Application of RNA-sequencing to identify biomarkers in broiler chickens prophylactic administered with antimicrobial agents.

Antimicrobial (AM) resistance is largely acknowledged as one of the biggest global health and food safety challenges and the overuse of AMs is known to generate resistance in bacteria that may affect both animals and humans. Poultry meat is the second most-produced meat in the European Union and in recent years consumers are becoming more concerned about food safety, traceability, and animal welfare in poultry rearing system, increasingly requiring meats from broilers reared without AMs. In the present study, we performed RNA sequencing to analyze 64 liver and 54 muscle transcriptomic profiles in broilers reared without treatment or treated with different classes of AMs. Moreover, we validated the most differentially expressed genes among the treated groups to detect putative novel biomarkers able to discriminate meats of broilers reared without AMs. The PDK4, IGFBP1, and RHOB genes were identified as putative novel hepatic biomarkers, discriminating broilers treated with AMs compared to broilers reared without treatments. The whole transcriptome changes revealed the liver as a valuable target organ for AM administration screening. In addition, our results suggest a leading effect of the coccidiostat when associated with AMs, influencing several biological processes. Our study showed that RNA sequencing is a powerful and valuable method to detect aberrant regulated genes and to identify biomarker candidates for AM misuse detection in farm animals. Further validation on larger sample size and a wider spectrum of AMs are needed to confirm the viability of the aforementioned biomarkers in poultry population.

Expression of corticosteroid hormone receptors, prereceptors, and molecular chaperones in hypothalamic-pituitary-adrenal axis and adipose tissue after the administration of growth promoters in veal calves.

The action of glucocorticoids on target tissues is regulated by the glucocorticoid and mineralocorticoid receptors (codified by the NR3C1 and NR3C2 gene, respectively). Moreover, the prereceptor system, represented by the hydroxysteroid 11-beta dehydrogenases (HSD11Bs), catalyzes the interconversion from active glucocorticoids into inactive compounds. This study aimed to determine whether the expression of the prereceptor system, the corticosteroid receptors, and the molecules regulating their intracellular trafficking (FKBP prolyl isomerase 4 and FKBP prolyl isomerase 5) could be regulated in the hypothalamic-pituitary-adrenal axis and in different type of adipose tissue of calves by the administration of dexamethasone in combination with estradiol or prednisolone. Research about the glucocorticoid effects on bovine target tissues may allow development of new diagnostic methods that use potential molecular biomarkers of glucocorticoid treatment. The administration of dexamethasone in combination with estradiol increased the gene expression of HSD11B1 (P < 0.01), HSD11B2 (P < 0.05), NR3C1 (P < 0.01), and NR3C2 (P < 0.01) in the adrenal glands; NR3C2 in the intramuscular adipose tissue (P < 0.01), and HSD11B1 in the subcutaneous adipose tissue (P < 0.01). Prednisolone administration increased the gene expression of HSD11B1 (P < 0.01), NR3C1 (P < 0.05), and NR3C2 (P < 0.05) in the adrenal glands and HSD11B1 (P < 0.01) in the subcutaneous adipose tissue. Interestingly, most of the examined tissues/organs showed a significant variation of FKBP5 gene expression after the administration of dexamethasone in combination with estradiol. So, these changes suggest that the FKBP5 gene expression could be a possible biomarker of the illegal dexamethasone administration in calves.

Dexamethasone and prednisolone treatment in beef cattle: influence on glycogen deposition and gene expression in the liver.

The illegal administration of glucocorticoids in livestock is problematic and identification of pathways in which these hormones are involved is critically important, and new direct or indirect biomarkers should be identified. In this work, glucocorticoid transcriptional effects on some genes involved in the glucose metabolism were studied in the bovine liver. This study was conducted on adult Charolais male cattle treated with long-term low dose dexamethasone or prednisolone. Gene expression analysis was conducted in the liver by qPCR, and the geNorm algorithm was applied to select optimal reference genes. In line with the literature, a significant overexpression of genes involved in the gluconeogenic pathway and glycogen synthesis was detected in the liver of dexamethasone-treated animals, but histological and biochemical examination showed hepatocyte glycogen depletion particularly in dexamethasone-treated animals. It possible to hypothesize that glucocorticoids or adrenal insufficiency due to glucocorticoids withdrawal inhibit the enzymatic activity of glycogen synthase and/or induce glycogen autophagy in bovine liver. In fact, markers of glycophagy as starch-binding domain-containing protein 1 and γ-aminobutyric acid receptor-associated protein-like 1 mRNAs were upregulated in the liver by glucocorticoids treatment. Furthermore, glycogen synthase kinase-3 beta gene was significantly overexpressed in dexamethasone-treated animals, and this protein is also implicated in liver autophagy modulation and glycogen synthesis inhibition. These results showed that glucocorticoids likley have dual roles in hepatic glycogen metabolism of cattle, and investigation of these pathways could help find treatment biomarkers.

Meat quality traits and canonical discriminant analysis to identify the use of illicit growth promoters in Charolais bulls.

The administration of anabolic agents in farm animals to improve meat production has been prohibited in EU, due to the potential risks to human health. Meat quality was investigated to detect the effects of illegal administration of dexamethasone or prednisolone or 17ß-estradiol on Charolais bulls. Three groups of 6 bulls were treated and 12 bulls were the control. Meat quality parameters were measured on live animals, carcasses and on samples of Longissimus thoracis and multivariate statistical data analysis was applied. In Charolais bulls, these parameters were affected by growth promoter administration and the multivariate canonical discriminant analysis was able to distinguish between treated and untreated animals mainly due to three electronic nose's parameters, 24 h carcass temperature and drip loss. Therefore, meat quality control and the multivariate analysis could be useful as a first screening to address targeted controls on farms suspected of illicit use of growth promoters.

Evaluation of nandrolone and ractopamine in the urine of veal calves: liquid chromatography-tandem mass spectrometry approach.

Under European legislation, the use of growth promoters is forbidden in food-producing livestock. The application of unofficial protocols with diverse combinations of veterinary drugs, administered in very low concentrations, hinders reliable detection and subsequent operative prevention. It was observed that nandrolone (anabolic steroid) and ractopamine (ß-adrenergic agonist) are occasionally administered to animals, but little is known about their synergic action when they are administered together. Two specific analytical methods based on liquid chromatography-tandem mass spectrometry have been developed, both of which include hydrolysis of the corresponding conjugates. For the nandrolone method, solid-phase extraction was necessary for the complete elimination of the interferences, while employment of the Quantitation Enhanced Data-Dependent scan mode during MS acquisition of ractopamine enabled the utilization of simple liquid-liquid extraction. The nandrolone method was linear in the range of 0.5-25 ng/mL, while the ractopamine calibration curve was constructed from 0.5 to 1000 ng/mL. The corresponding coefficients of correlations were >0.9907. The lower limit of quantification for both methods was 0.5 ng/mL, followed by overall recoveries >81%. Precisions expressed as relative standard deviations were <17%, while matrix effects were minimal. Urine samples taken at the slaughterhouse from veal calves enrolled in an experimental treatment consisting of intramuscular administration of ß-nandrolone-phenylpropionate accompanied with a ractopamine-enriched diet were analysed. Those methods might be useful for studying the elimination patterns of the administered compounds along with characterization of the main metabolic pathways. Copyright © 2016 John Wiley & Sons, Ltd.

Oxytocin precursor gene expression in bovine skeletal muscle is regulated by 17ß-oestradiol and dexamethasone.

Growth promoter administration, in livestock, potentially poses a major threat to public health, due to the potential endocrine and carcinogenic activity of residues, accumulating in edible tissues, such as skeletal muscle. Therefore, development of new screening tests and methods for the detection of illicit treatments of food animals would be useful. In this study the serum concentrations of oxytocin peptide were measured in beef cattle receiving 17ß oestradiol, dexamethasone or placebo over a period of 40 days. Changes in gene expression of oxytocin precursor in skeletal muscle were also examined in these animals. Serum analysis using an oxytocin EIA kit indicated a significant up-regulation of the biosynthesis of this nonapeptide only in cattle after 17ß oestradiol, but not after dexamethasone or placebo treatment. Quantitative PCR (qPCR) analysis showed a significant overexpression of the oxytocin precursor gene by 33.5 and 13.3-fold in cattle treated with 17ß oestradiol and dexamethasone, respectively, in comparison to placebo treated animals. Regulation of gene expression by some myogenic regulatory factors in skeletal muscle was also evaluated in these animal groups, confirming the activity of both growth promoters on this gene. To investigate the use of the oxytocin precursor gene as biomarker for 17ß oestradiol and dexamethasone treatment in beef cattle, an absolute quantification of this gene by qPCR was developed. A standard curve was generated and developed with TaqMan® technology and optimal criterion value, sensitivity and specificity of this screening method were established through ROC analysis. This analysis suggested that the up-regulation of oxytocin precursor gene expression in skeletal muscle tissue is a valid marker for detection of illicit 17ß oestradiol and/or dexamethasone use in beef cattle. This method may serve as a novel diagnostic tool in the screening phase, and, if introduced in routine testing, may significantly improve overall efficacy and success of the food screening process ordered by state authorities.

Gene expression profiling of thymus in beef cattle treated with prednisolone.

Glucocorticoids (GCs) are extensively used in livestock production, not only for their anti-inflammatory properties but also to improve the quality and quantity of meat in veal and beef production. In Italy, an increase in GC-positive cases has been observed in cattle since 2008, particularly prednisolone (PDN). Recent studies clearly demonstrate that both histopathological analysis and high-performance liquid chromatography tandem mass spectrometry (HPLC/MS-MS) were unable to detect PDN treatments. The aim of this study was to identify transcriptomic signatures of PDN administration in the thymus of experimentally treated animals by comparison with untreated controls, in order to identify gene expression changes or pathways alteration induced by the corticosteroid treatment. Microarray data analysis showed substantial modifications in thymus gene expression profiles after PDN treatment. Several of the 388 differentially expressed genes encoded pro-inflammatory and anti-inflammatory mediators or immune regulators which showed that PDN might have a role in the regulation of immunologic homeostasis, act on both innate and acquired components of the immunity and mainly induce the activation of immune tolerance and anti-inflammatory pathways. Thus, this study allowed to deepen the effects of PDN on the immune system and showed the potentiality of gene expression profiling by DNA-microarray as a powerful tool to complement the existing methods against the illegal use of growth promoting hormones, especially when working on samples collected after slaughtering.

Application of absolute qPCR as a screening method to detect illicit 17ß-oestradiol administration in male cattle.

It has been previously demonstrated that the progesterone receptor gene is up-regulated in the sex accessory glands of pre-pubertal and adult male bovines after 17ß-oestradiol treatment. In the present study, a qualitative screening method was optimised to detect 17ß-oestradiol treatment using absolute quantification by qPCR of the progesterone receptor gene to determine the amount of gene expression in bulbo-urethral glands. An external standard curve was generated and developed with TaqMan® technology. Based on two in vivo experiments, the decision limit CCα, sensitivity and specificity of this screening method were established. Trial 1 consisted of 32 Friesian veal calves divided into two groups: group A (n = 12), consisting of animals treated with four doses of 17ß-oestradiol (5 mg week(-1) per animal); and group B (n = 20), consisting of control animals. Trial 2 was performed on 26 Charolaise beef cattle that either received five doses of 17ß-oestradiol (group C; 20 mg week(-1) per animal; n = 6) or remained untreated (group D; n = 20). Further, progesterone receptor gene expression was evaluated in beef and veal calves for human consumption. A specific CCα on 20 Piedmontese control beef cattle was calculated to include these animals in a field investigation. Five out of 190 beef cattle and 26 out of 177 calves tested expressed the progesterone receptor gene above their respective CCα and they were classified as being suspected of 17ß-oestradiol treatment. Additionally, 58% of veal calves that tested suspect via qPCR exhibited histological lesions of the bulbo-urethral gland tissue, which are typical of oestrogen administration and are consistent with hyperplasia and metaplasia of the glandular epithelium.

Progesterone receptor up-regulation: a diagnostic tool for the illicit use of oestrogens in adult beef cattle.

The monitoring of gene regulation via mRNA levels to detect anabolic sex steroid administration in cattle is a novel approach to detecting the illicit treatment of livestock in meat production. A previous study revealed that progesterone receptor (PR) gene expression levels were increased in the bulbourethral glands and prostates of 17ß-oestradiol-treated prepubertal calves, suggesting that the PR can be used as a specific molecular biomarker for oestrogen treatment. The aim of this study was to verify the specificity and applicability of the PR to detect the illegal use of 17ß-oestradiol in sexually mature beef cattle. Accessory sex glands were sampled from 42 male beef cattle that were divided into six experimental groups, including two control groups, K1 and K2. Group A cattle were treated with 17ß-oestradiol (five weekly intramuscular doses of 20 mg), and group B cattle were treated with dexamethasone (40 daily doses of 0.7 mg per os). Group C cattle received an implant of Revalor-200 (200 mg of trenbolone acetate and 20 mg of 17ß-oestradiol), and group D cattle received Revalor-200 plus dexamethasone (0.7 mg daily per os). 17ß-Oestradiol, either alone or in combination with other steroids, up-regulated the PR gene and protein expression, even in the absence of detectable histological changes in the accessory sex glands, confirming the high sensitivity of PR gene expression as an indirect diagnostic screening tool to detect illicit oestrogen treatment in sexually mature male bovine.

Progesterone receptor gene expression in the accessory sex glands of veal calves.

This study investigated progesterone receptor (PR) cDNA expression in the testes, prostate and bulbourethral glands of prepubertal calves treated experimentally with high and low doses of 17beta-oestradiol and with testosterone. Tissue samples were examined histologically and immunohistochemically for PR. Western blot analysis and quantitative PCR against PR was performed on cDNA and protein extracted from the same tissues. Bulbourethral glands from animals treated with low and high dosages of 17beta-oestradiol had 39- and 429-fold increases of PR transcript, respectively, compared with controls. In the prostate there were 7.5- and 16-fold increases, respectively. Animals treated with testosterone showed no increases in PR transcript. The results demonstrate that 17beta-oestradiol specifically induces marked overexpression of the PR gene and protein, particularly in the bulbourethral gland.

A RIKILT yeast estrogen bioassay (REA) for estrogen residue detection in urine of calves experimentally treated with 17beta-estradiol.

17beta-Estradiol is one of the most powerful sex steroids illegally used in bovine production. The objective of this study was to evaluate the application and the specificity of the RIKILT yeast estrogen bioassay (REA) for the detection of molecules with estrogenic activities in the urine of calves experimentally treated with anabolics. Four groups of six calves each received an injection of 17beta-estradiol intramuscularly (group B), androsterone and gliburide (group A), and testosterone (group C) molecules at different dosage for 40 days. Group D was the control. The ability of the REA test to detect estrogenic activity in urine samples from all animals was assessed. All estrogen-treated animals (group B) showed as being positive up to 7 days after administration of the highest dosage of 17beta-estradiol, while the other three groups showed as being negative. The identity of estrogenic molecules in the urine of group B (17beta-estradiol, 17alpha-estradiol) was confirmed by gas chromatography-mass spectrometry (GC/MS). This is the first time the REA test has been applied to detect 17beta-estradiol in the urine of calves treated with the hormone in vivo. The technique may offer an advantageous laboratory method for the veterinary surveillance of illegal steroid use.

17beta-oestradiol-induced gene expression in cattle prostate: biomarkers to detect illegal use of growth promoters.

The effects of 17beta-oestradiol (E2) on gene expression in cultures of bovine primary prostate stromal cells (BPSCs) and prostate gland tissue were studied. In the first part of the study, BPSCs were grown in the presence of E2 from the first passage to the end of the experiment; a second group was treated in the same way but the treatment was suspended for 48 hours before the end of the experiment; a third group of BPSCs served as a control. In the second part of the study, five male veal calves, aged 130 days, were treated four times intramuscularly with 10 mg of E2 at intervals of two weeks and then euthanased two weeks after the last treatment. Quantitative PCR and immunohistochemistry were used to evaluate the expression of fibroblast growth factor (FGF) receptors (FGFRs), FGFs, progesterone receptor, androgen receptor and oestrogen receptor in BPSCs and prostate tissue. E2 induced a significant over-expression of progesterone receptor in both BPSCs and prostate tissue. There was also a marked up-regulation of FGFR types 1, 2 and 3 genes observed in the BPSCs.

Mucopolysaccharidosis VI in a Siamese/short-haired European cat.

A 3-year-old Siamese/short-haired European cat was referred for clinical disease characterized by dwarfism, facial dysmorphia, paralysis, small and curled ears, corneal clouding and large areas of alopecia. X-ray examination showed multiple bone dysplasia. On the basis of clinical features a form of mucopolysaccharidosis was suspected. The cat, killed at the owner's request, presented several severe skeletal deformities such as long caudal limbs, enlarged thorax with sunken breastbone, vertebral ankylosis in many spinal segments and visceral involvement. Histologically, the cat showed diffuse vacuolization and enlargement of cells in cartilage, bone and visceral organs. Ultrastructurally, membrane-bound vacuoles were filled with fibrillar and fluffy-material or concentrically whorled lamellae. Arylsulphatase B activity was 3.24 nm/mg/h in the affected cat and 30.6 in a normal age-matched control (NC). The L-iduronidase activity was slightly increased. Quantitation of total glycosaminoglycans (GAGs) revealed a 4.5-fold increase in the affected cat as compared with NC, while electrophoretic run of specific GAGs [chondroitin sulphate (CA); hyaluronan (HA); heparan sulphate (HS); dermatan sulphate (DS); keratan sulphate (KS)] performed on a cellulose acetate sheet, showed a striking increase in the DS band. On densitometric analysis of the electrophoretic run stained with Alcian Blue 8GX, the absorption of DS was eight-fold increased as compared with NC. The clinical and morphological features, and the biochemical findings, were consistent with the diagnosis of feline mucopolysaccharidosis VI.

Phenol hydroxylase from Acinetobacter radioresistens is a multicomponent enzyme. Purification and characterization of the reductase moiety.

This paper reports the isolation and characterization of phenol hydroxylase (PH) from a strain belonging to the Acinetobacter genus. An Acinetobacter radioresistens culture, grown on phenol as the only carbon and energy source, produced a multicomponent enzyme system, located in the cytoplasm and inducible by the substrate, that is responsible for phenol conversion into catechol. Because of the wide diffusion of phenol as a contaminant, the present work represents an initial step towards the biotechnological treatment of waste waters containing phenol. The reductase component of this PH system has been purified and isolated in large amounts as a single electrophoretic band. The protein contains a flavin cofactor (FAD) and an iron-sulfur cluster of the type [2Fe-2S]. The function of this reductase is to transfer reducing equivalents from NAD(P)H to the oxygenase component. In vitro, the electron acceptors can be cytochrome c as well as other molecules such as 2, 6-dichlorophenolindophenol, potassium ferricyanide, and Nitro Blue tetrazolium. The molecular mass of the reductase was determined to be 41 kDa by SDS/PAGE and 38.8 kDa by gel permeation; its isoelectric point is 5.8. The N-terminal sequence is similar to those of the reductases from A. calcoaceticus NCIB 8250 (10/12 identity) and Pseudomonas CF600 (8/12 identity) PHs, but much less similar (2/12 identity) to that of benzoate dioxygenase reductase from A. calcoaceticus BD413. Similarly, the internal peptide sequence of the A. radioresistens PH reductase displays a good level of identity (9/10) with both A. calcoaceticus NCIB 8250 and Pseudomonas CF600 PH reductase internal peptide sequences but a poorer similarity (3/10) to the internal peptide sequence of benzoate dioxygenase reductase from A. calcoaceticus BD413.

Beta-galactosidase deficiency in a Korat cat: a new form of feline GM1-gangliosidosis.

A 7-month-old Korat cat was referred for a slowly progressive neurological disease. Circulating monocytes and lymphocytes showed the presence of single or multiple empty vacuoles and blood leukocytes enzyme assay revealed a very low beta-galactosidase activity level (4.7 nmol/mg per h) as compared to unaffected parents and relatives. Histologically, the cat, euthanized at the owner request at 21 months of age, presented diffuse vacuolization and enlargement of neurons throughout the brain, spinal cord and peripheral ganglia, severe cerebellar neuronal cell loss, and moderate astrocytosis. Stored material was stained with periodic acid-Schiff on frozen sections and with the lectins Ricinus conmmunis agglutinin-I, concanavalin A and wheat germ agglutinin on paraffin-embedded sections. Ultrastructurally, neuronal vacuoles were filled with concentrically whorled lamellae and small membrane-bound vesicles. In the affected cat, beta-galactosidase activity was markedly reduced in brain (18.9%) and liver (33.25%), while total beta-hexosaminidase activity showed a remarkable increase. Quantitation of total gangliosides revealed a 3-fold increase in brain and 1.7-fold in liver of affected cat. High-performance thin layer chromatography (HPTLC) detected a striking increase of GM1-ganglioside. On densitometric analysis of HPTLC bands, the absorption of GM1-ganglioside band was 98.52% of all stained bands (GD1a, GD1b, GT1b). Based on clinical onset, morphological and histochemical features, and biochemical findings, the Korat cat GM1-gangliosidosis is comparable with the human type II (juvenile) form. However, clinical progression, survival time and level of beta-galactosidase deficiency do not completely fit with those of human type II GM1-gangliosidosis. The disease in the Korat cat is also different from other reported forms of feline GM1-gangliosidosis.