences of Four Mannheimia varigena Isolates from Cattle with Shipping Fever.

Genome Announc 2014 Feb 13;2(1). Epub 2014 Feb 13.

U.S. Department of Agriculture, ARS, U.S. Meat Animal Research Center (USMARC), Clay Center, Nebraska, USA.

Mannheimia varigena is an occasional respiratory pathogen of cattle and pigs. We present the first four complete closed genome sequences of this species.
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http://dx.doi.org/10.1128/genomeA.00088-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3924380PMC
February 2014

Complete Closed Genome Sequences of Three Bibersteinia trehalosi Nasopharyngeal Isolates from Cattle with Shipping Fever.

Genome Announc 2014 Feb 13;2(1). Epub 2014 Feb 13.

U.S. Department of Agriculture, ARS, U.S. Meat Animal Research Center (USMARC), Clay Center, Nebraska, USA.

Bibersteinia trehalosi is a respiratory pathogen affecting cattle and related ruminants worldwide. B. trehalosi is closely related to Mannheimia haemolytica and is often associated with bovine respiratory disease complex (BRDC), a polymicrobial multifactorial disease. We present three complete closed genome sequences of this species generated using an automated assembly pipeline.
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http://dx.doi.org/10.1128/genomeA.00084-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3924379PMC
February 2014

Small ruminant lentivirus genetic subgroups associate with sheep TMEM154 genotypes.

Vet Res 2013 Jul 29;44:64. Epub 2013 Jul 29.

United States Department of Agriculture (USDA) Agricultural Research Service (ARS), U,S, Meat Animal Research Center (USMARC), State Spur 18D, Clay Center, NE 68933, USA.

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.
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http://dx.doi.org/10.1186/1297-9716-44-64DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734121PMC
July 2013

Complete Closed Genome Sequences of Mannheimia haemolytica Serotypes A1 and A6, Isolated from Cattle.

Genome Announc 2013 May 16;1(3). Epub 2013 May 16.

USDA, ARS, U.S. Meat Animal Research Center, Clay Center, Nebraska, USA;

Mannheimia haemolytica is a respiratory pathogen affecting cattle and related ruminants worldwide. M. haemolytica is commonly associated with bovine respiratory disease complex (BRDC), a polymicrobial multifactorial disease. We present the first two complete closed genome sequences of this species, determined using an automated assembly pipeline requiring no manual finishing.
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http://dx.doi.org/10.1128/genomeA.00188-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3656199PMC
May 2013

Development and characterization of two porcine monocyte-derived macrophage cell lines.

Results Immunol 2013 ;3:26-32

USDA, ARS, U.S. Meat Animal Research Center (USMARC), Clay Center, NE 68933, United States.

Cell lines CΔ2+ and CΔ2- were developed from monocytes obtained from a 10-month-old, crossbred, female pig. These cells morphologically resembled macrophages, stained positively for α-naphthyl esterase and negatively for peroxidase. The cell lines were bactericidal and highly phagocytic. Both cell lines expressed the porcine cell-surface molecules MHCI, CD11b, CD14, CD16, CD172, and small amounts of CD2; however, only minimal amounts of CD163 were measured. The lines were negative for the mouse marker H2K, bovine CD2 control, and secondary antibody control. Additionally, cells tested negative for Bovine Viral Diarrhea Virus and Porcine Circovirus Type 2. Therefore, these cells resembled porcine macrophages based on morphology, cell-surface marker phenotype, and function and will be useful tools for studying porcine macrophage biology.
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http://dx.doi.org/10.1016/j.rinim.2013.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3631004PMC
January 2013

Genetic testing for TMEM154 mutations associated with lentivirus susceptibility in sheep.

PLoS One 2013 11;8(2):e55490. Epub 2013 Feb 11.

U.S. Meat Animal Research Center (USMARC), Clay Center, Nebraska, USA.

In sheep, small ruminant lentiviruses cause an incurable, progressive, lymphoproliferative disease that affects millions of animals worldwide. Known as ovine progressive pneumonia virus (OPPV) in the U.S., and Visna/Maedi virus (VMV) elsewhere, these viruses reduce an animal's health, productivity, and lifespan. Genetic variation in the ovine transmembrane protein 154 gene (TMEM154) has been previously associated with OPPV infection in U.S. sheep. Sheep with the ancestral TMEM154 haplotype encoding glutamate (E) at position 35, and either form of an N70I variant, were highly-susceptible compared to sheep homozygous for the K35 missense mutation. Our current overall aim was to characterize TMEM154 in sheep from around the world to develop an efficient genetic test for reduced susceptibility. The average frequency of TMEM154 E35 among 74 breeds was 0.51 and indicated that highly-susceptible alleles were present in most breeds around the world. Analysis of whole genome sequences from an international panel of 75 sheep revealed more than 1,300 previously unreported polymorphisms in a 62 kb region containing TMEM154 and confirmed that the most susceptible haplotypes were distributed worldwide. Novel missense mutations were discovered in the signal peptide (A13V) and the extracellular domains (E31Q, I74F, and I102T) of TMEM154. A matrix-assisted laser desorption/ionization-time-of flight mass spectrometry (MALDI-TOF MS) assay was developed to detect these and six previously reported missense and two deletion mutations in TMEM154. In blinded trials, the call rate for the eight most common coding polymorphisms was 99.4% for 499 sheep tested and 96.0% of the animals were assigned paired TMEM154 haplotypes (i.e., diplotypes). The widespread distribution of highly-susceptible TMEM154 alleles suggests that genetic testing and selection may improve the health and productivity of infected flocks.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0055490PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3569457PMC
September 2013

Reduced lentivirus susceptibility in sheep with TMEM154 mutations.

PLoS Genet 2012 Jan 26;8(1):e1002467. Epub 2012 Jan 26.

U.S. Meat Animal Research Center, Agriculture Research Service, United States Department of Agriculture, Clay Center, Nebraska, USA.

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.
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http://dx.doi.org/10.1371/journal.pgen.1002467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3266874PMC
January 2012

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

Appl Environ Microbiol 2010 Aug 11;76(15):5278-81. Epub 2010 Jun 11.

USDA, ARS, U.S. Meat Animal Research Center, P.O. Box 166, State Spur 18D, Clay Center, NE 68933, USA.

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.
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http://dx.doi.org/10.1128/AEM.00400-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2916454PMC
August 2010

The genome sequence of taurine cattle: a window to ruminant biology and evolution.

Science 2009 Apr;324(5926):522-8

To understand the biology and evolution of ruminants, the cattle genome was sequenced to about sevenfold coverage. The cattle genome contains a minimum of 22,000 genes, with a core set of 14,345 orthologs shared among seven mammalian species of which 1217 are absent or undetected in noneutherian (marsupial or monotreme) genomes. Cattle-specific evolutionary breakpoint regions in chromosomes have a higher density of segmental duplications, enrichment of repetitive elements, and species-specific variations in genes associated with lactation and immune responsiveness. Genes involved in metabolism are generally highly conserved, although five metabolic genes are deleted or extensively diverged from their human orthologs. The cattle genome sequence thus provides a resource for understanding mammalian evolution and accelerating livestock genetic improvement for milk and meat production.
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http://dx.doi.org/10.1126/science.1169588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943200PMC
April 2009

Use of bovine single nucleotide polymorphism markers to verify sample tracking in beef processing.

J Am Vet Med Assoc 2005 Apr;226(8):1311-4

USDA, Agricultural Research Service, US Meat Animal Research Center, State Spur 18D, PO Box 166, Clay Center, NE 68933-0166, USA.

Objective: To determine whether a selected set of 20 single nucleotide polymorphism (SNP) markers derived from beef cattle populations can be used to verify sample tracking in a commercial slaughter facility that processes primarily market (ie, culled) dairy cows.

Design: Prospective, blinded validation study.

Animals: 165 cows and 3 bulls from 18 states (82% Holstein, 8% other dairy breeds, and 10% beef breeds).

Procedure: Blood was collected by venipuncture from randomly chosen animals just prior to slaughter. The purported corresponding liver samples were collected during beef processing, and genotype profiles were obtained for each sample.

Results: On the basis of SNP allele frequencies in these cattle, the mean probability that 2 randomly selected individuals would possess identical genotypes at all 20 loci was 4.3 x 10(-8). Thus, the chance of a coincidental genotype match between 2 animals was 1 in 23 million. Genotype profiles confirmed appropriate matching for 152 of the 168 (90.5%) purported blood-liver sample pairs and revealed mismatching for 16 (9.5%) pairs. For the 16 mismatched sample pairs, 33% to 76% of the 20 SNP genotypes did not match (mean, 52%). Discordance that could be attributed to genotyping error was estimated to be < 1% on the basis of results for split samples.

Conclusions And Clinical Relevance: Results suggest that this selected set of 20 bovine SNP markers is sufficiently informative to verify accuracy of sample tracking in slaughter plants that process beef or dairy cattle. These or similar SNP markers may facilitate high-throughput, DNA-based, traceback programs designed to detect drug residues in tissues, control of animal diseases, and enhance food safety.
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http://dx.doi.org/10.2460/javma.2005.226.1311DOI Listing
April 2005

Gene expression profiling of bovine macrophages in response to Escherichia coli O157:H7 lipopolysaccharide.

Dev Comp Immunol 2004 May;28(6):635-45

USDA, ARS, Roman L. Hruska US Meat Animal Research Center (MARC), State Spur 18D, P.O. Box 166, Clay Center, Nebraska 68933-0166, USA.

The aim of this study was to identify changes in bovine macrophage gene expression in response to treatment with Escherichia coli 0157:H7 lipopolysaccharide (LPS), utilizing a human gene microarray. Bovine cDNA from control and LPS-treated primary macrophages hybridized to greater than 5644 (79.8%) of the non-control gene targets on a commercially available microarray containing greater than 7075 targets (Incyte Genomics, St. Louis, MO). Of these target sequences, 44 were differentially expressed upon exposure to LPS, including 18 genes not previously reported to exist in cattle. These included a pentaxin-related gene, CASP8, TNF-induced genes, interferon-induced genes, and inhibitors of apoptosis. Using the human microarray, cDNA from bovine LPS-treated and control macrophages consistently hybridized to targets known to be expressed constitutively by macrophages, as expected given the predicted cDNA sequence homology. That this human system was accurately estimating levels of bovine transcripts was further verified by real-time quantitative reverse transcriptase polymerase chain reaction (RTQ-PCR) using bovine-specific primers. This first report of bovine-human cross-species expression profiling by microarray hybridization demonstrates the utility of this technique in bovine gene expression and discovery.
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http://dx.doi.org/10.1016/j.dci.2003.10.002DOI Listing
May 2004
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