1 Introduction

Over the past few decades, the world has encountered several zoonotic spillovers beginning with multiple Influenza outbreaks in the 1900s up to the recent SARS-CoV-2 outbreak in 2019. The World Health Organization (WHO), Food and Agriculture Organization (FAO) of the United Nations, and World Organization for Animal Health (OIE) have identified the increasing incidence of emerging of infectious diseases (EID) capable of transmission from animals to humans (zoonoses) as a major threat to biosecurity and public health1, 2 (Fig. 1). Zoonotic spillovers, which constitute more than 60% of all EIDs3, often involve three primary components, namely, animals, humans and their immediate environment (Fig. 1). The consequences of these spillovers have proven to be devastating for environmental, social, economic, and political systems. The same has been demonstrated through the Ebola virus outbreak of 2014 and the recent SARS-CoV-2 outbreak of 2019, resulting in an economic loss of approximately 28 trillion USD in 2019–20204, 5. However, we are still at a nascent stage when it comes to understanding the diversity of potential zoonotic pathogens and the causes of their spillovers. A more rigorous evaluation of these aspects is critical to preempt future spillover incidents and install appropriate preventive measures. Modern sequencing technologies have contributed immensely to pathogen discovery and are increasingly being used not only towards surveillance for zoonotic pathogens, but also towards a more holistic understanding of disease biology and host–pathogen coevolution6. The purpose of this review is to discuss the role of recent developments in sequencing techniques for pathogen discovery, particularly concentrating on the development of non-invasive environmental sampling and its applications thereof (Fig. 2).

Figure 1:
figure 1

Bats serve as potential reservoirs for a wide variety of viruses across the globe. Along with the plausible cross-species transmission, they form the connecting link between different zoonotic spillovers. A In India and Bangladesh, bat urine and feces contaminate the date palm juice with Nipah virus thereby exposing the palm juice handlers to deadly pathogens. B In Southeast Asia, the same virus passes through an amplification host, i.e., pigs and transmit it to humans, ultimately leading to human–human transmission through aerosols. C In Australia, Hendra virus has been isolated from bats and has caused outbreaks in horses, sometimes further leading to human fatalities. D In Africa, despite being detected in bats and non-human primates, the theories on the origin of Ebola virus remain to be deciphered. The red ticks denote points, where non-invasive sampling can be instrumental in revealing the pathogen load and help inform public health professionals to take necessary interventions.

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Figure 2:
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Schematic representation of the different approaches which can be used for the collection and analysis of environmental samples from wildlife. The analytical tools depicted here aid in the investigation of genomic material of the pathogens present in environmental samples collected non-invasively.

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The mechanistic processes involved in pathogen spillover from zoonotic reservoirs remain a poorly understood phenomenon. Wolfe et al.7 proposed a multistage model of how zoonotic microbes become specialized human pathogens. In the initial stages of this transformation, one may document microbes to be restricted to specific animal groups excluding humans. The consecutive stages of transformation may involve primary transmission between animals and humans, followed by occasional or continuous human to human secondary transmission events7. Infections caused by the West Nile virus or Dengue virus are ideal examples of the presence of a sylvatic cycle (transmission cycle in the wild) involving transmission of microbes between zoonotic reservoirs and humans in the wild followed by repeated secondary transmissions between humans8. The final stage comprises pathogens causing diseases exclusively to humans as seen in the case of measles, mumps, and rubella7. Such a mechanism has evolved either through co-specialization events between microbes and host or through a transformation event from an animal-associated microbe to a specialist human microbe7. Owing to the low fidelity of RNA-dependent RNA polymerase, RNA viruses exhibit a high mutation rate, raising the possibility of altering host tropism and giving room for successful cross-species transmissions to take place further. The transmission of HIV from non-human primates to humans is one of the classical examples that shows how human–wildlife interactions shape disease dynamics9. Similarly, genomic, and serological evidence hints at bats serving as potential reservoirs of Ebola virus. Transmission cycle of Ebola virus between chimpanzees and humans in Africa has been well established. However, involvement of many intermediate hosts during spillover events may further complicate our understanding of the origin of the pathogen10,11,12.

Essentially, reservoirs are the habitats in which the infectious agent lives, grows and multiplies13. Identifying such reservoir species is beneficial to managing emerging infections14. There has been sufficient evidence that a larger population size of the reservoir species is associated with a higher diversity of viruses15, placing small mammals such as bats and rodents at a critical position to serve as reservoirs. The wider range of these species also correlates to a higher chance of cross-species spillovers. Increased contact rate between individual animals ultimately increases the risk of pathogen transmission to humans16. However, the need for rapid and reliable detection along with subsequent characterizations of novel microbes, particularly in the wild, remains one of the major challenges while studying zoonotic spillovers.

2 Approaches to Detect Pathogens from Wildlife

2.1 Classical Approaches

Conventionally, several invasive methods have been employed for the detection of pathogens in wildlife. Serological methods such as ELISA have enabled the detection of antibodies in blood samples, shedding light on the recent or past exposure of the host to a specific infectious agent17. Targeted PCR approaches, classical methods involving isolation of bacteria in culture media or of viruses using embryonated eggs, cell lines and cutting-edge genomics have significantly improved the chances of pathogen discovery18. Detection of genomic fragments in clinical samples from humans or wildlife, using unique pathogen markers can help decipher whether the infection is acute. Serology is useful in estimating the infection history, especially in cases, where the pathogen titre may be significantly low as in the case of Henipa virus infections in bats19, or if the infection is latent as applied to pseudorabies in wild swine20. However, cross reactivity of antibodies to related etiological agents and determination of an optimum threshold to distinguish between positive and negative reactions can result in incorrect estimates of antibody prevalence21. For pathogens circulating in blood or excreted through feces and urine, detection of genomic fragments or antigen is possible but the sensitivity of PCR-based approaches can affect the detection probability. Finally, development of primary bat cell lines for isolation of bat viruses and subsequent host-virus studies is lagging, because bats are enormously diverse, and a given species-specific virus may not infect cells obtained from another species. Controlling optimum temperatures and other physiological parameters resembling that of a natural reservoir as well as allowing efficient cell culture can also be challenging22. While performing blind passage of viruses in cell lines, one may encounter adaptive mutations which attenuates the virus strain as seen during the isolation of Tacaribe virus from Artibeus bats, which acquired point mutations after 20 passages in newborn mice22.

One of the major challenges in formulating novel diagnostic assays for wildlife is attributed to the limitations imposed by smaller sample size and the challenges of test validation. One may encounter poor or degraded nucleic acid in samples, inaccessibility of post-mortem samples, and issues with the capture–recapture of infected individuals in the wild. Long-term monitoring of susceptible individuals in the wild is extremely difficult, further raising two issues: (a) stages of disease progression and pathogen shedding cannot be studied longitudinally (b) in vitro molecular diagnostics developed in the lab may not necessarily reflect observations in the wild, because community interactions amongst species in the wild is unpredictable, making pathogen transmission dynamics even more complex. One must be careful while validating data on physiological stress markers which are potentially associated with any infection that may have a different profile for captive animals in comparison to free ranging ones23, 24. This also holds true for interpreting any stress or immunological profile through serum biomarkers during experimental infection studies.

2.2 The Need for Metagenomics

The gold standard for establishing the true association of an etiological agent with diseases is the isolation and culturing of microbes, followed by validation in accordance with Koch's postulates. There have been exceptions to this rule too; for instance, viruses could not be grown under in vitro conditions for the longest time. Pathogen discovery from wildlife has faced several setbacks, some of the factors contributing to the setback are the specific growth requirement of fastidious microbes, lack of specific cell lines, ecological dependencies on other microbial or non-microbial partners, and lack of pathogen-specific identification markers. The major problem associated with PCR-based surveillance is the lack of prior knowledge of the microbial entity.

Less than 2% of the total microbes can be cultured25. Thus, metagenomics has served as a boon to study the unculturable microbes in nature26. The evolution and diversity of microbial communities in which individual microbial species are strongly dependent on each other’s function in a given habitat, can be studied through the lens of metagenomics. It is a powerful unbiased tool which has helped in a very detailed investigation of the human microbiome27 and similar approaches could be employed to microbiome studies in the wild. Metagenomics has helped to acquire genetic data on the host as well, suggesting better theories on how individual differences can shape the virome diversity. A recent report by Brussel and Holmes shows that 30% of the bat virus discovery has been achieved with the aid of next-generation sequencing tools28, and metagenomics has become an important tool to identify and characterize pathogens of zoonotic potential in the wild29. For example, metagenomics contributed significantly towards the discovery of the SARS coronavirus30. Malayan pangolin coronaviruses which show high similarity in their receptor binding domain to that of SARS-CoV-2 were identified with the aid of metagenomic sequencing31.

2.3 Metagenomics Workflow

The workflow of any metagenomic study starts from sample collection, followed by extraction of nucleic acid, library preparation, sequencing and bioinformatic analyses. One of the major challenges in wildlife viromics is the computational complexity of analyzing vast amounts of data generated through next-generation sequencing platforms. Metagenomics can help to understand the virome composition in reservoirs. However, one should be careful while interpreting which pathogens persist for a longer period in reservoirs against certain microbes which are merely a component of diet. Bats show the presence of insect viruses and bacteriophages which are predominantly acquired through diet32. Whole-genome sequencing of microbes is extremely expensive. Therefore, amplicon sequencing based on the 16S rRNA gene is extensively used to determine taxonomic identities in a microbial community as it is evolutionarily stable. Amplicon sequence variants provide higher resolution data of 16S sequences. As opposed to all these technologies, metagenomics or whole microbiome/virome shotgun sequencing involves the amplification of all the DNA/RNA within a sample, increasing the probability of detecting any and every microbe present in the study system33. However, few challenges of using metagenomics for establishing the etiology of a disease can be posed by the limited access of such technologies in developing or under-developed countries. Depletion of the host DNA with an aim to enrich the microbial genomes, the lack of standardized laboratory protocols of extracting genomic material from varied sources, contamination and working complexity associated during library preparation are some of the major challenges of implementing metagenomics approaches34.

The DNA or RNA sequence can be used to extract complete or partially assembled genomes of pathogens and further employ genome annotation studies to predict the virulence and understand their molecular evolution. Deep sequencing of samples potentially containing diverse bacterial communities is based on consensus gene sequence, i.e., 16S ribosomal RNA. On the other hand, viral communities do not have any such conserved sequence. Hence, discovery of viruses rely majorly on metatranscriptomics for RNA viruses or metagenomics for DNA viruses. However, many studies have identified viruses based on family-specific gene sequences of RNA-dependent RNA polymerase or DNA-dependent DNA polymerase. Isolation of viruses has also been successful from urine or fecal samples of wildlife. Paramyxoviruses were isolated from urine samples of Pteropus species35 and close relatives of the SARS coronavirus from Chinese horseshoe bats36.

There have been issues related to the nature of the buffer to be used for collection of environmental samples. Majority of the studies utilize RNA LATER. for preserving the samples which prevents RNA degradation, but it is ideal to store samples in RNA shield buffer, with the aim to inactivate viruses at its source. Storage of the environmental samples in Viral Transport Media (VTM) gives an opportunity to isolate viruses by maintaining its viability and enables further study of the agent through molecular virological approaches.

2.4 Novel Approaches

To make pathogen quantification reliable, more sensitive, and specific, digital droplet PCR (ddPCR) is an effective tool for samples which have low abundance of nucleic acids. The costs associated with ddPCR is high, but the current need is to make it affordable and accessible to a wide range of labs globally. ddPCR helps in accurate and faster quantification of molecules, having each molecule in one microdroplet. ddPCR involves individual PCR reactions after the initial template has been diluted in individual wells and then enumerates the pathogen load based on PCR positive percentage among all reactions. Using 180 fecal samples, the detection of human-associated Bacteroidales genetic markers, BacHum and B. theta was demonstrated employing dd-PCR37, 38. Detection of HIV, Hepatitis B, Human Herpes virus and malarial parasite is also possible using ddPCR and blood samples; however, this method remains to be explored in the context of environmental sampling of wildlife, as sufficient information about zoonotic pathogens in wild animals is lacking.

Once discovered as adaptive immune systems in bacteria against phages, Microbial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (CRISPR-Cas) programmable endonucleases, the genome editing tool have also found its place in diagnosing emerging infectious diseases. SHERLOCK strategy It can be a revolutionary approach in rapid detection of EIDs in the wild as it contributed to the detection of attomolar concentrations of Dengue and Zika virus as well as pathogenic bacteria, such as drug resistant Klebsiella pneumoniae39. It combines the properties of RNA guided RNA detection, promiscuous activity of Cas 13a and isothermal amplification40. Lyophilization of SHERLOCK reagents enabling easy storage, reconstitution on paper-based strip test enabling portability to the field, costs going as low as $0.61/test, applicability to a broad sample range including urine and sera are some of the reasons that make it ideal for environmental sampling especially for the developing countries40. In addition, CRISPR-based methodologies have made it feasible to detect deadly hemorrhagic fevers caused by Ebola and Lassa virus. A modified protocol of the SHERLOCK strategy, called HUDSON, has made it possible to detect BSL-4 viruses without involving any nucleic acid extraction step through the heat inactivation step with TCEP: EDTA directly from non-invasive samples, such as saliva and urine41. The same could be employed for surveillance of bat roosts and rodent samples. CRISPR-based lateral flow assays have also become handy in detecting SARS-CoV-2 from RNA extracted from throat swabs42.

Among others, a rapid and robust approach utilizing high-throughput targeted proteomics assay, towards the clinical diagnostics of viruses such as the novel coronavirus (SARS-CoV-2) is of significant value. Coupling two techniques, turbulent flow chromatography and tandem mass spectroscopy (TFC–MS/MS), such a diagnostic test proves to have specificity towards the detection of SARS-CoV-2, while maintaining peptides from other coronavirus strains, rhinovirus, enterovirus, and influenza viruses undetected. With a higher analyte stability and sensitivity, this strategy proves to be more advantageous than the gold standard real-time RT-PCR. In situations which demand large-scale population testing in a shorter duration and the supply for real-time RT-PCR equipment is insufficient, targeted proteomics provides an alternative strategy to real-time RT-PCR and would be an interesting addition to the viral testing panels43.

The use of biosensors also offers the possibility and potential as a rapid, sensitive, and specific detection platform scalable to global surveillance of pathogens and their control. It does not rely on cold chain storage and can aid in sample detection without additional equipment44, 45.

Ideally, a combination of targeted approaches and metagenomics, complementing the drawbacks of each other should be adapted and brought to the forefront of screening potential pathogens, driving advances in the prediction and control of zoonotic spillovers.

3 Environmental Sampling: Non-invasive Strategies

Environmental sampling refers to the isolation, extraction, and identification of DNA from an environmental sample, such as skin, mucous, saliva, urine, feces, leaves, secretions, water, soil, and rotting bodies. It also includes sampling of abiotic sources, potentially fomites, supplementing an investigation of an outbreak when environmental reservoirs are implicated epidemiologically in disease transmission46 (Fig. 1). During the last few decades, environmental DNA (eDNA) sampling has enhanced our abilities to identify a plethora of pathogenic organisms from the wildlife and predict zoonotic disease and outbreaks, thus aiding in disease preparedness. It has proven to be a potential tool in the detection of common species, along with those which are endangered, invasive, or elusive,  and their microflora47. This non-invasive approach is significant in monitoring wildlife diseases and detecting infectious agents as eDNA allows one to sequence information obtained from the environment without encountering the target species and minimizes disruption of already fragile habitats48. It neither utilizes any on-site biosafety equipment which would have been required for dissection or blood sample collection nor is prior monitoring required to localize ideal trapping sites, such as in invasive sampling, thus making feces, urine and saliva an ideal source to understand microbial diversity in the wildlife49. In addition to being less labor-intensive and cost-effective, the process is rapid, allowing multiple microbial species detection. Furthermore, even certain infectious diseases in the wildlife which may cause extinction or loss of biodiversity of species, thereby impacting the ecological balance, can be detected, and managed at an early stage of disease spread. A quantitative analysis of the contents of eDNA aids in estimating the relative prevalence of microbes in natural systems besides its mere presence or absence.

eDNA sampling has been rendered as a vital tool for the early detection of infectious agents at a low density, which improves the success of pathogen eradication and decreases the costs of control and impact on public health. There have been enough examples to prove the use of eDNA as a promising non-invasive and sensitive tool for pathogenic surveillance in wildlife. For instance, non-invasive collection of feces in the wild is one of the most economical, non-hazardous ways of gathering information on the host taxonomy, their population structure as well as understanding the shedding patterns of various pathogens in the environment50 (Fig. 2). Recently, there has been much focus on the surveillance of SARS-CoV-2 in wastewater, relevant for early monitoring and subsequent measures that can be taken by government authorities in treating wastewater51.

Thus, in the coming years, a well-rounded network of eDNA surveillance can be envisioned to understand host–pathogen dynamics, transitioning eDNA from an emerging field to one at the forefront of disease transmission and public health.

There are several biases involved in non-invasive sampling. While implementing a pooled sampling strategy, where individual fecal pellets cannot be differentiated from one another. For instance, collecting bat guano from a cave or social animals performing communal desiccation, it is difficult to identify the infection status at an individual level. One practical solution is to investigate pooled sampling from a collection of individuals. Even while estimating the abundance, considering that eDNA has variable decay rates under different environmental conditions52, 53, it will result in biased estimates and hence be a limitation to this approach. Therefore, one should ideally collect fresh fecal samples and subject them to freezing temperatures immediately to investigate the pathogen diversity.

Giles et al.54 attempted to optimize non-invasive or under roost sampling of bat-borne viruses. Despite many research groups looking at the viral or other microbial diversity using bat guano and urine samples in Asia, Australia, and Africa; this was the first study presenting a theoretical model of what an ideal strategy for sampling under roost would look like and the various sampling biases one may encounter while calculating the actual prevalence of viruses from such field data (Fig. 2). As opposed to the approach, wherein the presence–absence data of diseases is tabulated from pooled samples and then tracking individual level data from the positive pools to estimate the true prevalence; the authors presented a theoretical model of an ideal sampling strategy and showed that a small-sheet design, increased number of sheets spread across the roost area, combined with ‘multi-stage’ pooling of urine at a spatial scale offers a better alternative to estimate virus presence both with high sensitivity and specificity54, 55. Such sample pooling methods prove to be advantageous by minimizing the number of tests, time and costs56. The same has found its applications in recent times as well. While the testing for SARS-CoV-2 using the RT-PCR tests has gained an overwhelming response and has now become a bottleneck, sample pooling strategies are an attractive alternative that increases testing throughput while maintaining high sensitivity57. While pooling of samples, as well as sample libraries for NGS platforms, uneven mixing of pools, may give a low number of sequencing reads58. Furstenau et al. provided a detailed overview of sample pooling to screen pathogens and has concluded that despite the merits, it is important to consider that choosing an optimized pooling approach is often influenced by the epidemiology of the infectious agent, availability of the specimen, and the accuracy and sensitivity of the assay56.

Considering the current advancement in sampling strategies, we have proposed various strategies which can be employed for environmental sample collection (Fig. 2). Plastic sheets can be spread across a larger area for under roost sampling of fresh bat guano samples and urine droplets can be pipetted out in screw cap vials containing RNA/DNA shield buffer and transported to investigation laboratories, maintaining a proper cold chain and three-layered packaging. Alternatively, one could also collect fecal pellets of small mammals such as rodents or birds along with sample collection of nearby abiotic environments to serve as appropriate controls, accounting for any environmental contamination. As far as companion animals and wet markets are concerned, rectal and oral swabs can be taken easily. However, standardization of protocols to collect saliva from potentially infected fruits and investigate the virome or microbiome in the same remains to be explored. Water bodies and ticks in the wild can provide a broader perspective of the different microbes co-circulating in the wildlife and can serve as alternate sources to track the different pathogens that can spread through the feco-oral route.

While developing countries in South Asia and Southeast Asia are known for their high population density, increased wildlife habitat fragmentation and innumerable human–wildlife interactions, they are not yet adequately equipped with facilities to carry out long-term disease surveillance studies, particularly in wildlife. Having 125 reference laboratories certified by World Organization for Animal Health (OIE) to screen one or more pathogen, a disproportionality exists in the distribution of the reference laboratories, with almost 62% of these labs being in Europe and North America and the remaining labs being located mostly in China, while some of them being dispersed across Australia, South America, Africa and Japan59. Moreover, places that serve as potential zoonotic hotspots require the involvement of local communities skilled in lab safety, biosafety and having sufficient knowledge such that they contribute to the efforts of estimating the incidence and prevalence of zoonotic diseases. Non-invasive sampling can play a significant role in developing countries for pathogen bio surveillance studies. Furthermore, training of local communities to collect environmental samples of urine, feces, or oral swabs of dead animals along with individuals equipped with ‘on field’ genomic technologies and subsequently proper communication with regional laboratories can aid in faster epidemiologic analyses and inform outbreak management teams.

4 Small Mammals are Important!

The two most diverse groups of mammals, bats and rodents, account for an overwhelming proportion of pathogens with zoonotic potential and have been implicated in numerous zoonotic outbreaks14. Order Chiroptera within mammals has been associated with deadly, high-risk pathogens, such as Ebola virus and Nipah virus (Fig. 1). A similar trend has also been observed for Order Rodentia, which constitutes the largest percentage of living mammals in the world60. Hantavirus Pulmonary syndrome, Lassa fever, Plague, and Leptospirosis are caused by rodents61. Given the enormous species diversity, it is expected that the associated microbiome and virome for these species would also be diverse. Bats and rodents, also known to be synanthropes, have eventually become accustomed to artificial habitats created by humans62. Rapid globalization, urbanization, anthropogenic activities and increase in wildlife–human interactions have provided ample opportunities for spillovers to take place. There can be direct and indirect ways of transmitting pathogens from wildlife to humans either mediated through (a) consumption of contaminated bushmeat, (b) inhalation of rodents/ bat excreta aerosols, (c) direct contact with pathogens shed on animate household objects or during activities such as farming and camping, (d) intermediate hosts such as pigs and horses, serving as amplifying hosts. Intermediate hosts play a significant role due to their close association with humans, primarily in animal farms or being consumed as a nutrient source63. In addition, many cultural practices can expose humans to pathogens of zoonotic origins. For instance, the presence of filovirus surface protein reactive antibodies in bat harvesters of Mimi village of Nagaland, India, indicated possible exposure to filoviruses, possibly from the bat’s meat obtained during the bat harvests64. Behaviorally, bats are known to chew fruits, leaves, insects, followed by the leftover chewed parts of the food fallen on the ground becoming infected with viruses from the saliva of bats. Other animals such as palm civets (potential intermediate hosts for SARS) and swine consume these contaminated fruits and carry forward the transmission cycle of the virus65. The Nipah virus outbreak in Malaysia, Ebola virus outbreak in Africa and Hendra virus outbreak in Australia involve an intermediate host and are associated with bat reservoirs through a similar transmission route66 (Fig. 1). On the contrary, it has also been observed that the Nipah virus outbreak in Bangladesh involves the direct transmission of viruses from bats to humans through the consumption of contaminated date palm juice67 (Fig. 1). Bats are also long-lived and the only volant mammals with a unique immune system which restricts viruses from showing overt pathologies while persistently evolving within each individual bat and dispersing across wide spatial ranges through their excreta, saliva, and interactions during movement68.

Similar to bats, rodents also play a key role as reservoirs for many medically important pathogens, such as Hantavirus, Arenavirus, Bartonella, Leptospira, Yersinia pestis69, 70. ‘Anthropocene defaunation’—owing to globalization, has reduced the diversity of larger mammals and consequently an increase in rodent population. This further increases the probability of rodent borne spillovers in the future.

5 Environmental Sampling, Metagenomics, and Reservoirs—Foundation Pillars for Disease Surveillance

While we talk about the increasing number of infectious diseases, it is crucial to tap into the microbiome and virome of wildlife (a) to understand the ecological parameters driving the evolution and functional importance of host associated microbes, and (b) to investigate the trends in emergence of novel pathogens or increased shedding rates of previously well-characterized pathogens. However, a major drawback is the lack of baseline data on diversity and prevalence of microbes in wildlife, particularly small mammals, such as bats and rodents. The virome refers to the collection of all viruses present in any organism71. The Global Virome project was launched with an aim to create an atlas of all the viruses that are present in the world, especially in low income or middle-income countries72. The economic losses associated with managing an outbreak outnumbers the amount of investment that would facilitate research in understanding the diversity of viruses which are present naturally in different biological systems. Investments in viral discovery can aid the Global Preparedness Monitoring Board (GPMB) to take necessary measures to mitigate pandemic risks. Such global projects would foster collaborations between countries and initiate exchange of ideas to solve gray areas in the domain of emerging infectious diseases.

Non-invasive sampling of wildlife, especially with a concentrated focus on small mammals such as bats and rodents can mediate important signals prior to any outbreak. Table 1 summarizes the wide range of questions explored using environmental sampling, in the context of reservoirs and infectious agents.

Table 1: Summary of studies done on environmental samples from bats and rodents in the context of disease ecology
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6 The Future

We propose a very detailed investigation approach and strongly recommend coordinated and collaborative efforts comprising epidemiologists, evolutionary biologists, and infectious disease experts to unravel the mystery of zoonotic spillovers for the future (Fig. 3). Currently, in South and Southeast Asian countries, advanced biosafety level facilities are present in restricted numbers. The ecological aspects of any wildlife associated disease have been neglected globally and such studies have almost been negligible in South Asia. While being widely known for its rich biodiversity and population explosion, India can potentially serve as an ideal model system to study cross-species transmission of pathogens (Fig. 4).Through this review, we looked at the wide range of questions one can address simply with the help of non-invasive sampling. We envision environmental sampling to be the ideal solution for informing public health professionals about any unnatural incidence of a disease. Early and rapid monitoring of any emerging infectious agent can help better prepare for the outbreak. Understanding what cues lead to pathogen spillover can help prevent the same. Investigation of genomic data from the field and various mathematical projections can help quantitate the disease burden and predict the pathogenicity of any EIDs.

Figure 3:
figure 3

Integrated approach is needed to investigate disease monitoring in the wild. We propose a coordinated investigation between multiple district level, regional, and local laboratories across India to predict and pre-empt spillovers in the wild.

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Figure 4:
figure 4

BatVir is a database software for all the bat viruses reported across the world. In this graphical representation, we show that there have been very few studies in India which have investigated viruses from bat reservoirs. No virome study has been done in India, which points out at the urgency of such studies in the country.

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The regional laboratories should be well equipped with state-of-the-art genomics facilities and should be able to carry out serosurveys (Fig. 3). However, the entire process of disease investigation must begin with environmental sampling at the field laboratories (Fig. 2). There should be dedicated teams to collect not only non-invasive samples from the wild, but also implement a routine screening of fecal, urine and saliva samples from local animal farms. Farfetched as it is, companion animals should also be screened monthly as they are the ones in closest proximity with humans. Investments need to be made enormously to improve the genomics facilities at the district level. Decentralizing surveillance efforts will make the detection of infectious agents much faster. Regional and District level laboratories should be able to carry out functional genomics studies independently and rely on the apex wildlife disease investigation lab only when the isolation of pathogens is required (Fig. 3). The idea of having one apex laboratory in India should not be encouraged; alternatively, five to six apex laboratories spanning the entire country would be significantly more beneficial. This would again be instrumental in reducing the workload of apex laboratories and prompt outbreak decisions at an early stage.

Discussions, collaborations, research, and outreach are the four foundational pillars that can help in policy making towards preventing zoonotic spillovers. Educating the young generation and engaging local communities can significantly reduce misinformation regarding ecologically important disease reservoirs, such as bats. Adding information about zoonoses in school curricula can generate interest in studying the field and hopefully disseminate the knowledge in their families who may be involved in professions (for instance—date palm sap handlers), where there is a high risk of getting exposed to pathogenic organisms. A major challenge is the time span in which we wish to see the change as age-old beliefs are deep-rooted in many societies. Model predictions and knowledge of bat or rodent diversity in various habitats is necessary to predict the hotspots of spillovers. A well-coordinated disease surveillance effort (Fig. 3) will certainly attract many non-governmental funders, providing more resources. Sharing of information and knowledge regarding any EID is of utmost importance within and outside the research community and calls for a pan-India virome project venture. Finally, pilot environmental sampling studies should be initiated using the current resources available in India which can lay down the proof of concept for any long-term disease monitoring efforts. With such information in hand, it will be possible to tackle the threat of emerging zoonotic spillovers, which in turn will shape the future of public health.