Dataset Information

Regucalcin expression profiles in veal calf testis: validation of histological and molecular tests to detect sex steroids illicit administration.

ABSTRACT:

Background

Sex steroids administration in meat producing animals is forbidden within the EU to preserve consumers' safety, but continuous monitoring to identify resurgence of their misuse is needed. Among biomarkers related to sex steroids abuse in veal calves the regucalcin (RGN) mRNA perturbations in testis have been described in RNAlater samples. To setup novel diagnostic method, to update current tests available in National Residue Control Plans (NRCPs) and in legal dispute when illicit practices on farm animals are suspected, the reliability of RGN profiling was assessed by histological and molecular techniques.

Methods

Formalin fixed paraffin embedded (FFPE) testis samples, chosen being the most effective preservation strategy adopted by histological NRCPs and allowing easier retrospective analysis if required by legal disputes, were analyzed from veal calves treated with nandrolone, 17?-estradiol and a cocktail of the two hormones. RGN levels were determined by quantitative Real Time PCR and Immunohistochemistry assays. Test performances were assessed and compared by multiple ROC curves.

Results

Both tests resulted sensitive and specific, allowing to enrich, in future field investigation, novel integrated diagnostic protocols needed to unveil sex steroid abuse.

Discussion

Developed RT-qPCR and IHC methods confirmed RGN as a useful and robust biomarker to detect illegal administration of sex steroid hormones in veal calves. The developed methods, successfully applied to ten years old FFPE blocks, could allow both retrospective analysis, when supplementary investigations are requested by authorities, and future implementation of current NRCPs.

SUBMITTER: Benedetto A

PROVIDER: S-EPMC7899017 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Publications

Regucalcin expression profiles in veal calf testis: validation of histological and molecular tests to detect sex steroids illicit administration.

Benedetto Alessandro A Biasibetti Elena E Beltramo Chiara C Audino Valentina V Peletto Simone S Bozzetta Elena Maria EM Pezzolato Marzia M

PeerJ 20210219

<h4>Background</h4>Sex steroids administration in meat producing animals is forbidden within the EU to preserve consumers' safety, but continuous monitoring to identify resurgence of their misuse is needed. Among biomarkers related to sex steroids abuse in veal calves the regucalcin (RGN) mRNA perturbations in testis have been described in RNA<i>later</i> samples. To setup novel diagnostic method, to update current tests available in National Residue Control Plans (NRCPs) and in legal dispute wh ...[more]

PMID: 33643712

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Project description:BackgroundAntibiotic therapy is commonly used in animal agriculture. Antibiotics excreted by the animals can contaminate farming environments, resulting in long term exposure of animals to sub-inhibitory levels of antibiotics. Little is known on the effect of this exposure on antibiotic resistance. In this study, we aimed to investigate the long term effects of sub-inhibitory levels of antibiotics on the gut microbiota composition and resistome of veal calves in vivo. Forty-two veal calves were randomly assigned to three groups. The first group (OTC-high) received therapeutic oral dosages of 1 g oxytetracycline (OTC), twice per day, during 5 days. The second group (OTC-low) received an oral dose of OTC of 100-200 μg per day during 7 weeks, mimicking animal exposure to environmental contamination. The third group (CTR) did not receive OTC, serving as unexposed control. Antibiotic residue levels were determined over time. The temporal effects on the gut microbiota and antibiotic resistance gene abundance was analysed by metagenomic sequencing.ResultsIn the therapeutic group, OTC levels exceeded MIC values. The low group remained at sub-inhibitory levels. The control group did not reach any significant OTC levels. 16S rRNA gene-based analysis revealed significant changes in the calf gut microbiota. Time-related changes accounted for most of the variation in the sequence data. Therapeutic application of OTC had transient effect, significantly impacting gut microbiota composition between day 0 and day 2. By metagenomic sequence analysis we identified six antibiotic resistance genes representing three gene classes (tetM, floR and mel) that differed in relative abundance between any of the intervention groups and the control. qPCR was used to validate observations made by metagenomic sequencing, revealing a peak of tetM abundance at day 28-35 in the OTC-high group. No increase in resistance genes abundance was seen in the OTC-low group.ConclusionsUnder the conditions tested, sub-therapeutic administration of OTC did not result in increased tetM resistance levels as observed in the therapeutic group.

Project description:The shift from cure toward prevention in veterinary medicine involves the implementation of biosecurity. In cattle farming, the application of biosecurity measures has been described to a limited degree, yet no data on biosecurity on veal farms is available. A high degree of commingling of veal calves from multiple farms causes frequent disease outbreaks, and thereby high antimicrobial usage and increased risk of antimicrobial resistance. Therefore, this study aimed to describe the current implementation of biosecurity on veal farms in Belgium. To this extent, a list of the most important calf diseases (n?=?34) was created, and risk factors and related biosecurity measures for these diseases were determined and included in a questionnaire. Herd visits and face-to-face interviews were conducted on 20 randomly selected veal farms, comprising 8.3% of the target population. A categorical principal component and clustering analysis were performed to determine the influence of the veal companies on the farms' biosecurity level. Awareness of biosecurity was very low among the farmers. All farms used an "all-in, all-out" production system with calves originating from multiple farms without quarantine. On average, farms were filled in 11.4 days (range 2-52). The degree of commingling for these farms was 1.24, meaning that, on average, 124 calves originated from 100 farms. Veterinarians wore farm-specific boots on eight farms (40%) and farm-specific clothes on six farms (30%), while technical advisors wore farm-specific boots on six farms (30%) and farm-specific clothes on four farms (20%). Disinfection footbaths were only used in five farms (25%) despite being present in all farms in the sample. Concerning internal biosecurity, none of the farmers isolated sick animals; only one farmer (5%) had a physically separated hospital pen, and only 11 farmers (55%) both cleaned and disinfected the stables after each production cycle. In most farms, animals were of comparable age. Healthy calves generally remained in the same compartment during the entire production cycle, limiting the risks associated with the movement of animals. No influence of the integrations on the biosecurity level could be determined. It can be concluded that a few biosecurity measures, such as "'all-in, all-out" and compartmentation, are implemented relatively well, while other measures, such as good cleaning and disinfection and proper entrance measures for visitors and personnel can easily be improved. The improvement of measures regarding the introduction of animals of different origins will require more fundamental changes in the veal industry.

Dataset Information

Regucalcin expression profiles in veal calf testis: validation of histological and molecular tests to detect sex steroids illicit administration.

Background

Methods

Results

Discussion

Publications

Regucalcin expression profiles in veal calf testis: validation of histological and molecular tests to detect sex steroids illicit administration.

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