Identification Of Animal Species Research Articles

Encephalomyocarditis virus is potentially derived from eastern bent-wing bats living in East Asian countries

Bats are reservoir hosts of many zoonotic viruses and identification of viruses that they carry is important. This study aimed to use high throughput screening to identify the viruses in fecal guano of Taiwanese insectivorous bats caves in order to obtain more information on bat-derived pathogenic viruses in East Asia. Guano samples were collected from two caves in Taiwan, pooled, and then subjected to Multiplex PCR-based next generation sequencing for viral identification. Subsequently, encephalomyocarditis virus (EMCV) sequence was detected and confirmed by reverse transcription PCR. EMCV is considered as rodent virus and thus, animal species identification through cytochrome oxidase I (COI) barcoding was further done to identify the viral source. Finally, determination of distribution and verification of the presence of EMCV in guano obtained from Japanese and South Korean caves was also done. We concluded that the guano collected was not contaminated with the excrement of rodents which were reported and presumed to live in Taiwan. Also, EMCV genome fragments were found in guanos of Japanese and South Korean caves. It is possible that the eastern bent-wing bat (Miniopterus fuliginosus) is one of the natural hosts of EMCV in East Asia.

BARKODING DNA BURUNG ELANG (FAMILI ACCIPITRIDAE) DI INDONESIA

The cytochrome c oxidase subunit I (COI) gene is a reprensentative of all the protein-coding genes of the mitochondrial DNA genome that has been widely used as an animal species identification tool. In this study, 86 sequences of DNA barcodes of members of the family Accipitridae in Indonesia including Nisaetus bartelsi, Nisaetus cirrhatus, Haliaeetus leucogaster, Spilornis cheela, Haliastur indus, and 11 sequences from Genbank were examined. Each species was confirmed through the Basic Local Alignment Search Tool (BLAST). The construction of phylogeny trees based on COI gene sequences was performed by the Neighbors-joining method where the calculation of the genetic distance matrix with the Kimura 2-parameter model was implemented in pairwise distance calculation in the Mega version 6.05 programe. The results of the analysis showed that the divergence within species ranged from 0 to 0.3% (0.13 ± 0.12%), between species ranged from 1.6 to 18.5% (12.8 ± 3.73%), between genera ranged from 13 to 18.6%, and the average in the Accipitridae Family was 11.8%. Therefore, it could form clusters in each species cohesively and clearly separated between the taxa analyzed.

Hair histology as a tool for forensic identification of some domestic animal species.

Animal hair examination at a criminal scene may provide valuable information in forensic investigations. However, local reference databases for animal hair identification are rare. In the present study, we provide differential histological analysis of hair of some domestic animals in Upper Egypt. For this purpose, guard hair of large ruminants (buffalo, camel and cow), small ruminants (sheep and goat), equine (horse and donkey) and canine (dog and cat) were collected and comparative analysis was performed by light microscopy. Based on the hair cuticle scale pattern, type and diameter of the medulla, and the pigmentation, characteristic differential features of each animal species were identified. The cuticle scale pattern was imbricate in all tested animals except in donkey, in which coronal scales were identified. The cuticle scale margin type, shape and the distance in between were characteristic for each animal species. The hair medulla was continuous in most of the tested animal species with the exception of sheep, in which fragmental medulla was detected. The diameter of the hair medulla and the margins differ according to the animal species. Hair shaft pigmentation were not detected in all tested animals with the exception of camel and buffalo, in which granules and streak-like pigmentation were detected. In conclusion, the present study provides a first-step towards preparation of a complete local reference database for animal hair identification that can be used in forensic investigations.

Tackling critical parameters in metazoan meta-barcoding experiments: a preliminary study based on coxI DNA barcode.

Nowadays DNA meta-barcoding is a powerful instrument capable of quickly discovering the biodiversity of an environmental sample by integrating the DNA barcoding approach with High Throughput Sequencing technologies. It mainly consists of the parallel reading of informative genomic fragment/s able to discriminate living entities. Although this approach has been widely studied, it still needs optimization in some necessary steps requested in its advanced accomplishment. A fundamental element concerns the standardization of bioinformatic analyses pipelines. The aim of the present study was to underline a number of critical parameters of laboratory material preparation and taxonomic assignment pipelines in DNA meta-barcoding experiments using the cytochrome oxidase subunit-I (coxI) barcode region, known as a suitable molecular marker for animal species identification. We compared nine taxonomic assignment pipelines, including a custom in-house method, based on Hidden Markov Models. Moreover, we evaluated the potential influence of universal primers amplification bias in qPCR, as well as the correlation between GC content with taxonomic assignment results. The pipelines were tested on a community of known terrestrial invertebrates collected by pitfall traps from a chestnut forest in Italy. Although the present analysis was not exhaustive and needs additional investigation, our results suggest some potential improvements in laboratory material preparation and the introduction of additional parameters in taxonomic assignment pipelines. These include the correct setup of OTU clustering threshold, the calibration of GC content affecting sequencing quality and taxonomic classification, as well as the evaluation of PCR primers amplification bias on the final biodiversity pattern. Thus, careful attention and further validation/optimization of the above-mentioned variables would be required in a DNA meta-barcoding experimental routine.

PCR-RFLP and Sequence Analysis of Hair Cytochrome b Gene for Identification of Animal Species

PCR-RFLP and Sequence Analysis of Hair Cytochrome b Gene for Identification of Animal Species

DNA Barcoding for the Identification and Authentication of Animal Species in Traditional Medicine.

Animal-based traditional medicine not only plays a significant role in therapeutic practices worldwide but also provides a potential compound library for drug discovery. However, persistent hunting and illegal trade markedly threaten numerous medicinal animal species, and increasing demand further provokes the emergence of various adulterants. As the conventional methods are difficult and time-consuming to detect processed products or identify animal species with similar morphology, developing novel authentication methods for animal-based traditional medicine represents an urgent need. During the last decade, DNA barcoding offers an accurate and efficient strategy that can identify existing species and discover unknown species via analysis of sequence variation in a standardized region of DNA. Recent studies have shown that DNA barcoding as well as minibarcoding and metabarcoding is capable of identifying animal species and discriminating the authentics from the adulterants in various types of traditional medicines, including raw materials, processed products, and complex preparations. These techniques can also be used to detect the unlabelled and threatened animal species in traditional medicine. Here, we review the recent progress of DNA barcoding for the identification and authentication of animal species used in traditional medicine, which provides a reference for quality control and trade supervision of animal-based traditional medicine.

A PCR Method That Can Be Further Developed into PCR-RFLP Assay for Eight Animal Species Identification.

There are many PCR-based methods for animal species identification; however, their detection numbers are limited or could not identify unknown species. We set out to solve this problem by developing a universal primer PCR assay for simultaneous identification of eight animal species, including goat, sheep, deer, buffalo, cattle, yak, pig, and camel. In this assay, the variable lengths of mitochondrial DNA were amplified using a pair of universal primers. PCR amplifications yielded 760 bp, 737 bp, 537 bp, 486 bp, 481 bp, 464 bp, 429 bp, and 359 bp length fragments for goat, sheep, deer, buffalo, cattle, yak, pig, and camel, respectively. This primer pair had no cross-reaction with other common domestic animals and fish. The limit of detection varied from 0.01 to 0.05 ng of genomic DNA for eight animal species in a 20 µl PCR mixture. Each PCR product could be further digested into fragments with variable sizes and qualitative analysis by SspI restriction enzyme. This developed PCR-RFLP assay was sufficient to distinguish all targeted species. Compared with the previous published related methods, this approach is simple, with high throughput, fast processing rates, and more cost-effective for routine identification of meat in foodstuffs.

Group Sparse Representation Approach for Recognition of Cattle on Muzzle Point Images

The usage of computer vision adds a new paradigm in the field of animal biometric, and has recently received more attention due to the growing importance of identification and tracking of animal species or individual animals. Biometric characteristics help to develop a better representation and a better identification of different animal species and individual animals. In this work, we propose an effective approach for automatic cattle recognition based on the multiple features of muzzle points and the cattle face images. The proposed method deals the cattle recognition problem as a classification problem among the multiple linear regression models and provides a new theory for the recognition of individual cattle. The group sparse signal representation based classification offers the key to addressing this problem using L2-minimization. In this paper, a comparative study among the well-established handcrafted texture feature extraction techniques and the appearance-based feature extraction techniques is also presented. A detailed set of experimental results on muzzle point image database is also carried to prove the theory. Our method has achieved 93.87% identification accuracy which demonstrates the superiority of the proposed method than the other existing machine learning based recognition algorithms.

Simple and fast multiplex PCR method for detection of species origin in meat products.

Identification of animal species is one of the major concerns in food regulatory control and quality assurance system. Different approaches have been used for species identification in animal origin of feedstuff. This study aimed to develop a multiplex PCR approach to detect the origin of meat and meat products. Specific primers were designed based on the conserved region of mitochondrial Cytochrome C Oxidase subunit I (COX1) gene. This method could successfully distinguish the origin of the pig, camel, sheep, donkey, goat, cow, and chicken in one single reaction. Since PCR products derived from each species represent unique molecular weight, the amplified products could be identified by electrophoresis and analyzed based on their size. Due to the synchronized amplification of segments within a single PCR reaction, multiplex PCR is considered to be a simple, fast, and inexpensive technique that can be applied for identification of meat products in food industries. Nowadays, this technique has been considered as a practical method to identify the species origin, which could further applied for animal feedstuffs identification.

Development of a simultaneous identification method for 13 animal species using two multiplex real-time PCR assays and melting curve analysis.

We developed a simple and rapid method for animal species identification in the forensic science field based on mitochondrial DNA using two multiplex real-time PCRs and analysis of the resultant SYBR Green I melting curves. This method was designed to identify nine domestic animals simultaneously (dog, cat, rabbit, cattle, pig, chicken, goat, sheep and horse) and four wild animals (deer, raccoon-dog, monkey and bear) by comparing the different melting temperatures of the amplicons produced from samples originating from each species. For this analysis, we targeted various mitochondrial genes, including those encoding cytochrome b (cytb), NADH dehydrogenase 5 (ND5), cytochrome c oxidase 3 (COX3), tRNA-ND5, and tRNA-ATP synthase 8 (ATP8). For practical applications, this study presents a validation of this assay including its specificity, sensitivity and robustness. The limits of detection in the multiplex reactions were 10 pg for eight of the nine animals, excluding horse (1 pg for horse). The method was able to correctly identify the animal species from artificial forensic samples including blood stains, saliva, hair and bone, and samples digested in artificial gastric fluid, and for 17 forensic casework samples. The data from the multiplex real-time PCR assays are obtainable only 30 min after DNA extraction of the samples, making the assays useful for screening samples containing DNA from unknown animal origin in the forensic field.

Species Identification of Fox-, Mink-, Dog-, and Rabbit-Derived Ingredients by Multiplex PCR and Real-Time PCR Assay.

Various detection methods have been developed to date for identification of animal species. New techniques based on PCR approach have raised the hope of developing better identification methods, which can overcome the limitations of the existing methods. PCR-based methods used the mitochondrial DNA (mtDNA) as well as nuclear DNA sequences. In this study, by targeting nuclear DNA, multiplex PCR and real-time PCR methods were developed to assist with qualitative and quantitative analysis. The multiplex PCR was found to simultaneously and effectively distinguish four species (fox, dog, mink, and rabbit) ingredients by the different sizes of electrophoretic bands: 480, 317, 220, and 209bp. Real-time fluorescent PCR's amplification profiles and standard curves showed good quantitative measurement responses and linearity, as indicated by good repeatability and coefficient of determination R 2>0.99. The quantitative results of quaternary DNA mixtures including mink, fox, dog, and rabbit DNA are in line with our expectations: R.D. (relative deviation) varied between 1.98 and 12.23% and R.S.D. (relative standard deviation) varied between 3.06 and 11.51%, both of which are well within the acceptance criterion of ≤25%. Combining the two methods is suitable for the rapid identification and accurate quantification of fox-, dog-, mink-, and rabbit-derived ingredients in the animal products.

Loop-Mediated Isothermal Amplification (LAMP): A Rapid and Sensitive Tool for Quality Assessment of Meat Products.

Loop-mediated isothermal amplification (LAMP) is a novel method that amplifies target nucleic acids under isothermal conditions. It is a rapid, specific, and sensitive method, which does not require costly thermal cyclers for the detection of nucleic acids. Thus, it is suitable for on-site detection assays under low-resource settings. It can also be integrated on compact lab-on-a-chip devices for the development of micro-total analysis systems. This review discusses LAMP-based methods, as well as LAMP-based centrifugal, microfluidic, and other fluid-handling devices, which have been developed for the assessment of meat quality parameters that are related to the presence or absence of nucleic acids, for example, animal species identification and microbiological quality. Advances in improving the rapidity, specificity, and sensitivity of LAMP techniques for the assessment of these meat quality parameters are also discussed in this review.

Detection and quantification of beef and pork materials in meat products by duplex droplet digital PCR.

Meat products often consist of meat from multiple animal species, and inaccurate food product adulteration and mislabeling can negatively affect consumers. Therefore, a cost-effective and reliable method for identification and quantification of animal species in meat products is required. In this study, we developed a duplex droplet digital PCR (dddPCR) detection and quantification system to simultaneously identify and quantify the source of meat in samples containing a mixture of beef (Bos taurus) and pork (Sus scrofa) in a single digital PCR reaction tube. Mixed meat samples of known composition were used to test the accuracy and applicability of this method. The limit of detection (LOD) and the limit of quantification (LOQ) of this detection and quantification system were also identified. We conclude that our dddPCR detection and quantification system is suitable for quality control and routine analyses of meat products.

Towards automatic wild animal monitoring: Identification of animal species in camera-trap images using very deep convolutional neural networks

Non-intrusive monitoring of animals in the wild is possible using camera trapping networks. The cameras are triggered by sensors in order to disturb the animals as little as possible. This approach produces a high volume of data (in the order of thousands or millions of images) that demands laborious work to analyze both useless (incorrect detections, which are the most) and useful (images with presence of animals). In this work, we show that as soon as some obstacles are overcome, deep neural networks can cope with the problem of the automated species classification appropriately. As case of study, the most common 26 of 48 species from the Snapshot Serengeti (SSe) dataset were selected and the potential of the Very Deep Convolutional neural networks framework for the species identification task was analyzed. In the worst-case scenario (unbalanced training dataset containing empty images) the method reached 35.4% Top-1 and 60.4% Top-5 accuracy. For the best scenario (balanced dataset, images containing foreground animals only, and manually segmented) the accuracy reached a 88.9% Top-1 and 98.1% Top-5, respectively. To the best of our knowledge, this is the first published attempt on solving the automatic species recognition on the SSe dataset. In addition, a comparison with other approaches on a different dataset was carried out, showing that the architectures used in this work outperformed previous approaches. The limitations of the method, drawbacks, as well as new challenges in automatic camera-trap species classification are widely discussed.

Validation of a DNA biochip for species identification in food forensic science

Authentication of food product ingredients by food control agencies relies on analytical laboratory methods for animal species identification. Such methods must be validated if their results are to be used as acceptable evidence in a court of law. Following the validation guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), we carried out a validation process for the Low Cost and Density (LCD) Array (MEAT 5.0 version) kit for food forensics based on a DNA biochip technology that simultaneously detects 24 animal species. Mixtures of the animal species the kit is designed to detect were prepared at different concentrations and tested on raw/pasteurized, and heated meat and milk matrices. The LCD Array showed high specificity and high sensitivity and it appeared to be robust and repeatable. As such, it provides a useful tool for detecting common adulterants in food items of animal origin.

Molecular identification of cetaceans from the West Atlantic using the E3-I5 region of COI.

Molecular identification is very useful in cases where morphology-based species identification is not possible. Examples for its application in cetaceans include the identification of carcasses of stranded animals in advanced state of decomposition and body parts that are illegally traded. One DNA region that is often used for molecular identification is the Folmer region of the mitochondrial gene cytochrome c oxidase subunit I (COI) (locus 48 to 705 bp). This locus has been used for the identification of several animal species, including whales and dolphins. The goal of the present study was to evaluate the usefulness of another region of COI, the E3-I5 (locus 685 to locus 1179; 495 bp) as a marker for identification of cetaceans from northeastern Canada and northeastern Brazil. The identification markers were successfully obtained for seven cetacean species after performing percent identity and Basic Local Alignment Search Tool analyses. The obtained markers are now publicly available and are useful for the identification of the endangered blue whale (Balaenoptera musculus), common minke whale (B. acutorostrata), vulnerable sperm whale (Physeter macrocephalus), harbor porpoise (Phocoena phocoena), common bottlenose dolphin (Tursiops truncatus), Guiana dolphin (Sotalia guianensis), and melon-headed whale (Peponocephala electra).

Using DNA barcodes to identify forensically important species of Diptera in Espírito Santo State, Brazil

ABSTRACT Accurate insect specimen identification is usually a crucial first step in a forensic entomological analysis. It is traditionally done by morphological classification using identification keys. However, due to sensibility limitations in the identification of animal species based only on their morphology, new methods have been developed, including species identification by DNA barcodes. The objective of this study was to identify forensically important species of Diptera in Espirito Santo state using DNA barcodes. For this, adult flies were collected in Espirito Santo, Southeast Region of Brazil. After DNA extraction, COI gene was amplified and sequenced. All sequences were matched to BOLD platform and alternatively to GenBank MegaBLAST. As result, 281 adult flies were collected and identified morphologically. From these, 36% of samples were classified as Calliphoridae, 34% of Muscidae and 30% of Sarcophagidae. Approximately 10% of all collected samples were analyzes by DNA. It was possible to identify only 35.7% of tested samples, probably due to lack of samples deposited in databases. Therefore, more efforts should be made to deposit a greater variety of dipterous in databases to allow the use of this technique in forensic routine, especially in BOLD.

Real-time PCR is a potential tool to determine the origin of milk used in cheese production

Traceability of foods has become very important problem respect to food quality and typicalness of foods. Cheeses' milk origin cannot be identified by the consumer and they are sold at different prices under various product names. Recently, this has caused the problem of 'adulteration'. The aim of this study is to determine the amount and origin of milk used in cheese production by using real-time PCR which has been used for identification of animal species in dairy products. In this study, 90 different cheeses offered for sale in Turkey of 30 brands from various sources such as cow, sheep and goat have been studied. At the end of the study, only 36.67% of samples were determined to be produced from 100% cow milk. In the remaining 13.33%, a mixture of goat and sheep milk have been found. Only one sample of sheep cheese was produced from 100% sheep milk. In goat cheese samples, 16.67% of cheese was produced from 100% sheep milk and the origin of only 10% was identified as 100% cow milk. No linear relationship could be determined between chemical composition, fatty acids ratios and amount and origin of cheeses (P < 0.05).

Identification of Animal Species and Foreign Tissues in Ready-to-Sell Fresh Processed Meat Products

The demand for ready-to-eat meat products has recently increased, and routine controls are inevitable to provide food safety. Therefore, a total of 250 fresh processed beef meat products collected from local markets and restaurants in the districts of Istanbul mostly with low purchasing power were investigated by histological and biomolecular methods. Chicken tissue was found in 62 (24.8%) of the analysed samples and horse tissue was found in 2 (0.8%) samples, while pork tissue was not detected. Additionally, foreign tissues were detected in 70 (28%) of 250 samples. PCR assays combined with histological examinations can be used as an important method in establishing food safety by determining the deliberate or accidental adulterations of meat products.

8.Animal Species Identification through High Resolution Melting Real Time PCR (HRM) of the Mitochondrial 16S rRNA Gene

Abstract Animal species identification has received growing attention, regarding genetic diversity and food traceability. The objective of this study is to apply a universal primer of part of the mitochondrial 16S rRNA gene analysis using the PCR-RFLP and HRM methods for identification of species origin in cattle, chicken, horse, sheep, pig, buffalo, and goat. PCR product size was 512 bp. The PCR product of 16S rRNA was digested with two restriction enzymes (BclI and MseI); sufficient to easily generate analyzable species-specific restriction profiles that could distinguish the unambiguity of all targeted species. The HRM method successfully identified all species by shape of melting temperature, and proved to be of higher resolution, and a more cost effective, alternative method compared with other identification techniques.