Haemonchus contortus infection induces a variable immune response in resistant and susceptible Pelibuey sheep.

The immune response and phenotypic characteristics of Pelibuey lambs were analysed after the induction of a Haemonchus contortus trickle infection. Male lambs (n = 29; 20 kg live weight) were infected with 100 H. contortus infective larvae per kg of live weight on day 3, 5 and 7 of the experiment. The number of eggs per gram (epg), seven haematological parameters and the immunoglobulin A (IgA) level were analysed for 56 experimental days. In addition, histopathological samples from the fundic abomasal region and the relative expression of 10 immune-related genes from 15 infected and three non-infected lambs were analysed at day 0 and 49 of the experiment. The epg count and some haematological parameters (leucocytes, red blood cells, haemoglobin and total protein) with statistically significant differences (P < 0.01) were used to identify nine resistant and 20 susceptible lambs (1166 ±â€¯1071 and 3171 ±â€¯1463 epg, respectively). Moreover, acute infiltration of immune cells and parasitic granuloma formation were observed in susceptible lambs; the resistant group had moderate inflammatory cell infiltration. With respect to relative gene expression, resistant lambs showed upregulation (P < 0.001) of 10 genes, from 2.2 to 15.99 fold. Moreover, there was a strong indirect correlation (P < 0.05) between the epg count and interleukin 5 (IL5) gene expression. By contrast, there was an average 0.34 fold downregulation in nine of the immune-related genes (P ≤ 0.05) in susceptible lambs (the only exception was Fc fragment of IgE receptor Ia [FCER1A] upregulation). In addition, there was a direct correlation (P ≤ 0.05) between the epg count and the expression of IL8, which encodes an inflammatory chemokine. In conclusion, this study showed differential IL5 and IL8 gene expression during haemonchosis in resistant and susceptible Pelibuey lambs, respectively, together with a variable immune response based on histopathological and haematological parameters.

Extracellular and intracellular zilpaterol and clenbuterol quantification in Hep G2 liver cells by UPLC-PDA and UPLC-MS/MS.

Zilpaterol and Clenbuterol are ß-adrenergic agonists that have been widely used to feed cattle. Although the use of Zilpaterol has been approved, Clenbuterol is still used illegally at unknown doses. However, the research of both substances has been based mainly on the evaluation of residues. To our knowledge, this is the first time that a cellular model using Hep G2 cells treated with Zilpaterol and Clenbuterol is presented as an alternative approach to quantify both drugs at the cellular level. Thus, a complete analytical methodology has been developed for the accurate quantitation of these ß-adrenergic agonists in both cellular compartments. We propose the use of ultra-performance liquid chromatography with photodiode array detector (UPLC-PDA) for extracellular determinations while UPLC coupled to a tandem mass spectrometer (UPLC-MS/MS) for intracellular analysis. The methods were fully validated in terms of selectivity, linearity, accuracy, and precision, limits of detection and quantitation (LOD and LOQ, respectively), stability, carryover, and matrix effect. The method for intracellular content was linear ranging from 0.25 to 8 ng/mL while for extracellular content, the concentration of Zilpaterol and Clenbuterol ranged from 0.125 to 4 µg/mL, with correlation coefficients of R > 0.98 and >0.99, respectively. The combination of the two methodologies in the cellular model showed intracellular concentrations of 0.344 ± 0.06 µg/mL and 2.483 ± 0.36 µg/mL for Zilpaterol and Clenbuterol, respectively. Extracellular concentration was 0.728 ± 0.14 µg/mL and 0.822 ± 0.11 µg/mL for Zilpaterol and Clenbuterol, respectively. This work shows the potential applications of cellular modelling in the study of toxicity for the mentioned drugs.