Wild animal nursing degree evaluation technology based on fecal tetracycline drug-resistant genes

Abstract

The invention constructs a multi-index system for quantitatively evaluating the interference degree of wild animals to human activities and the relationship between the wild animals and human relatives on the basis of the tetracycline drug-resistant genes in feces of the wild animals. According to the system, a quantitative evaluation system is established by analyzing abundance, diversity and expression potential (average expression quantity of a target gene) of tetracycline drug-resistant genes in a wild animal manure microbiome and coupling a human activity interference degree gradient of a sampling site. The innovation of the method lies in that the expression potential of the drug-resistant gene is introduced as a key index, the limitation of only dependence on abundance and diversity of the drug-resistant gene is broken through, and multi-index evaluation is realized; taking a lowest interference degree (such as natural wild animals) or a lowest tetracycline drug-resistant gene index sample as a reference, and setting a threshold value based on polymorphic characteristics and research requirements to construct a multi-stage evaluation system; according to the method, quantitative evaluation of the molecular level is achieved, a support tool is provided for re-introduction planning of endangered species, habitat ecological monitoring, wild animal protection area management and the like, the scientificity and timeliness of a wild animal protection strategy are improved, and the method is a beneficial supplement of a traditional method based on investigation and macroscopic index evaluation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of wildlife conservation, and particularly relates to a method for quantifying the degree of disturbance of wild animals (including mammals, birds, etc.) by human activities or the relationship between wild animals and humans by analyzing the abundance and diversity characteristics of tetracycline resistance genes (Tetra-RGs) in the feces of wild animals. The core innovation of the method lies in the fact that tetracycline resistance genes have two sources, natural and artificial. The use of tetracycline drugs for breeding livestock and poultry and human treatment enhances the environmental resistance gene abundance, and the fecal tetracycline resistance gene abundance is affected by the diet of wild animals, thereby characterizing the degree of disturbance of wild animals by human activities or the relationship between wild animals and humans. This characterization technology is different from traditional large-scale surveys of wild animal behavior, population and habitat surveys, which require a lot of manpower and time. The characterization can be performed without disturbance by collecting the feces of target wild animals or multiple wild animals in one survey. The characterization method has been quantitatively verified in the evaluation of the release progress of Przewalski's horses. BACKGROUND

[0002] With the continuous expansion of the influence of human activities on natural ecosystems, leading to habitat fragmentation of wild animals, the survival of wild animals has been seriously threatened. How to quantify and characterize the influence of human activities on wild animals or threats has become a frontier of conservation biology research. Traditional evaluation systems (such as behavioral observation and habitat quality assessment) mainly rely on the adaptive response of wild animals to habitat changes or assess the ecological environmental carrying capacity based on habitat quality parameters. Population surveys based on survival rate or reproductive success rate assess the biodiversity maintenance ability from the perspective of population dynamics. Building a rapid evaluation system for the degree of disturbance of wild animals by human activities and their relationship with humans is a useful supplement to traditional evaluation methods, which is beneficial to timely evaluate the degree of influence of habitat disturbance and food shortage on wild animals, and provide decision-making basis for timely developing protection and conservation measures and countermeasures.

[0003] The use of antibiotics in aquaculture and medical industries leads to the excretion of unmetabolized antibiotics and their induced resistance genes into the environment, which enhances the environmental resistance. When wild animals consume water and food contaminated by antibiotics and their resistance genes, their intestinal microbes are exposed to environmental residues of antibiotics and their resistance genes, leading to an increase in the abundance of resistance genes in their feces. Tetracycline antibiotics are derived from soil microorganisms, and tetracycline antibiotics and their resistance genes exist in the natural environment. After industrial production, they are now widely used in livestock and aquaculture, and historically used for human disease treatment. The concentration of tetracycline antibiotics and their resistance genes in the environment of human activity area is high. Compared with other antibiotic resistance genes (such as artificially synthesized quinolones), the selection of tetracycline resistance genes as a biological indicator not only has natural continuity (i.e. natural background value) but also has the effect of human interference proliferation, so it can be used to continuously quantify the degree of wild animals affected by human activities and their relationship with humans. At the same time, there are 46 known tetracycline resistance genes widely distributed in different environmental media and animal feces, which can be used for diversity analysis, enriching the connotation of tetracycline resistance genes as a biological indicator, and can be used to further interpret the degree of wild animals affected by human activities and their relationship with humans in and across ecosystems. For example, whether the migration path of migratory birds and their habitat, stopover and wintering ground selection are affected by human activities, and the process of wilding of flagship species such as giant pandas and Przewalski's horses. The mature gene chip and high-throughput sequencing technology can provide reliable analysis and detection of tetracycline resistance genes in different environmental media and feces, and provide technical support for the use of tetracycline resistance genes as a quantitative indicator of the degree of wild animals affected by human activities and their relationship with humans. SUMMARY

[0004] The patent proposes an evaluation method for quantitatively characterizing the degree of disturbance of wild animals by human activities and the relationship between wild animals and humans based on tetracycline resistance genes in wild animal feces. The core of the method is to use the abundance, diversity and expression potential of tetracycline resistance genes in the microbiome of wild animal feces as key biological indicators, and to construct a quantitative system for its response by coupling the gradient of the degree of human activity disturbance. The core of the invention is an evaluation method system, not a gene detection technology itself. Its innovation lies in the multi-index system with the abundance, diversity and expression potential of tetracycline resistance genes in wild animal feces as the core, which quantifies the disturbance intensity of human activities on wild animals and the relationship between wild animals and humans. This method is suitable for a variety of wild animals that can obtain fresh feces within a week, including but not limited to wild mammals (such as giant pandas and Przewalski's horses), wild birds (such as migratory birds), and wild reptiles. In addition to directly applying the evaluation method to the characterization of the degree of disturbance of wild animals by human activities and the relationship between wild animals and humans, it is also suitable for the evaluation of wild animal food source and water source abundance, human activity pollution impact, and invasive grazing in nature reserve management, providing decision-making basis for timely development of conservation measures and countermeasures in nature reserves.

[0005] To achieve the above-mentioned purpose of the present patent, the following scheme is adopted:

[0006] (1) Sampling strategy based on the gradient of human activity disturbance: In the habitat of wild animals, sampling strips are set up according to the distance from human activity areas, contaminated water bodies are set up in the opposite direction, or migratory birds are selected according to their migration paths, resting places and wintering grounds, etc., or conservation of wild animals is designed according to their conservation centers, wildlife parks and natural reserves for wild animals, etc. According to the collection site of wild animal feces, soil and / or surface water are collected for analysis of drug resistance genes.

[0007] (2) Analysis of tetracycline resistance genes: The existing gene chip or high-throughput sequencing technology is used to quantitatively analyze the differences in tetracycline resistance gene abundance, diversity and expression potential (defined as the ratio of the sum of the abundance of quantified tetracycline resistance genes to the number of detected ones) of feces and environmental media (soil and / or surface water) samples. The expression potential of ARGs in wild animal feces and environmental media samples is calculated according to the following formula (1) to standardize the abundance of tetracycline resistance genes.

[0008] (1)。

[0009] (3) Evaluation system construction: coupling the human activity disturbance degree gradient in step (1) with the tetracycline resistance gene indicators in step (2), taking the sample with the lowest human activity disturbance degree and the lowest tetracycline resistance gene indicators as the reference point (or non-human breeding wild animals), establishing a multi-level human activity disturbance degree gradient according to the polymorphism characteristics and specific research target requirements, quantifying the influence degree of wild animals affected by human activities and the relationship between wild animals and humans, and the management purpose of the nature reserve.

[0010] Figure 1 shows the wild Przewalski's horse.

[0011] Figure 1 Figure 2 shows the technical method flowchart.

[0012] Figure 2 Figure 3 shows the relative abundance of tetracycline resistance genes in fecal samples of Przewalski's horses in different human intervention intensity environments, such as captivity (Xinjiang Wild Horse Breeding Center), semi-captivity (Xinjiang Tianshan Wildlife Park) and wild release (Xinjiang Kalamaili Nature Reserve), with the fecal sample of Mongolian wild horse in the nature reserve as the reference benchmark.

[0013] Figure 3 Figure 4 shows the expression potential of quantified drug resistance genes (box plot) and tetracycline resistance genes (dot) in fecal samples of Przewalski's horses under different human intervention intensity, with the fecal sample of Mongolian wild horse in the nature reserve as the reference benchmark.

[0014] Figure 4 Figure 5 shows the beta-diversity analysis of tetracycline resistance genes (expression potential) in fecal samples of Przewalski's horses under different human intervention intensity, with the fecal sample of Mongolian wild horse in the nature reserve as the reference benchmark. DETAILED DESCRIPTION

[0015] The examples provided in the present application are intended to illustrate the practical application of the method and do not constitute a limitation on the scope of protection of the present application. Therefore, non-essential improvements and adjustments made by those skilled in the art based on the description of the present application still fall within the scope of protection of the present application.

[0016] Example 1: Evaluation of the wild process of Przewalski's horse

[0017] (1) Sample collection and drug resistance gene detection

[0018] Fresh fecal samples of Przewalski's horses were collected in Xinjiang Wild Horse Breeding Center, Tianshan Wildlife Park and Kalamaili Mountain Nature Reserve with different degrees of human disturbance, and fecal samples of Mongolian wild horses were collected in the nature reserve as a control group, to construct a human intervention intensity gradient of Przewalski's horses. In this example, gene chip high-throughput quantitative technology PCR (HT-qPCR) was used to detect the relative abundance and diversity of tetracycline resistance genes in the samples.

[0019] (2) Tetracycline resistance gene feature analysis

[0020] (a) Relative abundance analysis of tetracycline resistance genes: The relative abundance of tetracycline resistance genes in Przewalski's horse feces showed a significant gradient change, i.e., the highest in captive environment, the second highest in semi-captive environment, and the lowest in natural reserve wild release Przewalski's horse feces, which was close to the control group of natural reserve Mongolian wild horse feces. Figure 2 ).

[0021] (b) Analysis of tetracycline resistance gene expression potential: The tetracycline resistance gene expression potential of Przewalski's horse feces showed a decreasing trend in the order of captive > semi-captive > natural reserve wild release, and the tetracycline resistance gene expression potential of Przewalski's horse feces in natural reserve wild release was close to that of completely wild Mongolian wild horse, showing a significant wilding process. Figure 3 ).

[0022] (c) Gene β-diversity analysis: Using principal coordinate analysis (PCoA) based on Bray-Curtis distance (Bayesian distance), the tetracycline resistance gene expression potential of different wilding released Przewalski's horse feces showed community structure differences, i.e., the captive and semi-captive Przewalski's horse feces samples partially aggregated and overlapped, the semi-captive and wild release Przewalski's horse feces samples partially overlapped but separated from the captive feces samples, and all wilding Przewalski's horse feces samples were significantly separated from the wild Mongolian wild horse control group feces samples, which clearly revealed the significant wilding process. Figure 4 ).

[0023] (3) Quantitative index construction and evaluation system design

[0024] Based on the above analysis results and the requirements of this example, a quantitative evaluation system for the intensity of human intervention in the three stages of Przewalski's horse conservation, display, and wilding was constructed, i.e.:

[0025] High-intensity intervention (captive conservation): The relative abundance, diversity, and expression potential of tetracycline resistance genes are the highest;

[0026] Medium-intensity intervention (semi-captive display): The above indexes are at the median value;

[0027] Low-intensity intervention (wild release): The above indexes are the lowest and close to the wild Mongolian wild horse control group.

[0028] This system objectively evaluates the response of Przewalski's horse to human intervention in the process of conservation, display, and wilding, and provides clear quantitative indicators, which verifies the application of the evaluation method in the practice of wild animal management.

[0029] The detection method of the tetracycline resistance gene in this example: High-throughput quantitative PCR (HT-qPCR) is used to detect tetracycline resistance genes and other resistance genes, and the specific experimental process is as follows:

[0030] (1) DNA extraction: Genomic DNA of Przewalski's horse and Mongolian wild horse fecal samples was obtained using a soil genomic extraction kit. After measuring the fecal DNA concentration using a spectrophotometer, the DNA concentration was homogenized to 20 ng / μL using sterile water. -1 ;

[0031] (2) Target gene screening: This example selected 323 target ARGs, covering 9 categories, including tetracyclines (46), the remaining categories including: beta-lactams (54), MLSB (macrolide-lincosamide-streptogramin B, 45), aminoglycosides (36), vancomycins (32), fluoroquinolones (13), sulfonamides (10), synergists (trimethoprim, 19), chloramphenicols (3), multiple drug resistance (57), and unclassified (8).

[0032] (3) Gene amplification: The intelligent chip system was used for high-throughput detection of drug-resistant genes, and a 5148-hole chip was used to realize target gene amplification and quantification. The PCR thermal cycling program was set as: 95℃ initial denaturation for 10 minutes, 95℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds, a total of 40 cycles. Data acquisition was completed through the corresponding software, and the cycle number <31 was set as the effective detection threshold.

[0033] The above experiments show that the present application quantifies the influence of human activities on wild animals at the molecular level through the abundance, diversity and expression potential indicators of tetracycline-resistant genes in wild animal feces. The applicability of this method is verified by taking Przewalski's horse as an example. This evaluation method is not only suitable for the introduction of wild animals into the wild process, but also can be widely used in ecological monitoring, wild animal habitat assessment and nature reserve management, etc.

Claims

1. A quantitative assessment method for wildlife conservation based on fecal tetracycline resistance genes, characterized in that: The following steps are involved: (1) Analyze the abundance, diversity, and expression potential of tetracycline resistance genes in wild animal feces; (2) Couple the human activity interference intensity gradient at the sampling site and compare the above indicators in the target fecal samples; (3) Based on the comparison results, take the fecal sample with the lowest human interference intensity and the smallest tetracycline resistance gene abundance at the sampling site as the benchmark, and quantitatively assess the degree of human interference on wild animals and their closeness to humans by coupling the human interference degree gradient through sorting all fecal samples.

2. The evaluation method according to claim 1, wherein: Step (3) includes setting up a gradient quantitative evaluation system of multi-level interference intensity based on the polymorphic distribution of the above indicators of fecal tetracycline resistance genes and combining it with research needs.

3. The evaluation method according to claim 1, wherein: The target wild animals include but are not limited to wild mammals, wild birds, wild reptiles, etc.

4. The evaluation method according to claim 1, wherein: This method can be applied to areas including, but not limited to, grazing, planting, reclamation, and wildlife reserve construction and management to evaluate the effectiveness of wildlife conservation measures or guide the optimization of management strategies. An example of quantitatively assessing the degree of human intervention based on fecal tetracycline resistance genes at each stage of Przewalski's horse conservation, display, and rewilding has been provided.

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