V.S.R. Dukkipati

Parturition in dairy cows temporarily alters the expression of genes in circulating neutrophils

Extensive metabolic and physiologic changes occur during the peripartum, concurrent with a high incidence of infectious disease. Immune dysfunction is a likely contributor to the increased risk of disease at this time. Studies using high-yielding, total mixed ration-fed cows have indicated that neutrophil function is perturbed over the transition period; however, this reported dysfunction has yet to be investigated in moderate-yielding, grazing dairy cows. Therefore, we investigated changes in the expression of genes involved in neutrophil function. Blood was collected from cows at 5 time points over the transition period: precalving (-1wk; n=46), day of calving (d 0; n=46), and postcalving at wk 1 (n=46), wk 2 (n=45), and wk 4 (n=43). Neutrophils were isolated by differential centrifugation and gene expression was investigated. Quantitative reverse transcriptase PCR with custom-designed primer pairs and Roche Universal Probe Library (Roche, Basel, Switzerland) chemistry, combined with microfluidics integrated fluidic circuit chips (96.96 Dynamic Array, San Francisco, CA) were used to investigate the expression of 78 genes involved in neutrophil function and 18 endogenous control genes. Statistical significance between time points was determined using a repeated measures ANOVA. Genes that were differentially expressed over the transition period included those involved in neutrophil adhesion (SELL, ITGB2, and ITGBX), mediation of the immune response (TLR4, HLA-DRA, and CXCR2), maturation, cell cycle progression, apoptosis (MCL1, BCL2, FASLG, and RIPK1), and control of gene expression (PPARG, PPARD, and STAT3). We noted reduced gene expression of proinflammatory cytokines (IFNG, TNF, IL12, and CCL2) on the day of calving, whereas anti-inflammatory cytokine gene expression (IL10) was upregulated. Increased gene expression of antimicrobial peptides (BNBD4, DEFB10, and DEFB1) occurred on the day of calving. Collectively, transcription profiles are indicative of functional changes in neutrophils of grazing dairy cows over the transition period and align with studies in cows of conventional total mixed ration systems. This altered function may predispose cows to disease over the transition period and is likely to be a natural change in function due to parturition.

Association of microsatellite polymorphisms with immune responses to a killed Mycobacterium avium subsp. paratuberculosis vaccine in Merino sheep

Abstract AIM: To study the association of polymorphisms at five micro-satellite loci with immune responses to a killed Mycobacterium avium subsp. paratuberculosis (Map) vaccine. METHODS: Merino sheep (504 vaccinates and 430 unvaccinated controls) from a long-term Johnes vaccine trial undertaken on three different properties in the Central Tablelands of New South Wales, Australia, were genotyped for five micro-satellite markers located in three immunologically significant chromosome regions. The marker loci included three from the major histocompatibility complex (MHC), namely DYMS1, OLADRB and SMHCC1; and one each from the solute carrier family 11 member 1 (SLC11A1), OVINRA1, and the interferon-γ (IFN-γ), o(IFN)-γ, gene regions. Associations between immune responses and genetic polymorphisms at the marker loci were examined by analysing both allelic and genotypic effects. RESULTS: The o(IFN)-γ locus had only two alleles, whereas the other four loci exhibited extensive polymorphism, with the number of alleles ranging from 10 (OVINRA1) to 21 (DYMS1), resulting in 30–92 genotypes per locus. Heterozygosities varied between 37% (o(IFN)-γ) and 87% (SMHCC1), while information on polymorphic contents ranged from 0.31 (o(IFN)-γ) to 0.87 (DYMS1). Each of the three properties exhibited unique allelic and genotypic frequencies. Analysis of immune response data revealed strong antibody and IFN-γ responses as early as 2 months post-vaccination. Immune responses in control animals on all three properties remained consistently low, except for slightly elevated IFN-γ responses at a few time-points on two properties, concomitant with exposure to natural infection. Genotype-phenotype association analyses revealed a number of marker geno types/alleles to be significantly associated with antibody and IFN-γ responses. However, the effects of only five genotypes (one each at DYMS1, OLADRB, SMHCC1, OVINRA1 and o(IFN)-γ) and three alleles (one each at o(IFN)-γ, DYMS1 and OLADRB) on IFN-γ responses were consistent across the three properties. CONCLUSION: Considering the significance of IFN-γ responses in protection against Map, it is possible that the genotypes/alleles identified might have a role in protective immune responses to natural Map infections, and further studies are warranted to confirm this.

Alteration of Electroencephalographic Responses to Castration in Cats by Administration of Opioids

The aim of this study was to investigate the effect of opioids on electroencephalogram (EEG) indices of nociception in cats undergoing castration. Cats were randomly assigned to receive one of the four treatments (n=8); 0.2 mg/kg morphine, 0.005 mg/ kg fentanyl, 0.01 mg/kg buprenorphine or 0.2 mg/kg butorphanol, administered subcutaneously (SC) at the time of pre-anesthetic medication. Anesthesia was induced with intravenous propofol and maintained with halothane in oxygen. EEG was recorded continuously in a three electrode montage. Median frequency (F50), total power (PTOT) and 95% spectral edge frequency (F95) derived from the EEG power spectra recorded prior to skin incision (baseline) were compared with those recorded during the ligation of the spermatic cords of both testicles. During the ligation of testicle 1, the mean F50 of cats that received buprenorphine and butorphanol was significantly (p0.05). These results indicate that opioid analgesics, acting at different opioid receptors with variable affinity, produce changes in the EEG responses that reflect their anti-nociceptive efficacy. This study demonstrates the usefulness of the EEG as a valid tool for evaluating analgesic efficacy in cats, as shown in other species of animals in previous studies.

Effects of precalving body condition and prepartum feeding level on gene expression in circulating neutrophils

Extensive metabolic, physiological, and immunological changes are associated with calving and the onset of lactation. As a result, cows transitioning between pregnancy and lactation are at a greater risk of metabolic and infectious diseases. The ability of neutrophils to mount an effective immune response to an infection is critical for its resolution, and increasing evidence indicates that precalving nutrition affects postpartum neutrophil function. The objectives of the current study were to investigate the effect of 2 precalving body condition scores (BCS; 4 vs. 5 on a 10-point scale) and 2 levels of feeding (75 vs. 125% of estimated maintenance requirements) on gene expression in circulating neutrophils. We isolated RNA from the neutrophils of cows (n = 45) at 5 time points over the transition period: precalving (-1 wk), day of calving (d 0), and postcalving at wk 1, 2, and 4. Quantitative reverse transcriptase PCR with custom-designed primer pairs and Roche Universal Probe Library (Roche, Basel, Switzerland) chemistry, combined with microfluidics integrated fluidic circuit chips (96.96 dynamic array), were used to quantify the expression of 78 genes involved in neutrophil function and 18 endogenous control genes. Statistical significance between time points was determined using repeated measures ANOVA with Tukey-Kramer multiple-testing correction to determine treatment effects among weeks. Precalving BCS altered the inflammatory state of neutrophils, with significant increases in overall gene expression of antimicrobial peptides (BNBD4 and DEFB10) and the anti-inflammatory cytokine IL10, and significantly decreased expression of proinflammatory cytokine IL23A in thinner cows (BCS 4) compared with cows calving at BCS 5. Feeding level had a time-dependent effect on gene expression; for example, increased expression of genes involved in leukotriene synthesis (PLA2G4A and ALOX5AP) occurred only at 1 wk postcalving in cows overfed (125% of requirements) precalving compared with those offered 75% of maintenance requirements. Results indicate that precalving body condition and changes in prepartum energy lead to altered gene expression of circulating neutrophils, highlighting the importance of transition cow nutrition for peripartum health.

Recovery of intact IgG in the gastrointestinal tract of the growing rat following ingestion of an ovine serum immunoglobulin.

The aim of this study was to determine whether orally ingested ovine serum IgG partly resists digestion in the growing rat. Fifteen Sprague-Dawley male rats were allocated to one of three diets for a 3-week study: a control diet (CON) and two test diets containing either freeze-dried ovine serum immunoglobulin (FDOI) or inactivated ovine serum immunoglobulin (IOI). Samples of stomach chyme and intestinal digesta from the ad libitum-fed rats were subjected to ELISA and Western blot analysis. Amounts of intact ovine IgG for the FDOI diet were found to be 13.9, 20.0, 34.1, 13.0 and 36.9 μg in the total wet digesta from the stomach chyme, duodenal, jejunal, ileal and colonic digesta respectively. Qualitative detection by Western blot revealed the presence of intact ovine serum IgG with a ~150 kDa MW. This was detected in all of the gut segments (stomach chyme, duodenal, jejunal, ileal and colonic digesta) for growing rats fed the FDOI diet. No ovine IgG was detected in the chyme or digesta from rats fed the CON or the IOI diets. Ovine serum IgG partly resisted digestion in the growing rat fed the FDOI diet and was found throughout the digestive tract. These results provide a basis to explain the reported biological effects of orally administered immunoglobulin.

Effect of circulating exosomes from transition cows on Madin-Darby bovine kidney cell function

The greatest risk of metabolic and infectious disease in dairy cows is during the transition from pregnancy to lactating (i.e., the transition period). The objective of this experiment was to determine the effects of extracellular vesicles (microvesicles involved in cell-to-cell signaling) isolated from transition cows on target cell function. We previously identified differences in the protein profiles of exosomes isolated from cows divergent in metabolic health status. Therefore, we hypothesized that these exosomes would affect target tissues differently. To investigate this, 2 groups of cows (n = 5/group) were selected based on the concentration of β-hydroxybutyrate and fatty acids in plasma and triacylglycerol concentration in liver at wk 1 and 2 postcalving. Cows with high concentrations of β-hydroxybutyrate, fatty acids, and triacylglycerol were considered at increased risk of clinical disease during the transition period (high-risk group; n = 5) and were compared with cows that had low concentrations of the selected health indicators (low-risk group; n = 5). At 2 time points during the transition period (postcalving at wk 1 and 4), blood was sampled and plasma exosomes were isolated from the high-risk and low-risk cows. The exosomes were applied at concentrations of 10 and 1 µg/mL to 5 × 103 Madin-Darby bovine kidney cells grown to 50% confluence in 96-well plates. Results indicate a numerical increase in cell proliferation when exosomes from high-risk cows were applied compared with those from low-risk cows. Consistent with an effect on cell proliferation, quantitative reverse transcriptase PCR indicated a trend for upregulation of 3 proinflammatory genes (granulocyte colony-stimulating factor, ciliary neurotrophic factor, and CD27 ligand) with the application of high-risk exosomes, which are involved in cellular growth and survival. Proteomic analysis indicated 2 proteins in the low-risk group that were not identified in the high-risk group (endoplasmin and catalase), which may also be indicative of the metabolic state of origin. It is likely that the metabolic state of the transition cow affects cellular function through exosomal messaging; however, more in-depth research into cross-talk between exosomes and target cells is required to determine whether exosomes influence Madin-Darby bovine kidney cells in this manner.

Corrigendum to “Parturition in dairy cows temporarily alters the expression of genes in circulating neutrophils” (J. Dairy Sci. 99:6470–6483)

In Table 3 (page 6478), the means and standard errors of the difference (SED) for 9 of the parameters were tabulated as log values, rather than in the original units. All P-values and Tukey comparisons are correct. The corrected table is shown below, with corrected parameters and values in bold. The authors regret the errors.

Technical note: Evaluation of endogenous control gene expression in bovine neutrophils by reverse-transcription quantitative PCR using microfluidics gene expression arrays

Reverse-transcription quantitative-PCR (RT-qPCR) is commonly used for assessing the cellular response to changes in physiologic and pathologic conditions. The selection of stable endogenous control genes is an important step of any RT-qPCR study, as expression can vary depending on the experimental environment. Our objective was to identify endogenous control genes in circulating neutrophils isolated from cows during the peripartum period. To do this, we used microfluidics gene expression arrays (Fluidigm, San Francisco, CA) for RT-qPCR analysis. Selection of the endogenous control genes was based on previous research investigating gene expression in neutrophils. The selected genes included ACTB, B2M, G6PD, GAPDH, GCH1, GOLGA5, OSBPL2, PGK1, RPL13A, RPL19, RPS9, SDHA, SMUG1, SNRPA, TBP, UXT, and YWHAZ. Four genes (GAPDH, GOLGA5, PGK1, and UXT) did not provide satisfactory quantification results using the selected method and were therefore excluded from the analyses. The suitability of the remaining 13 genes for use as endogenous control genes was assessed using geNorm and Normfinder. The gene pair with the greatest stability using geNorm was RPL13A and RPL19, whereas Normfinder ranked RPL19 and YWHAZ as the most stable pair. The 2 genes deemed most suitable for the experimental design were RPL19 and YWHAZ, which were selected for subsequent gene expression analysis. This study highlights that genes used as endogenous controls for relative quantification should be assessed on an experimental basis, even if the genes have been used in previous research.

Experimental infection of New Zealand Merino sheep with a suspension of Mycobacterium avium subspecies paratuberculosis (Map) strain Telford: Kinetics of the immune response, histopathology and Map culture.

A long-term study was undertaken to monitor immune responses, faecal cultures and clinical disease in sheep experimentally infected with Mycobacterium avium subspecies paratuberculosis (Map) strain Telford. New Zealand Merino lambs (N=56) were challenged with three oral doses of Map suspension. The lambs were weighed and faecal and blood samples obtained at different time-points. At 63 weeks post-challenge, surviving sheep were euthanised and samples of liver, ileo-caecal valve and mesenteric lymph node were collected for histopathology and Map culture. High IFN-γ and antibody responses were evident as early as 8 weeks post-C1 which persisted until the end of the trial. Approximately 92% of the sheep shed Map in faeces at 36 weeks post-challenge, with the prevalence decreasing to around 40% at the end of the trial. Thirteen sheep progressively lost weight and were euthanised between weeks 32 and 58 post-challenge. Nearly 58% of surviving sheep exhibited histo-pathological lesions in at least one of the three tissues sampled, while 42% showed acid-fast bacilli in at least one tissue. A positive Map culture in at least one tissue was obtained from approximately 85% of sheep. These results indicate that the three doses of Map challenge were highly effective in establishing Johnes disease in NZ Merino lambs.