Interesting differentiation of malicious poisoning in a wild leopard

Introduction

Human wildlife conflict has reached its peak owing to the loss of habitat, habitat fragmentation and invasion by development infrastructure. The incidences of predation of livestock have increased due to the presence of the carnivores near human settlements. The predation of livestock leads to financial losses to the village community. Incidences of poisoning of the predated livestock as revenge have come to light in recent times. Malicious poisoning of the carcass often leads to massive deaths as scavengers are unintentionally poisoned (Radhakrishnan, 2018) [9] . From the conservation point of view, malicious poisoning of carcass is a threat to biodiversity as it can lead to large scale mortality. Under the Indian Wildlife Protection Act, 1972 all endangered animals are protected. The cases of poisoning in Indian wildlife are often under reported due to non-availability of laboratories for detection, decomposition of the carcass, improper packaging/ handling of the samples and long waiting period in the laboratories. There are reports of poisoning of a variety of wildlife with a range of chemicals ranging from insecticides, pesticides, chemical toxins, rodenticides etc. Leopards are distributed throughout the Indian subcontinent and despite the stern competition for resources have adapted to survive at the wild-human interface. In recent times, leopardhuman conflict has reached its pinnacle due to habitat loss, limited prey base, deforestation and encroachment of development infrastructure in protected areas. Leopards cause economic hardship by predation of the livestock and dogs. The cases of revenge poisoning have been encountered in the country but poorly documented (Gupta et al., 2021) [3] . This case report deals with the differential diagnosis of poisoning of leopards using molecular and forensic analysis.

Case Presentation

A carcass of a sub-adult male leopard was found during the forest patrolling at Semadoh under Sipna Division of Melghat Tiger Reserve, Amravati, Maharashtra, a systematic post mortem examination was carried out to probe the cause of death. The gross lesions were noted and samples were collected for further investigation. The samples were submitted to Wildlife research & Training Centre, Gorewada, Nagpur for diagnosis. The major findings included congestion of the lungs, heart, intestine, kidney and spleen along with necrotic foci on the liver and kidney. The serosal and mucosal surfaces of the intestine showed typical haemorrhagic bands. A day later a carcass of a female adult leopard and civet cat was recovered from the same area. The carcass was sent to Wildlife Research & Training Centre, Gorewada, Nagpur for the post mortem examination. The findings were similar to the findings reported in the male sub adult leopard. The stomach and intestinal content were sent for the forensic analysis. The description of lesions prompted a possibility of Feline Panleukopenia, hence intestinal content and visceral organs were utilised for the diagnosis of the disease. During the post mortem examination undigested meat was discovered in the stomach of the female leopard. The sample was collected for the species identification.

The samples were transported on ice to the laboratory and DNA was isolated using commercially available DNA stool kit (Mfg. Qiagen) and blood and tissue Kit (Mfg. Qiagen). The DNA was quantified using Qubit and a PCR test for Feline Panleukopenia was performed using protocol described by Sheikh et al., 2017 [13] . Also, qPCR was performed as described by Singh et al., 2013 [14] . The DNA recovered from undigested meat in the stomach was utilised for the species identification using cytochrome b based primers (Singh & Verma, 2003) [15] . The amplicon so obtained was purified using commercial kits and sequenced using Gene Analyser 3500 (Mfg. Appied Biosystems). The consensus sequence was built using the Mega X software and subjected to nBlast analysis using the NCBI database.

Result:

The PCR and qPCR test was negative for Feline Panleukopenia; the species identification PCR of the recovered meat provided an amplification of approximately 440 bp. The consensus sequence on blast analysis was found similar to goat (Capra hircus). The findings were communicated to the law enforcement agencies of the forest department. The team conducted search at the site where the carcasses were discovered. The search led to discovery of a decomposed goat carcass. The field investigation and enquiry revealed malicious poisoning of the kill by a farmer to seek revenge. The forensic lab confirmed the presence of zinc phosphide in the stomach contents of the male and female leopard.

Discussion

Leopards (Panthera pardus) are one of the most adaptable large carnivores and are widely distributed across India. They have been increasingly coming into conflict with humans due to habitat fragmentation, depletion of natural prey, and encroachment of developmental infrastructure (Athreya et al., 2013) [1] . Livestock depredation by leopards is a significant issue in many parts of India, particularly in states such as Maharashtra, Uttarakhand, Himachal Pradesh, and Madhya Pradesh (Odden et al., 2014) [7] . Studies indicate that leopards exhibit a high degree of behavioural plasticity, enabling them to thrive even in human-dominated landscapes, often preying upon domestic livestock, including goats, cattle, and dogs (Kolipaka et al., 2017) [8] . The economic burden of livestock loss often leads to retaliatory actions by local communities, including poisoning, electrocution, and snaring. Leopards, being highly elusive, are difficult to track, and indirect evidence such as pugmarks, scat, and camera trap data are often used to monitor their movements (Dutta et al., 2021) [2] . While government compensation schemes exist, delays in processing claims and inadequate compensation frequently drive local communities to take matters into their own hands. Revenge killing of large carnivores is a persistent issue in India, primarily executed through poisoning, electrocution, and snaring. Poisoning remains one of the most common and detrimental methods, as it not only targets the predator but also results in collateral damage to scavengers such as vultures, hyenas, and other carnivores (Radhakrishnan, 2018b) [10] . Poisoning involves the use of various chemicals such as organophosphates, carbamates, zinc phosphide, and rodenticides to lace carcasses. Zinc phosphide, identified in the current case, is a potent rodenticide known to cause severe pulmonary and gastrointestinal damage (Gupta et al., 2021b) [4] . The indiscriminate use of poison disrupts the ecological balance and poses a significant threat to non-target species. Electrocution occurs when farmers and poachers install highvoltage electric fences or tamper with power lines to electrocute wild animals. Electrocution incidents have been frequently reported in states like Karnataka and Madhya Pradesh, where power lines run through forest fringes (Sivakumar et al., 2019) [11] . Snaring involves the use of metal wire snares, commonly set for small game but inadvertently trapping leopards and other large carnivores. Injuries caused by snares often lead to prolonged suffering and eventual death due to infections and starvation (Gubbi et al., 2019) [6] .

In wildlife forensics, differentiating between infectious diseases and poisoning is crucial for accurate diagnosis and legal investigation. In the present case, differential diagnosis was necessary to rule out infectious causes, particularly Feline Panleukopenia Virus (FPV), which shares overlapping clinical signs such as haemorrhagic enteritis and multi-organ congestion (Steinel et al., 2001) [12] . However, molecular diagnostics, including PCR and qPCR assays, confirmed the absence of FPV, strengthening the hypothesis of toxicological causation. Species identification through molecular forensic methods plays a pivotal role in wildlife crime investigations. In this case, DNA extracted from the undigested meat in the female leopard's stomach was amplified using cytochrome bbased primers, confirming the presence of goat (Capra hircus) DNA (Singh & Verma, 2003) [15] . This finding was critical in linking the poisoned goat carcass to the leopard's mortality, strengthening the forensic evidence against the perpetrators. The use of molecular techniques such as DNA barcoding and species-specific PCR assays enables precise identification of prey species, aiding in forensic investigations and legal proceedings (Iyengar, 2014) [5] . The integration of molecular forensic tools with traditional necropsy findings provides a robust framework for investigating wildlife crimes and enforcing conservation laws.

Conclusion

This case highlights the urgent need for improved forensic capabilities in India to detect and document poisoning cases in wildlife. The findings underscore the importance of rapid and systematic necropsy procedures, molecular diagnostics, and toxicological analysis in differentiating between natural and anthropogenic causes of wildlife mortality. Strengthening law enforcement, enhancing awareness among local communities, and promoting coexistence strategies are critical steps toward mitigating human-wildlife conflicts and preventing retaliatory killings.

Acknowledgement

AcknowledgementThe authors acknowledge the kind approval of the Principal Chief Conservator of Forest (Wildlife) & Chief Wildlife Warden, Maharashtra State, for carrying out the research work. Field Director, Melghat Tiger Reserve and DCF, Sipna Division for the submission of samples to WRTC for analysis. Forest Development Corporation of Maharashtra Ltd., Nagpur and Office of the Divisional Manager, Gorewada Project, Nagpur, for the facilities provided to undertake the research. Director, Wildlife Research & Training Centre to facilitate the research work at WRTC, Gorewada, Nagpur.

References

1. Athreya V, Odden M, Linnell JD, Karanth KU. Big cats in our backyards: persistence of large carnivores in a human-dominated landscape in India. PLoS One. 2013;8(3):e57872.
2. Dutta T, Sharma S, Malviya M, Sinha P. Movement patterns and conflict dynamics of leopards in humandominated landscapes. J Wildl Res. 2021;45(2):123-136.
3. Gupta SK, Singh P, Yellapu S. Molecular investigation of three leopards death: a case of suspicious revenge killing. Forensic Science International: Genetics. 2021 Mar 1; 51:102425.
4. Gupta A, Sharma R, Singh R. Wildlife poisoning in India: A forensic perspective. J Forensic Sci. 2021;66(5):1895-1903.
5. Iyengar A. Forensic DNA analysis for animal protection and biodiversity conservation: A review. J Nat Conserv. 2014;22(3):195-205.
6. Gubbi S, Poornesha HC, Madhusudan MD. Impact of vehicular traffic on the use of highway edges by large mammals in a South Indian wildlife reserve. Curr Sci. 2019;116(8):1267-1272.
7. Odden M, Athreya V, Linnell JDC. Predation of livestock and wild prey by leopards in a human-dominated landscape in western Maharashtra, India. J Wildl Manag. 2014;78(3):504-513.
8. Kolipaka SS, Persoon GA, Prins HH. Conservation through coexistence: Human-leopard interactions in rural India. Biodivers Conserv. 2017;26(10):2397-2413.
9. Radhakrishnan S. Poisoning and its impact on wildlife conservation: A review. Environ Toxicol Pharmacol. 2018;62:74-81.
10. Radhakrishnan S. A note on wildlife poisoning cases from Kerala, South India. European journal of wildlife research. 2018 Oct; 64(5):58.
11. Sivakumar K, Johnsingh AJT, Sathyakumar S. Wildlife electrocution in India: An emerging conservation challenge. Environ Conserv. 2019;46(4):312-320.
12. Steinel A, Munson L, Van Vuuren M, Truyen U. Genetic characterization of feline parvovirus sequences from various carnivores. J Gen Virol. 2001;82(9):2323-2329.
13. Sheikh MO, Rashid PM, Marouf AS, Raheem ZH, Manjunath S, Janga SC. Molecular typing of canine parvovirus from Sulaimani, Iraq and phylogenetic analysis using partial VP2 gene. Bulg. J. Vet. Med. 2017;20(3):225-235.
14. Singh D, Verma AK, Kumar A, Srivastava M, Singh SK, Tripathi AK, et al. Detection of canine parvovirus by polymerase chain reaction assay and its prevalence in dogs in and around Mathura, Uttar Pradesh, India. Amer. J. Biochem. Mol. Biol. 2013; 3(2):264-70.
15. Singh R, Verma SK. Cytochrome b gene sequence variation and species identification in Indian mammals. Mol Ecol. 2003;12(5):1115-1121.