TP53 GENE VARIATION IN ELEPHANTS

May 2018Table of Contents
TOC o “1-3” h z u ABSTRACT PAGEREF _Toc530201334 h 5INTRODUCTION PAGEREF _Toc530201335 h 6Background Information PAGEREF _Toc530201336 h 6Problem Statement and Justification PAGEREF _Toc530201337 h 8Hypothesis PAGEREF _Toc530201338 h 8Objectives. PAGEREF _Toc530201339 h 9General Objective PAGEREF _Toc530201340 h 9Specific Objectives PAGEREF _Toc530201341 h 9Significance of the study PAGEREF _Toc530201342 h 11LITERATURE REVIEW PAGEREF _Toc530201343 h 12cancer epidemiology PAGEREF _Toc530201344 h 15comparative oncology PAGEREF _Toc530201345 h 15TP53 & functions PAGEREF _Toc530201346 h 15Cancer in elephants PAGEREF _Toc530201347 h 15METHODOLOGY PAGEREF _Toc530201348 h 16Work done on Secondary data PAGEREF _Toc530201349 h 16Work was done on Primary data PAGEREF _Toc530201350 h 16Methodology Triad for the Proposed Study PAGEREF _Toc530201351 h 16Detailed Procedure: Data Collection PAGEREF _Toc530201352 h 17A: Secondary Data PAGEREF _Toc530201353 h 17B: Primary Data PAGEREF _Toc530201354 h 17Study area PAGEREF _Toc530201355 h 18Sample size determination PAGEREF _Toc530201356 h 18Sampling design PAGEREF _Toc530201357 h 18Molecular and Bioinformatics study. PAGEREF _Toc530201358 h 19Data analysis: Statistical tests and Hypothesis Testing PAGEREF _Toc530201359 h 20Ethical considerations PAGEREF _Toc530201360 h 20REFERENCES PAGEREF _Toc530201361 h 21

ABBREVIATIONS AND ACRONYMS TP53;: Tumor protein 53
SNP:; Single Nucleotide Polymorphism.
TP21; :Tumor protein 21
DNA; Deoxyribonucleic Acid
cdk; Cyclin dependent Kinase
CDC; Cell division cycle
H0: Null Hypothesis;
H1; Alternative hypothesis
C; Characteristic
(mdm2); mouse double minute 2
CO2; Carbon Dioxide
mRNA; Messenger ribonucleic Acid
LFS; Li-Fraumeni syndrome
NCBI; National Centre for Biotech Information
ENSEMBL; European molecular biology genome database.

BLAST; Basic local alignment search tools
(SCA); Statistical Computing Analysis
PCR; Polymerase chain reaction
ABI; Applied Biosystems Institute
(MMNR); Maasai Mara National Reserve
ABSTRACTCancer is a life-threatening critical disorder which increases the morbidity and mortality of any metazoan species. Although different therapeutic modalities are indicated for managing various types of cancer, eradication of cancer remains a significant challenge across healthcare professionals and scientists. Multicellular and large-sized organisms better mitigate the risk of cancer compared to small-sized and unicellular organisms a theory known as Peto’s Paradox. The African Elephant (Loxodonta Africana) remain cancer resistant, with an estimated cancer mortality of 4.81% (95% CI, 3.14%-6.49%). Understanding the complex mechanisms that govern predisposition and resistance to cancer is an area of active research interest. Hence, there has been a constant search for novel therapeutic strategies based on natural tumor-suppressing mechanisms that could alleviate cancer or eliminate the risk of cancer. This proposed research aims to explore the genetic diversity of TP53 gene in three distinct elephant populations in Kenya and to evaluate its implications in cancer resistance (which differs in phenotype, genotype, and geographic distribution) and changes in amino acid sequence that will lead to functional variation, taking into account the domains of bioinformatics, cladistics, and molecular biology. This study will examine 35-52 Individual elephants per herd in 3 distinct elephant populations across Kenya with a total population of 26,000 African elephants namely; Maasai Mara (Approx.3,000), Tsavo East (Approx.15,000) and Samburu National Parks (Approx.8,000). Drop down Biopsy darting technique will be used to collect elephant tissue samples through random stratified sampling. Molecular Biology techniques will be used as follows;DNA extraction will be carried out using (PureLink® Genomic DNA Mini Kit, Thermo scientific.), PCR will be carried out using Rotor Gene Q Machine (Qiagen), DNA Sequencing will be? using (ABI Prism Genetic Analyzer®). Data Analysis will be performed using; genome assembly LoxAfr3 for sequence analyses, BLAST, Multiple alignments using (ENSEML and NCBI Databases) and SNP Analysis using fast Structure software. This study will determine; Geographical variation in the African elephant TP53 gene, single nucleotide polymorphisms and detection of changes in amino acid sequence with functional variation. The respective domains will be explored to address the research questions in a comprehensive manner. The findings of the proposed research will also be beneficial in understanding the dynamics of gene polymorphisms and geographic distribution of the TP53 genotypes in humans. Such an exhaustive research framework is intended to ensure the reliability and reproducibility of the proposed study.
1.0 INTRODUCTION1.1Background Information Cancer is a life-threatening critical disorder which increases the morbidity and mortality of any metazoan species. Over the past two decades, there has been a significant increase in the prevalence of all-cause cancer across humans. Hence, cancer has surfaced and remains as a global concern for healthcare professionals and patients. The disease is featured by uncontrolled division of cells which often invades nearby and distant tissues of the body by metastasis. Hence, the hallmark feature of cancer cells is their inability to undergo programmed cell death (apoptosis) in the event of any damage. The damaging cues are speculated to induce mutation in the DNA of at-risk cells and alter the expression of tumor suppressor genes. Tumor suppressor genes (such as TP53 and TP21) express the notable tumor suppressor proteins p53 and p21. These proteins prevent the progression of cell cycle in cells those are at-risk of developing cancer. They act by inhibiting the cell cycle at different checkpoints based on their suppressive potential on the cdk-cyclin complex. To recall, cdk-cyclin complex is protein complex that helps in the progression of the cell cycle. Reference?As a result, the cell transforms from the G1/G0 phase to the M phase where it undergoes mitosis and cell division. As a result, the respective individual is predisposed to the risk of cancer. Different factors or cues predispose the risk of cancer in humans and related species (Abegglen et al. 1850-1860). Such factors include ionizing radiation, genetic predisposition, environmental toxicants, and lifestyle factors. Similarly, the risk of cancer varies from one individual to another and from one biological species to another. Such variations in the prevalence of cancer have elicited significant interests among researchers in exploring novel tumor suppressing mechanisms. Reference?It is speculated that identification of novel tumor suppressing mechanisms and polymorphisms in tumor suppressor genes hold the key to overcoming this dreadful disease. Recently, different studies have elucidated novel tumor suppressing mechanisms across a wide array of metazoan species. One such mechanism involves the identification and characterization of the TP53 gene in different elephant families. The major interest pivot around African elephants as such elephant species exhibit a lower prevalence of cancer compared to their Asian counterparts (Abegglen et al. 1850-1860). Abegglen et al., year?Such findings suggest that cancer is not only prevalent in humans but almost all metazoan species. Understanding the complex mechanisms that govern the predisposition and resistance to carcinogenesis across different metazoan species is an area of active research interest. Hence, there has been a constant search for novel therapeutic strategies based on natural tumor-suppressing mechanisms that could alleviate cancer or eliminate the risk of cancer. The search for novel tumor suppressing mechanisms in other metazoan species apart from humans is still in its infancy. Hence, the study of tumor-suppressing mechanisms in wildlife and related metazoan species will not only help to discover novel strategies against cancer but will also help to study the selective forces and evolutionary processes that led to the variation in such mechanisms across different metazoan species including humans (Abegglen et al. 1850-1860).
***you cannot have only one reference for this subsection!!!**always acknowledge the source of information1.2 Problem Statement and JustificationElephants remain cancer resistant, with an estimated cancer mortality of 4.81% (95% CI, 3.14%-6.49%), (Lisa et al., 2015). Although different therapeutic modalities are indicated for managing various types of cancer, eradication of cancer remains a significant challenge across healthcare professionals and scientists. Novel therapeutic modalities have certainly prolonged the survival rate of patients affected with different types of cancer. However, evidence on such modalities remains either inconclusive or questionable. The lack of multifaceted studies at the cellular and systemic level is one of the major bottlenecks that limit conclusive evidence on novel therapeutic modalities (Abegglen et al. 1850-1860) and no such studies have been conducted on African Elephants in Africa. Elephant TP53 gene has been demonstrated to destroy various forms Cancer, mMainly of Osteosarcoma, prostate Cancer, and Glioblastoma, (Shciffman,et al ., 2015) and novel therapeutic modalities should try to address such polymorphisms at the genetic level. In-depth analysis of comparative genomics might hold the promise of overcoming cancer in the near future. Currently, only a few of such modalities have explored as interventions at the molecular or chromosomal level. Since the role of mutations and single nucleotide polymorphisms have been implicated in the genesis of cancer, novel therapeutic modalities should try to address such polymorphisms at the genetic level.
1.3 Hypothesis (Hypotheses)The broad hypothesies forof the proposed study are;
i. There is geographical variation in the TP53 gene isolated from distinct Elephant populations in Kenya.
ii. There is single nucleotide polymorphisms in the TP53 gene in distinct Elephant populations in Kenya
iii. There is change in amino acid sequence that will lead to functional variation in TP53 gene in silico.
1.4 Objectives.1.4.1 General Objective;To characterize the genetic diversity of TP53 gene in three distinct elephant populations in Kenya and to evaluate its implications in cancer resistance.
1.4.2 Specific Objectives;To determine geographical variation in the TP53 gene isolated from three distinct Elephant populations in Kenya.
To identify single nucleotide polymorphisms in the TP53 gene in distinct Elephant populations in Kenya.
To detect changes in amino acid sequence that will lead to functional variation in in compare functional variation in TP53 gene in silico.

Significance of the study Cancer is a life-threatening critical disorder which increases the risk of morbidity and mortality in humans. Although different therapies are indicated for managing cancer patients, eradication of cancer remains a significant global challenge. To recall, the lack of multifaceted studies at the cellular and the systemic level is one of the major bottlenecks that limit conclusive evidence on novel therapeutic modalities that aim to manage cancer in humans. Such assumption is the guiding philosophy and motivation for conducting this proposed research. The proposed study will try to integrate the findings of cancer research both at the molecular and systemic level. The study will also elucidate the probable gene-environment interactions those led to the genesis of cancer in metazoan species due to variations. The findings of the proposed study could also help to understand the geographical variations in the prevalence of cancer. Such findings could help to mitigate the risk of cancer across at-risk populations and reveal different unexplored facets that could help to develop robust therapeutic modalities for cancer.

LITERATURE REVIEWCancer is recognized as the second largest cause of all-cause mortality in the United States. Studies suggest that almost 33% of the U.S. population is diagnosed with cancer at least once over their entire lifetime. Studies also suggest that 25% of the U.S. population dies due to cancer per year. In spite of radical and technological advancements in therapeutic modalities, management of cancer remains a clinical dilemma. It is speculated that an in-depth analysis of comparative genomics might hold the promise of overcoming the risk of cancer in the near future. Comparative genomics is an extension of the evolutionary theory that provides insight into the role of evolution in mediating cancer suppression across different species ref?. Merlo, Pepper & Reid et al., year? (924-935) stated that cancer results from multicellularity and represents a unique example of multilevel selection. The author stated that progression of cancer occurs through somatic evolution where genetic and epigenetic instability mediates fitness variation in the cells of at-risk individuals. It is contended that during the entire lifetime of an organism such cells accumulate mutation that predisposes the risk of malignancy. However, cancer is long recognized as a disorder that stems from gene-environment and it is inherited. Moreover, somatic cells within a tumor need to fulfil three conditions before they are predisposed to cancer. First of all, such cells should exhibit variation within a given population ref?.In fact, any tumor constitutes a heterogeneous cell population that are capable of undergoing somatic and epigenetic variationsref?. Secondly, the variations that take place should be inherited. Studies suggest that both genetic and epigenetic variations are heritable when a mother cell divides into daughter cells. Finally, such cells must exhibit differential survival and reproduction. In simple words, such cells must exhibit differential fitness under environmental selection pressures. Mutations are often deleterious to an organism and might predispose the risk of cancer across at-risk individuals. However, certain mutations are also favorable to the organism. Studies suggest that certain epigenetic and genetic mutations can increase the survival of cells and produces a reproductive advantage in them in comparison to other cells. As a result, such cells are more predisposed to the risk of malignancy compared to the cells undergoing deleterious mutations. Genetics and epigenetic changes induce eight hallmarks in cancer cells that increase their fitness over the healthy somatic cells. These hallmarks include self-sufficient growth signals through protein-protein interactions, marked insensitivity to anti-growth signals, significant reduction in the rate of apoptosis, significant increase in angiogenesis, unlimited potential for replication due to telomere stabilization, suppression of the immune system and avoidance of death signals, modifications in the metabolic capacity, and capability of invading new tissue through metastasis (Hanahan & Weinberg 646-674).
Somatic evolution within the mutant cell populations could lead to different forms of malignancy. Studies suggest that selection pressures not only apply at the cellular levels but they are also applicable at the organism level. As a result, somatic cells have evolved to generate different tumor suppressing mechanisms such as checkpoints in the cell-cycle or by inducing apoptosis in cells that are at-risk of malignancy. Such safeguards prevent the potential of somatic mutations to become carcinogenic. One of the important safeguards that mitigate the risk of cancer is the damage-sensing and repair ability of DNA to carcinogens. On the other hand, premature senescence and apoptotic mechanisms override such safeguards if mutations in DNA are not removed. Hence, maintaining the integrity of DNA in somatic cells is an essential prerequisite for all unicellular and multicellular organisms. As a result, the odds of somatic mutation are significantly higher in multicellular organisms. Hence, the question arises “why evolution has favored multicellular and large-sized organisms?” There are various advantages of a multicellular and large-sized animal compared to unicellular and small-sized ones. Evidence suggests that all organisms have evolved to ensure a physiological steady state. At the same time, evolution has endowed an organism to remain away from the state of thermodynamic equilibrium which is marked by physical death and termination of physiological functions in a body. For example, entropy is a key determinant that hastens an organism towards thermodynamic equilibrium. To recall, entropy is defined as the degree of randomness or disorderliness in a system. Such assumptions reflect that the rate and degree of entropy would be much higher in multicellular organisms (just like the anticipated number of somatic mutations) compared to their unicellular counterparts.
In reality, Peto’s (n.p.) assumption that the risk of cancer in large- sized and multicellular organisms is significantly lower than that in small-sized and unicellular organisms. This assumption is popular as “Peto’s paradox.” Likewise, Domazet-Loso and Tautz (66) highlighted that multicellular organisms exhibit higher cooperation in eliminating selfish cells for preventing the risk of cancer. Peto (n.p) contended that the body size of a human is manifold (1000 times) than that of mice. Hence, the probability of deleterious or somatic mutations in epithelial cells of humans was 304 to 306 times. Such mutational load would have significantly increased the prevalence of cancer than what is witnessed in humans. On the contrary, the prevalence of cancer in humans is almost same as the prevalence of cancer in their murine counterparts. Such observations do support Peto’s Paradox that multicellular and large-sized organisms are better suited to mitigate the risk of cancer compared to small-sized and unicellular organisms.
One of the striking features of the TP53 gene is its capacity to exist as various paralogs and homologs within an across different species of metazoans. TP53 gene expresses p53, which is commonly referred to as the tumor suppressor protein or tumor suppressor cellular antigen. TP53 or any other tumor suppression gene is defined as those genes that can increase the risk or progression of cancer if they are deleted or present in mutated versions. The evolutionary history and geographical variation in the TP53 gene from distinct Elephant populations have raised significant interests among scientists and healthcare professionals. Such interests have stemmed from their potential in mitigating different forms of malignancy compared to humans and other mammalian species. This variant of the elephant species is known to feature 20 copies (40 alleles) of the TP53 gene. It is speculated that such high load of TP53 gene might be associated with reduced incidence of cancer as witnessed across this elephant species. Studies further suggest that the risk of Loxodonta africana is even lower than their Asian counterparts. Casola (2016) elaborated the possible tumor suppressing mechanisms in different elephant species. The authors highlighted that the possible tumor suppressing mechanisms in different elephant species and pachyderms could stem from the genetic polymorphisms in their TP53 genes.
Genetic polymorphisms such as deleterious mutations and gene duplication in different alleles of the TP53 gene have been strongly associated with the genesis or mitigation of different types of malignancies. However, genetic and geographic polymorphisms in the paralogs of theTP53 gene across different metazoan species have rarely been explored. Casola (2016) highlighted two possible mechanisms through TP53 paralogs could mitigate the risk of cancer. Both these mechanisms pivot around the induction of the p53-mediated apoptotic pathways in response to DNA damage. It was contended that the retropseudogenes of the TP53 gene encode such p53 fragments that act as decoy receptors for the full-length p53 protein. However, the retrogenes of TP53 emerged in elephants after there was an evolutionary split between elephants and hyrax. Such findings fascinated Schiffman and the author considered such phenomenon “as natural protection that evolved over millions of years against diseases like cancer.” The study showed that the mortality rate in elephants due to all-cause cancer was significantly lower than the human mortality rates due to all-cause cancer 4.1% versus 11% to 25%, p<0.05). The authors further explored the dynamics of damage and repair in the DNA of healthy elephant cell lines, healthy human cell lines, and cells from patients who exhibited Li-Fraumeni syndrome. To recall, individuals with Li-Fraumeni syndrome have a compromised copy of the TP53 gene. The authors induced DNA damage in the respective cells by bombarding them with ionizing radiations and doxorubicin. However, the authors noted that there was no significant difference in the DNA damage-repair mechanisms between carcinoma-induced elephant and human cell lines compared to the cells of LFS patients and reflected that elephant cells exhibited higher rate of apoptosis than the other cell lines.
Introduce subsections to be precise, such as:cancer epidemiologycomparative oncologyTP53 & functionsCancer in elephants
METHODOLOGYWork done on Secondary dataThe work that has been done for retrieving secondary data pivots around evidence-based literature. Different keywords were used to access available evidence for undertaking the initial literature review. The initial literature review motivated me to conduct a systematic review. The studies that will be collated in the systematic review will be thematically analyzed.
Work was done on Primary data
The primary data that have been retrieved is based on bioinformatics studies. Structural Characterization of variation in the TP53 gene isolated from a distinct African Elephant. A BLAST analysis reflected that the length of the p53 protein varied between 386 amino acids (Feline species) to 393 amino acids (humans). The length of p53 in Loxodonta africana was estimated to be 392 amino acids. Hence, the protein database reflected that the TP53 gene in Loxodonta africana exhibited one deletion mutation. However, such deletion mutation in a single allele might not predispose the risk of cancer in Loxodonta africana. These findings suggest that either such deletions were balanced by alleles that are not mutated or paralogs that could offset the harmful effects of deleterious or deletion mutations in the TP53 gene of Loxodonta africana.
Methodology Triad for the Proposed Study
The methodology triangulation will include analyses of primary and secondary data which will be integrated to address the primary and secondary research questions that are formulated for the proposed study. Hence, primary and secondary data will be integrated to report the findings of the study.
Detailed Procedure: Data Collection
A: Secondary DataThe secondary data will be collected from a systemic review of the evidence-based literature and different databases that document appropriate data (For example; the Elephant Electronic database will be accessed for gathering health data on African and Asian elephants). The retrieved articles will be sorted under different themes for conducting a thematic analysis. Moreover, the findings of the different studies will be used to compare and critique the primary data of the proposed research. Finally, the systematic review of literature will help to consider new and novel primary data that was not planned before the initiation of the study.
B: Primary Data
Biopsy Darting Technique
Modern use of biopsy darts to get tissue samples from wild animals is on the rise as it enables scientists to collect the samples without the risks and expenses of capturing live animals (Dominic et al ,2016). Usually, tissue samples are collected from live wild animals following chemical immobilization or physical restraints in case of small animals.
Chemical immobilization is usually stressful, carries anesthetic risks and is expensive to undertake in many wildlife species especially African Elephant that are over 12,000 Kg.
When fired, the biopsy dart is supposed to hit the animal on preferred parts of the body with thick muscles such as the thigh, shoulder and neck. The dart cuts the skin and tissue then falls to the ground after the animal moves around. The skin tissue sample is then held onto the biopsy needle when successful .Modern Biopsy darts are used to collect samples from the threatened animals, and hence the success of the darting is very important since researchers may not re-dart the same animal as they are free-ranging Populations. The biopsy dart, Dan-inject, is a built-in CO2 pressure gauge oriented on the dart rifle facing the shooter, along with a pressure control valve, allowing the pressure setting to be monitored and adjusted, and red laser facilitate quick accurate aiming. The pink color of the dart tails makes it easy to find.
Study areaKenya has approximately 39,000 elephants according to Kenya wildlife service survey 2017.The study will be conducted in three distinct National Parks in Kenya ;Masai Mara National Reserve (MMNR) in South-Western part of Kenya .The reserve with approximately 3,000 African Elephants. The Samburu National Park, a home to approximately 8000 African elephants and the Tsavo East National Park with approximately 14,000 African Elephants. The distance between the parks are as follow, Laikipia to Tsavo National park is approximately 619km, Tsavo to Maasai Mara is approximately 730 km and finally Laikipia to Maasai Mara National park is approximately 551 km. We will dart he thigh and shoulder parts because of adequate musculature hence higher chances of getting enough tissue and less risk of hitting vital organs. These 3 sites are chosen as they are distinct, represents half the country Elephant population and form the stronghold of African Elephant in Kenya.
Sample size determinationElephants live in of herds of 30 – 50 related individuals (Dorst,et al., 1970) and African elephants will be randomly selected in a stratified manner in 3 distinct parks
26,000/50=520 herds
Selecting (20-30%) of study population per distinct site will be representative.
520*0.2/3=35
520*0.3/3=52
35-52 Individual African elephant herd will be eligible to be included in the study
Sampling designThe Skin biopsy samples will be collected through random stratified sampling from 35-52 individual elephant in related herds from each of the three distinct national parks using sterile needles for each elephant. The research is meant to collect skin tissue samples to determine geographical variation in the TP53 gene isolated from three distinct Elephant populations in Kenya. We will use 1.5 ml Dan-inject darts attached to Dan-inject biopsy needles which will be fired to each elephant by Dan-inject® (Denmark) long range projector (Dominic et al,2016 ).After each darting, tissue samples will be retrieved from the biopsy needles and preserved in cryovials filled with 70% ethanol and will be transported to Kenya wildlife service laboratories for storage.
Bioinformatics studies
The bioinformatics studies will include identification and characterization of the target gene. The target gene selected for the proposed study is reflected as ENSEMBL Id reference sequence ENSLAFG00000007483 (Michael,Lindsey Katelyn & Mika 4-5). BLAST will be conducted to explore the nucleotide sequence of the target gene. Primers will be designed according to (Lisa et al, 2015) from the coding sequence Ensembl–Elephant loxAfr3 (loxAfr3:GL010074.1) Genomic sequence SuperContig scaffold_47:11688025-11694358 ENSLAFT00000007484 and sent for commercial Synthesis ready for PCR (Inqaba,Biotech pty, SA).Molecular and Bioinformatics study.DNA will be extracted from the 35-52 individual tissues biopsy targeting pure genomic DNA as the elute using (PureLink® Genomic DNA Mini Kit, Thermo scientific.).End point PCR will be conducted using the designed primers (Lisa et al, 2018) to amplify the specific TP53 gene of interest.This will be followed by gel electrophoresis to visualize the DNA bands of interest and to clean the Band for DNA sequencing using the ABI Prism Genetic Analyzer (Thermo,Scientific Inc). The NCBI Nucleotide BLAST search will be performed and an alignment will be done again other known genetic sequences in the database. Sequence identity will be evaluated to ensure we are only dealing with the reference TP53 gene ENSLAFG00000007483 (Michael, Lindsey Katelyn & Mika 4-5).The result that will be obtained from the Bioinformatics studies will allow for the basis of the laboratory experimentation through DNA Extraction and Sequencing of the TP53 gene from the 3 distinct geographic locales in Kenya.
Have subsections that are in line with/ will lead to achievement of the study objectives.Be specific with key methods that will generate the results egDNA extraction; PCR amplification; DNA sequencing; BLAST-NCBI; Phylogenetic analysis; SNP analysis etc Data analysis: Statistical tests and Hypothesis Testing Single Nucleotide Polymorphism (SNP) Analysis, geographical variation and changes in amino acid sequence, statistical analysis that will be undertaken in the proposed study will be conducted by the using fastStructure software (Anil Raj et al, 2014).
Ethical considerations
Since the proposed study will involve data from animals appropriate permissions will be obtained from the animal ethics committees, Kenya wildlife service before initiation of the study. Moreover, since the study will involve secondary data and the interventions will be mostly non-invasive the chances of risk are minimal across the study participants. On the hand, care should be excised to ensure confidentiality of data that are provided by Government stakeholders and Ministries of Forest and Animal Welfare. Dissemination or unauthorized access to such data should be subjected to litigations.
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