Episodes of clinical mastitis and its relationship with duration of treatment and seasonality in crossbred cows maintained in organized dairy farm

Aim: Present study aimed to evaluate the different episodes of clinical mastitis (CM) and influence of duration of treatment and seasonality on the occurrence of different episodes of CM in crossbred cows. Materials and Methods: A total of 1194 lactation data of crossbred CM cows were collected from mastitis treatment record from 2002 to 2012. Data of CM cows were classified into types of episodes (pattern of repeated or multiple episodes occurrence) and number of episodes (magnitude of multiple cases). Types of episodes were divided as single (clinical cure by a single episode of treatment), relapse (retreatment of the same cow within 21 days), recurrence (new CM at least 21 days after treatment), and both (relapse and recurrence). The season was classified as winter (December to March), summer (April to June), rainy (July to September), and autumn (October to November). The difference between incidences of different types of CM episodes and the association between number or type of CM episodes with duration of treatment and seasons of CM occurrence were analyzed by Chi-square test. Results: Among 1194 animals suffered with CM, 53, 16, and 18% had the single episode, relapse, and recurrence, respectively; while 13% suffered by both relapse and recurrence. We estimated the duration of treatment and found 80% of the cows treated 1-8 days, in which 65% treated for 1-4 days, while 35% cows were treated for 5-8 days. Further, 12% cows treated for 9-15 days and 7.5% cows treated >15 days. The relationship between duration of treatment and different episodes of CM revealed that 1-8 days treated cows were mostly cured by the single episode with less relapse and recurrence. In contrast, the incidences of recurrence and relapse episodes were higher in cows treated for more than 9 days. The highest incidence of relapse was noticed in winter (36%) than other seasons (10-28%), while the recurrence was less during autumn (9%) compared to other seasons (20-40%). Conclusion: Cows those suffered by both relapse and recurrence were more susceptible to CM, and they need to be culled from farm to control the transmission of infections. Although the influence of seasonality was difficult to understand, the higher magnitude of relapse and recurrence during winter suggested the adverse effects of cold stress on treatment outcome

RNAi Screening in Primary Human Hepatocytes of Genes Implicated in Genome-Wide Association Studies for Roles in Type 2 Diabetes Identifies Roles for CAMK1D and CDKAL1, among Others, in Hepatic Glucose Regulation

<div><p>Genome-wide association (GWA) studies have described a large number of new candidate genes that contribute to of Type 2 Diabetes (T2D). In some cases, small clusters of genes are implicated, rather than a single gene, and in all cases, the genetic contribution is not defined through the effects on a specific organ, such as the pancreas or liver. There is a significant need to develop and use human cell-based models to examine the effects these genes may have on glucose regulation. We describe the development of a primary human hepatocyte model that adjusts glucose disposition according to hormonal signals. This model was used to determine whether candidate genes identified in GWA studies regulate hepatic glucose disposition through siRNAs corresponding to the list of identified genes. We find that several genes affect the storage of glucose as glycogen (glycolytic response) and/or affect the utilization of pyruvate, the critical step in gluconeogenesis. Of the genes that affect both of these processes, CAMK1D, TSPAN8 and KIF11 affect the localization of a mediator of both gluconeogenesis and glycolysis regulation, CRTC2, to the nucleus in response to glucagon. In addition, the gene CDKAL1 was observed to affect glycogen storage, and molecular experiments using mutant forms of CDK5, a putative target of CDKAL1, in HepG2 cells show that this is mediated by coordinate regulation of CDK5 and PKA on MEK, which ultimately regulates the phosphorylation of ribosomal protein S6, a critical step in the insulin signaling pathway.</p></div

Effect of siRNAs that affect hepatic glucose regulation on glucagon-induced translocation of CRTC2.

<p>Primary hepatocytes treated with siRNAs for 48 hr and 100 nM glucagon for 2 hr. Images show staining of primary human hepatocytes with antibodies to exogenous CRTC2. Images are single fields of hepatocytes treated with (A) NTC (non-targeting control) or (B) GCGR (glucagon receptor) and glucagon treatment. Location of nuclei were determined by staining with DAPI and used to calculate the extent of CRTC1 localization to the nucleus. Nuclei are shown in blue and CRTC2 indirect immunofluorescence is shown in yellow. C. Effect of siRNAs on CRTC2 levels in the nucleus. Images were analyzed and scaled for CRTC2 staining levels in nuclei. Histograms that quantify the extent of CRTC2 nuclear localization for siRNA-treated cells are shown as labeled when treated with glucagon (blue bars) or untreated (green bars). Nuclear CRTC2 levels are in arbitrary units after transformation to a log scale.</p

Effect of the expression of wild type and mutant forms of CDK5 on the phosphorylation of ribosomal protein S6.

<p>Effect of inhibitors of PKA PKC and RSK on RpS6 phosphorylation. Images are HepG2 cells tranduced with lentiviral vectors expressing forms of CDK5, as indicated at the top of each column. Cells were cultured in William's E medium without additional glucagon or insulin. Cultures from each line were treated with protein kinase inhibitors, as indicated by row. DNA is shown in blue and phosphorylated ribosomal protein S6 is shown in red. Treatment with inhibitors was for two hours prior to fixation and staining.</p

The hepatocyte cell culture system and the measurement of perturbations to hepatic glucose regulation.

<p>A. Diagram of the <i>in vitro</i> hepatocyte cell culture system. Primary hepatocytes are plated on an α 1-collagen matrix at a high density. 24 hr later, growth-factor reduced matrigel is added to the culture. B. Indirect immunofluorescence of primary hepatocytes showing nuclei in blue and β-catenin in red. β-catenin lines the cell-cell junctions and highlights the bile canaliculi, deposits of bile acids produced by hepatocytes. C. Indirect immunofluorescence of hepatoctytes stained for nuclei (blue), β-tubulin (green) and actin (red).</p

Quantitative fluorescence imaging of hepatocyte glycogen levels.

<p>A. Fluorescent image of PAS-stained hepatocytes. Staining levels reflect glycogen content. B. Image analysis of PAS-stained hepatocytes. C Quantification of glycogen levels of siRNA-treated hepatocytes by image analysis. Hepatocytes were treated with insulin for 1 hr in medium with a physiological glucose concentration.</p

Media pyruvate levels are dependent on gluconeogenic activity of primary hepatocytes.

<p>Measurement of pyruvate levels in hepatocyte cell culture media following insulin treatment. Sensitivity of the assay is increased as exogenous pyruvate levels are reduced.</p

Statistically significant SNPs identified in GWA studies and linked genes.

<p>Statistically significant SNPs identified in GWA studies and linked genes.</p

Effect of siRNAs on hepatocyte glucose regulation.

<p>siRNAs corresponding to the T2D-replicated GWA loci were transfected into primary hepatocytes. The effects on glycogen accumulation and pyruvate utilization were measured in separate assays.</p