Development of Electrochemical DNA Biosensor for Equine Hindgut Acidosis Detection.

The pH drop in the hindgut of the horse is caused by lactic acid-producing bacteria which are abundant when a horse's feeding regime is excessively carbohydrate rich. This drop in pH below six causes hindgut acidosis and may lead to laminitis. Lactic acid-producing bacteria Streptococcus equinus and Mitsuokella jalaludinii have been found to produce high amounts of L-lactate and D-lactate, respectively. Early detection of increased levels of these bacteria could allow the horse owner to tailor the horse's diet to avoid hindgut acidosis and subsequent laminitis. Therefore, 16s ribosomal ribonucleic acid (rRNA) sequences were identified and modified to obtain target single stranded deoxyribonucleic acid (DNA) from these bacteria. Complementary single stranded DNAs were designed from the modified target sequences to form capture probes. Binding between capture probe and target single stranded deoxyribonucleic acid (ssDNA) in solution has been studied by gel electrophoresis. Among pairs of different capture probes and target single stranded DNA, hybridization of Streptococcus equinus capture probe 1 (SECP1) and Streptococcus equinus target 1 (SET1) was portrayed as gel electrophoresis. Adsorptive stripping voltammetry was utilized to study the binding of thiol modified SECP1 over gold on glass substrates and these studies showed a consistent binding signal of thiol modified SECP1 and their hybridization with SET1 over the gold working electrode. Cyclic voltammetry and electrochemical impedance spectroscopy were employed to examine the binding of thiol modified SECP1 on the gold working electrode and hybridization of thiol modified SECP1 with the target single stranded DNA. Both demonstrated the gold working electrode surface was modified with a capture probe layer and hybridization of the thiol bound ssDNA probe with target DNA was indicated. Therefore, the proposed electrochemical biosensor has the potential to be used for the detection of the non-synthetic bacterial DNA target responsible for equine hindgut acidosis.

Regulation of veterinary point-of-care testing in the European Union, the United States of America and Japan.

Point-of-care testing (POCT) is used to detect diseases and other conditions or to monitor therapeutic procedures. In veterinary medicine, POCT not only helps during the prevention, diagnosis and treatment of animal diseases but it also has a direct impact on human health by safeguarding food supplies and preventing zoonoses. Despite its importance, the regulation of the quality, safety and effectiveness of POCT products is rarely discussed. This review reveals that the level of regulatory surveillance of veterinary POCT products in the European Union (EU), the United States of America and Japan is strikingly different, ranging from no regulation (EU) to comprehensive regulation, which is comparable to the procedures for the regulation of human in vitro medical devices (Japan). Details about the licensing procedures in these three locations, discussion of their strengths and weaknesses, and suggestions for possible future development of the regulation of these products are also provided.

Les tests utilisables sur le lieu d'intervention (POCT) permettent de détecter des maladies ou d'autres affections et d'assurer un suivi des procédures thérapeutiques appliquées. En médecine vétérinaire, les POCT apportent un soutien utile lors de la prévention, du diagnostic et du traitement des maladies animales, en plus d'avoir un impact direct sur la santé humaine en participant à la sécurité de l'approvisionnement alimentaire et à la prévention des zoonoses. La réglementation applicable aux produits utilisés pour les POCT, afin d'en garantir la qualité, la sécurité et l'efficacité, fait rarement l'objet de débats, bien qu'il s'agisse d'une question importante. L'examen présenté par les auteurs révèle que le niveau de la surveillance réglementaire exercée sur les produits vétérinaires utilisés pour les POCT dans l'Union européenne (UE), les états-Unis d'Amérique et le Japon est extrêmement variable, allant d'une absence totale de réglementation (UE) à une réglementation exhaustive comparable aux procédures appliquées pour les dispositifs in vitro utilisés en médecine humaine (Japon). Les auteurs décrivent en détail les procédures d'autorisation de mise sur le marché dans ces trois pays, examinent leurs atouts et points faibles respectifs et font quelques propositions pour faire évoluer la réglementation de ces produits à l'avenir.

Las pruebas realizadas en el punto de consulta, o «pruebas de diagnóstico inmediato¼, sirven para detectar enfermedades y otros trastornos o para seguir de cerca los resultados de procedimientos terapéuticos. En medicina veterinaria, estas pruebas no solo ayudan a prevenir, diagnosticar y tratar enfermedades animales, sino que también tienen una influencia directa en la salud humana, en la medida en que permiten proteger los suministros alimentarios y prevenir zoonosis. A pesar de su importancia, rara vez se aborda el tema de la regulación de la calidad, seguridad y eficacia de los productos que contienen pruebas de diagnóstico inmediato. Los autores revelan aquí llamativas disparidades en los regímenes de vigilancia reglamentaria que aplican a las pruebas de diagnóstico veterinario inmediato la Unión Europea (UE), los Estados Unidos de América (EE.UU.) y el Japón, regímenes que van desde la inexistencia de reglamentos (UE) hasta una regulación exhaustiva comparable a los procedimientos aplicados a los dispositivos médicos in vitro (Japón). Los autores también exponen en detalle los procedimientos de obtención de licencia vigentes en estos tres contextos, examinan sus ventajas e inconvenientes y formulan propuestas para el futuro desarrollo de la reglamentación aplicada a los antedichos productos.

Diagnostics in the veterinary field: The role in health surveillance and disease identification.

An international workshop, held in Wiesbaden, Germany on 15-17 May 2019 provided an overview of existing and new methods and approaches to diagnostics in animal health and their benefits and challenges. The variability in quality and authority review of test kits across the world is a concern for the reliability of test results and the decisions that are based on the diagnostic data. In countries or regions without regulatory oversight, there is an urgent need for international harmonisation of quality requirements and licensing procedures. This would increase the validity of the diagnostic methods and allow mutual recognition of test results within the network of official control laboratories and amongst animal health officials. Regional cooperation, as well as the OIE Laboratory Network, should be used to support licensing procedures, pool resources for serum and sample banks, survey outbreak responses, and coordinate research and development of new veterinary diagnostics. The end-users must have clear information on a test's performance, limitations, and interpretation of results.

Multilaboratory Evaluation of a Novel Lateral Flow Immunochromatographic Assay for Confirming Isolation of Mycobacterium bovis from Veterinary Diagnostic Specimens.

A novel lateral flow immunochromatographic device (LFD) was evaluated in several veterinary diagnostic laboratories. It was confirmed to be specific for Mycobacterium bovis and M.caprae cells. The performance of the novel LFD was assessed relative to the confirmatory tests routinely applied after culture (spoligotyping or quantitative PCR [qPCR]) in each laboratory; liquid (MGIT or BacT/Alert) and/or solid (Stonebrink, Coletsos, or Lowenstein-Jensen) cultures were tested. In comparison to spoligotyping of acid-fast-positive MGIT cultures, percent agreement between positive LFD and spoligotyping results was excellent in two United Kingdom laboratories (97.7 to 100%) but lower in the Spanish context (76%), where spoligotyping was applied to MGIT cultures previously confirmed to be positive for M. tuberculosis complex (MTBC) by qPCR. Certain spoligotypes of M. bovis and M. caprae were not detected by the LFD in Spanish MGIT cultures. Compared to qPCR confirmation, the agreement between positive LFD and qPCR results was 42.3% and 50% for BacT/Alert and MGIT liquid cultures, respectively, and for solid cultures, it ranged from 11.1 to 89.2%, depending on the solid medium employed (Coletsos, 11.1%; Lowenstein-Jensen, 55.6%; Stonebrinks, 89.2%). Correlation between the novel LFD and BD MGIT TBc Identification test results was excellent when 190 MGIT cultures were tested (r = 0.9791; P < 0.0001), with the added benefit that M. bovis was differentiated from another MTBC species in one MGIT culture by the novel LFD. This multilaboratory evaluation demonstrated the novel LFD's potential utility as a rapid test to confirm isolation of M. bovis and M. caprae from veterinary specimens following culture.

An indirect enzyme-linked immunosorbent assay for the identification of antibodies to Senecavirus A in swine.

BACKGROUND: Senecavirus A (SVA), a member of the family Picornaviridae, genus Senecavirus, is a recently identified single-stranded RNA virus closely related to members of the Cardiovirus genus. SVA was originally identified as a cell culture contaminant and was not associated with disease until 2007 when it was first observed in pigs with Idiopathic Vesicular Disease (IVD). Vesicular disease is sporadically observed in swine, is not debilitating, but is significant due to its resemblance to foreign animal diseases, such as foot-and-mouth disease (FMD), whose presence would be economically devastating to the United States. IVD disrupts swine production until foreign animal diseases can be ruled out. Identification and characterization of SVA as a cause of IVD will help to quickly rule out infection by foreign animal diseases. METHODS: We have developed and characterized an indirect ELISA assay to specifically identify serum antibodies to SVA. Viral protein 1, 2 and 3 (VP1, VP2, VP3) were expressed, isolated, and purified from E. coli and used to coat plates for an indirect ELISA. Sera from pigs with and without IVD symptoms as well as a time course following animals from an infected farm, were analyzed to determine the antibody responses to VP1, VP2, and VP3. RESULTS: Antibody responses to VP2 were higher than VP1 and VP3 and showed high affinity binding on an avidity ELISA. ROC analysis of the SVA VP2 ELISA showed a sensitivity of 94.2% and a specificity of 89.7%. Compared to IFA, the quantitative ELISA showed an 89% agreement in negative samples and positive samples from 4-60 days after appearance of clinical signs. Immune sera positive for FMDV, encephalomyocarditis virus, and porcine epidemic diarrhea virus antibodies did not cross-react. CONCLUSIONS: A simple ELISA based on detection of antibodies to SVA VP2 will help to differentially diagnose IVD due to SVA and rule out the presence of economically devastating foreign animal diseases.

A review of the current state of antimicrobial susceptibility test methods for Brachyspira.

The re-emergence of swine dysentery (Brachyspira-associated muco-haemorrhagic colitis) since the late 2000s has illuminated diagnostic challenges associated with this genus. The methods used to detect, identify, and characterize Brachyspira from clinical samples have not been standardized, and laboratories frequently rely heavily on in-house techniques. Particularly concerning is the lack of standardized methods for determining and interpreting the antimicrobial susceptibility of Brachyspira spp. The integration of laboratory data into a treatment plan is a critical component of prudent antimicrobial usage. Therefore, the lack of standardized methods is an important limitation to the evidence-based use of antimicrobials. This review will focus on describing the methodological limitations and inconsistencies between current susceptibility testing schemes employed for Brachyspira, provide an overview of what we do know about the susceptibility of these organisms, and suggest future directions to improve and standardize diagnostic strategies.

Opportunities and challenges for the application of microfluidic technologies in point-of-care veterinary diagnostics.

There is a growing need for low-cost, rapid and reliable diagnostic results in veterinary medicine. Point-of-care (POC) tests have tremendous advantages over existing laboratory-based tests, due to their intrinsic low-cost and rapidity. A considerable number of POC tests are presently available, mostly in dipstick or lateral flow formats, allowing cost-effective and decentralised diagnosis of a wide range of infectious diseases and public health related threats. Although, extremely useful, these tests come with some limitations. Recent advances in the field of microfluidics have brought about new and exciting opportunities for human health diagnostics, and there is now great potential for these new technologies to be applied in the field of veterinary diagnostics. This review appraises currently available POC tests in veterinary medicine, taking into consideration their usefulness and limitations, whilst exploring possible applications for new and emerging technologies, in order to widen and improve the range of POC tests available.

Outcome of a one-week intensive training workshop for veterinary diagnostic laboratory workers in Liberia.

There is a huge unmet need for veterinary diagnostic laboratory services in developing nations such as Liberia. One way of bridging the service gap is for visiting experts to provide veterinary laboratory training to technicians in a central location in a short-course format. An intensive 1-week training workshop was organized for 18 student and faculty participants from the College of Agriculture and Integrated Development Studies (CAIDS) at Cuttington University in rural central Liberia. The training was designed and delivered by the non-governmental organization Veterinarians Without Borders US and funded through a Farmer-to-Farmer grant provided by the United States Agency for International Development. Although at the start of training none of the students had any veterinary laboratory experience, by the end of the course over 80% of the students were able to discuss appropriate care and use of a microscope and name at least three important components of laboratory record keeping; over 60% were able to describe how to make and stain a blood smear and how to perform a passive fecal flotation; and over 30% were able to describe what a packed cell volume is and how it is measured and name at least three criteria for classifying bacteria. The intensive training workshop greatly improved the knowledge of trainees about veterinary diagnostic laboratory techniques. The training provided initial skills to students and faculty who are awaiting the arrival of additional grant-funded laboratory equipment to continue their training.

Comparison of atlantoaxial and lumbosacral cerebrospinal fluid centesis techniques in South American camelids.

BACKGROUND: Iatrogenic blood contamination during cerebrospinal fluid (CSF) centesis is common, which can limit the diagnostic usefulness of the sample. A novel ultrasound-guided CSF collection technique is described in horses, by which CSF is obtained from the atlantoaxial (AA) space. HYPOTHESIS/OBJECTIVES: To compare ultrasound-guided AA centesis with lumbosacral (LS) centesis in South American camelids (SAC). The hypotheses were that AA centesis would yield samples with less blood contamination although being technically more challenging than LS centesis. ANIMALS: Eight clinically healthy adult SAC from a university-owned teaching herd. METHODS: Single-blinded, randomized, 4-way, 4-period crossover study in which 2 veterinarians each performed both centesis techniques on each animal once. Cytological sample analysis was performed, and the technical difficulty of sample acquisition was assessed. RESULTS: The CSF was collected successfully and without complications by either technique during all collection attempts. Aspects of technical difficulty and concentrations of CSF analytes did not vary significantly between techniques. Median total nucleated cell and red blood cell counts were 1/µL and 0.5/µL and 167.5/µL and 155/µL for AA and LS techniques, respectively. The median total protein concentration was 32.9 mg/dL and 38 mg/dL for AA and LS centeses. A median of 1 attempt was necessary for both centesis techniques and the median number of needle repositioning events was 1 for AA and 0 for LS. CONCLUSION AND CLINICAL IMPORTANCE: Depending on clinical circumstances, ultrasound-guided AA centesis appears to be an acceptable alternative to other techniques for collection of CSF from SAC.

Lateral flow paired with RT-LAMP: A speedy solution for Influenza A virus detection in swine.

Influenza A Virus in swine (IAV-S) is a zoonotic pathogen that is nearly ubiquitous in commercial swine in the USA. Swine possess sialic acid receptors that allow co-infection of human and avian viruses with the potential of pandemic reassortment. We aimed to develop a fast and robust testing method for IAV-S detection on swine farms. Two primers of the RT-LAMP assay were labeled for use in a lateral flow readout. A commercially available lateral flow kit was used to read the amplicon product. With a runtime of ∼ 45 minutes, the limit of detection for the assay is comparable with an RT-qPCR Cq less than 35, with a sensitivity of 83.5 % and a specificity of 89.6 %. This assay allows veterinarians and producers with limited access to diagnostic services to perform and detect Matrix gene amplification on-site with low equipment costs. The time from sample collection to detection is less than one hour, making this method an accessible, convenient, and affordable tool to prevent the spread of zoonotic disease.

Diagnosis of Ruminant Viral Diseases with Loop-Mediated Isothermal Amplification.

Infectious diseases in livestock industry are major problems for animal health, food safety, and the economy. Zoonotic diseases from farm animals are significant threat to human population as well. These are notifiable diseases listed by the World Organization for Animal Health (OIE). Rapid diagnostic methods can help keep infectious diseases under control in herds. Loop-mediated isothermal amplification (LAMP) is a simple and rapid nucleic acid amplification method that is studied widely for detection of many infectious diseases in the field. LAMP allows biosensing of target DNA or RNA under isothermal conditions with high specificity in a short period of time. An untrained user can analyze results based on color change or turbidity. Here we review LAMP assays to diagnose OIE notifiable ruminant viral diseases in literature highlighting properties of LAMP method considering what is expected from an efficient, field usable diagnostic test.

RT-LAMP as Diagnostic Tool for Influenza-A Virus Detection in Swine.

Point-of-care diagnostic technologies are becoming more widely available for production species. Here, we describe the application of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect the matrix (M) gene of influenza A virus in swine (IAV-S). M-specific LAMP primers were designed based on M gene sequences from IAV-S isolated in the USA between 2017 and 2020. The LAMP assay was incubated at 65 °C for 30 min, with the fluorescent signal read every 20 s. The assay's limit of detection (LOD) was 20 M gene copies for direct LAMP of the matrix gene standard, and 100 M gene copies when using spiked extraction kits. The LOD was 1000 M genes when using cell culture samples. Detection in clinical samples showed a sensitivity of 94.3% and a specificity of 94.9%. These results show that the influenza M gene RT-LAMP assay can detect the presence of IAV in research laboratory conditions. With the appropriate fluorescent reader and heat block, the assay could be quickly validated as a low-cost, rapid, IAV-S screening tool for use on farms or in clinical diagnostic labs.

A study on the occurrence of Burkholderia cepacia complex in ultrasound gels used in different veterinary clinical settings in India.

Burkholderia cepacia complex (Bcc) organisms are emerging multidrug-resistant pathogens. They are opportunistic and cause severe diseases in humans that may result in fatal outcomes. They are mainly reported as nosocomial pathogens, and transmission often occurs through contaminated pharmaceutical products. From 1993 to 2019, 14 Bcc outbreaks caused by contaminated ultrasound gels (USGs) have been reported in several countries, including India. We screened a total of 63 samples of USGs from various veterinary and human clinical care centers across 17 states of India and isolated 32 Bcc strains of Burkholderia cenocepacia (46.8%), B. cepacia (31.3%), B. pseudomultivorans (18.8%) and B. contaminans (3.1%) species. Some isolates were co-existent in a single ultrasound gel sample. The isolation from unopened gel bottles revealed the intrinsic contamination from manufacturing sites. The MALDI-TOF analysis to identify the Bcc at the species level was supported by the partial sequencing of the recA gene for accurate species identification. The phylogenetic analysis revealed that isolates shared clades with human clinical isolates, which is an important situation because of the possible infections of Bcc by USGs both in humans and animals. The pulsed field gel electrophoresis (PFGE) typing identified the genetic variation among the Bcc isolates present in the USGs. The findings indicated USGs as the potential source of Bcc species.

Dried Blood Spots technology for veterinary applications and biological investigations: technical aspects, retrospective analysis, ongoing status and future perspectives.

Dried Blood Spots (DBS) technology has become a valuable tool in medical studies, however, in veterinary and biological research DBS technology applications are still limited. Up-to-date no review has comprehensively integrated all the evidence existing across the fields, technologies and animal species. In this paper we summarize the current applications of DBS technology in the mentioned areas, and provide a scope of different types of dried sample carriers (cellulose and non-cellulose), sampling devices, applicable methods for analyte extraction and detection. Mammals, birds, insects and other species are represented as the study objects. Besides the blood, the review considers a variety of specimens, such as milk, saliva, tissue samples and others. The main applications of dried samples highlighted in the review include epidemiological surveys and monitoring for infections agents or specific antibodies for disease/vaccination control in households and wildlife. Besides the genetic investigations, the paper describes detection of environmental contaminants, pregnancy diagnosis and many other useful applications of animal dried samples. The paper also analyses dried sample stability and storage conditions for antibodies, viruses and other substances. Finally, recent developments and future research for DBS technology in veterinary medicine and biological sciences are discussed.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry in veterinary medicine: Recent advances (2019-present).

Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) has become a valuable laboratory tool for rapid diagnostics, research, and exploration in veterinary medicine. While instrument acquisition costs are high for the technology, cost per sample is very low, the method requires minimal sample preparation, and analysis is easily conducted by end-users requiring minimal training. Matrix-assisted laser desorption ionization-time-of-flight MS has found widespread application for the rapid identification of microorganisms, diagnosis of dermatophytes and parasites, protein/lipid profiling, molecular diagnostics, and the technique demonstrates significant promise for 2D chemical mapping of tissue sections collected postmortem. In this review, an overview of the MALDI-TOF technique will be reported and manuscripts outlining current uses of the technology for veterinary science since 2019 will be summarized. The article concludes by discussing gaps in knowledge and areas of future growth.

Development of a novel real-time RT-PCR assay to detect Seneca Valley virus-1 associated with emerging cases of vesicular disease in pigs.

Seneca Valley virus 1 (SVV-1) can cause vesicular disease that is clinically indistinguishable from foot-and-mouth disease, vesicular stomatitis and swine vesicular disease. SVV-1-associated disease has been identified in pigs in several countries, namely USA, Canada, Brazil and China. Diagnostic tests are required to reliably detect this emerging virus, and this report describes the development and evaluation of a novel real-time (r) reverse-transcription (RT) PCR assay (rRT-PCR), targeting the viral polymerase gene (3D) of SVV-1. This new assay detected all historical and contemporary SVV-1 isolates examined (n=8), while no cross-reactivity was observed with nucleic acid templates prepared from other vesicular disease viruses or common swine pathogens. The analytical sensitivity of the rRT-PCR was 0.79 TCID50/ml and the limit of detection was equivalent using two different rRT-PCR master-mixes. The performance of the test was further evaluated using pig nasal (n=25) and rectal swab samples (n=25), where concordant results compared to virus sequencing were generated for 43/50 samples. The availability of this assay, will enable laboratories to rapidly detect SVV-1 in cases of vesicular disease in pigs, negated for notifiable diseases, and could enable existing knowledge gaps to be investigated surrounding the natural epidemiology of SVV-1.

Optimal Use of Vaccines for Control of Influenza A Virus in Swine.

Influenza A virus in swine (IAV-S) is one of the most important infectious disease agents of swine in North America. In addition to the economic burden of IAV-S to the swine industry, the zoonotic potential of IAV-S sometimes leads to serious public health concerns. Adjuvanted, inactivated vaccines have been licensed in the United States for over 20 years, and there is also widespread usage of autogenous/custom IAV-S vaccines. Vaccination induces neutralizing antibodies and protection against infection with very similar strains. However, IAV-S strains are so diverse and prone to mutation that these vaccines often have disappointing efficacy in the field. This scientific review was developed to help veterinarians and others to identify the best available IAV-S vaccine for a particular infected herd. We describe key principles of IAV-S structure and replication, protective immunity, currently available vaccines, and vaccine technologies that show promise for the future. We discuss strategies to optimize the use of available IAV-S vaccines, based on information gathered from modern diagnostics and surveillance programs. Improvements in IAV-S immunization strategies, in both the short term and long term, will benefit swine health and productivity and potentially reduce risks to public health.

SNAP Assay Technology.

The most widely used immunoassay configuration is the enzyme-linked immunosorbent assay (ELISA) because the procedure produces highly sensitive and specific results and generally is easy to use. By definition, ELISAs are immunoassays used to detect a substance (typically an antigen or antibody) in which an enzyme is attached (conjugated) to one of the reactants and an enzymatic reaction is used to amplify the signal if the substance is present. Optimized ELISAs include several steps that are performed in sequence using a defined protocol that typically includes application of sample and an enzyme-conjugated antibody or antigen to an immobilized reagent, followed by wash and enzyme reaction steps. The SNAP assay is an in-clinic device that performs each of the ELISA steps in a timed sequential fashion with little consumer interface. The components and mechanical mechanism of the assay device are described. Detailed descriptions of features of the assay, which minimize nonspecific binding and enhance the ability to read results from weak-positive samples, are given. Basic principles used in assays with fundamentally different reaction mechanisms, namely, antigen-detection, antibody-detection, and competitive assays are given. Applications of ELISA technology, which led to the development of several multianalyte SNAP tests capable of testing for up to 6 analytes using a single-sample and a single-SNAP device are described.

The potential of circulating extracellular small RNAs (smexRNA) in veterinary diagnostics-Identifying biomarker signatures by multivariate data analysis.

Worldwide growth and performance-enhancing substances are used in cattle husbandry to increase productivity. In certain countries however e.g., in the EU, these practices are forbidden to prevent the consumers from potential health risks of substance residues in food. To maximize economic profit, 'black sheep' among farmers might circumvent the detection methods used in routine controls, which highlights the need for an innovative and reliable detection method. Transcriptomics is a promising new approach in the discovery of veterinary medicine biomarkers and also a missing puzzle piece, as up to date, metabolomics and proteomics are paramount. Due to increased stability and easy sampling, circulating extracellular small RNAs (smexRNAs) in bovine plasma were small RNA-sequenced and their potential to serve as biomarker candidates was evaluated using multivariate data analysis tools. After running the data evaluation pipeline, the proportion of miRNAs (microRNAs) and piRNAs (PIWI-interacting small non-coding RNAs) on the total sequenced reads was calculated. Additionally, top 10 signatures were compared which revealed that the readcount data sets were highly affected by the most abundant miRNA and piRNA profiles. To evaluate the discriminative power of multivariate data analyses to identify animals after veterinary drug application on the basis of smexRNAs, OPLS-DA was performed. In summary, the quality of miRNA models using all mapped reads for both treatment groups (animals treated with steroid hormones or the ß-agonist clenbuterol) is predominant to those generated with combined data sets or piRNAs alone. Using multivariate projection methodologies like OPLS-DA have proven the best potential to generate discriminative miRNA models, supported by small RNA-Seq data. Based on the presented comparative OPLS-DA, miRNAs are the favorable smexRNA biomarker candidates in the research field of veterinary drug abuse.