The study focused on two geographic regions of Nepal, the Terai and the Mid-hills. The lowland Terai runs along the country’s southern border and is home to five national parks: Shuklaphanta National Park (ShNP), Bardia National Park (BNP), Banke National Park (BaNP), Chitwan National Park (CNP) and Parsa National Park (PNP). Between them, these protected areas total 3402 km2 and accommodate most of Nepal’s tigers. Inside the national parks, the landscape is a matrix of grassy floodplains and tropical forests, but outside these areas, the region is widely settled and heavily cultivated. The Mid-hills lie north of the Terai and range between 600 and 4800 m. Here, the majority of settlements are found in the valleys, while much of the hillside remain densely forested. The region includes a large portion of Annapurna Conservation Area (ACA). However, unlike the national parks, people are not excluded from living or working in this area.
The samples analyzed in this study were collected opportunistically and archived in the molecular lab at the Biodiversity Conservation Center, Sauraha, in CNP. They included sera from 28 tigers and 20 leopards. Captures took place between 2011 and 2021 and were carried out jointly by the Department of National Parks and Wildlife Conservation (DNPWC) and the National Trust for Nature Conservation (NTNC). Almost all (47/48) captures were conducted as part of routine wildlife management and conflict resolution. The only exception to this was a single tiger, which was captured as part of a collaring study in 2021. Animals were immobilized using a standard protocol of medetomidine (0.07 mg/kg) and ketamine (3 mg/kg) delivered intramuscularly via dart gun or hand injection. Blood samples were collected during immobilization, and the separated serum was archived at −20 °C.
Most tigers (26/28) were captured in and around the Chitwan–Parsa Complex (CPC; 27°46′ N, 84°53′ E) which includes CNP, PNP and the surrounding buffer zones. The remaining two tigers were captured in or around the Bardia–Banke Complex (BBC; 28°33′ N, 81°65′ E) which includes BNP, BaNP and the surrounding buffers zones. Meanwhile, most leopards (17/20) were captured in the central Mid-hill districts, two were captured in the CPC and one was captured in Koshi Thappu Wildlife Reserve (KTWR; 26°65′ N, 87°00′ E).
Prior to testing, all samples were heat inactivated at 56 °C for 30 min. A modified version of a previously described serum neutralization test [52 (link)] was used to screen the heat-inactivated samples. This protocol uses an engineered cell line, HEK293dogSLAM, which expresses the canine signaling lymphocytic activation molecule (SLAM-F1, the receptor used by CDV for cell entry) and a replication deficient vesicular stomatitis virus pseudotype expressing hemagglutinin and fusion surface glycoproteins from the Onderstepoort strain of CDV. The referenced protocol was adapted by substituting green fluorescent protein (GFP) for luciferase as the marker for infection [12 (link)]. Cell lines HEK293dogSLAM and HEK293T were supplied by the University of Glasgow. Tests were read by examining wells using an inverted fluorescent microscope under low power. Dilutions which achieved a 90% reduction in the number of infected cells compared to the mean count of four serum-free controls were considered to have effectively neutralized the virus.
All samples were tested in quadruplicate using a 1:16 serum dilution along with a panel of 20 dog sera as positive controls. Samples which tested positive on the initial assay were re-tested to determine the titer using four-fold serial serum dilutions from 1:16 to 1:16,384. The Spearman–Karber method [53 ] was then used to calculate the final titer. Samples with a titer of 1:16 or greater were considered positive. Seroprevalence was calculated as the number of animals testing positive, which was divided by the total number of animals tested and expressed with 95% binomial confidence intervals. Population comparisons were conducted using a two-proportions z-test in R [54 ]. Results were reviewed in conjunction with the clinical histories for the individuals concerned.
Mortality records from the Central Zoo in Lalitpur and in CNP were reviewed for the period September 2019 to September 2022 to identify additional tigers and leopards that had suffered fatal neurological disease, which was characterized by seizures.
The samples analyzed in this study were collected opportunistically and archived in the molecular lab at the Biodiversity Conservation Center, Sauraha, in CNP. They included sera from 28 tigers and 20 leopards. Captures took place between 2011 and 2021 and were carried out jointly by the Department of National Parks and Wildlife Conservation (DNPWC) and the National Trust for Nature Conservation (NTNC). Almost all (47/48) captures were conducted as part of routine wildlife management and conflict resolution. The only exception to this was a single tiger, which was captured as part of a collaring study in 2021. Animals were immobilized using a standard protocol of medetomidine (0.07 mg/kg) and ketamine (3 mg/kg) delivered intramuscularly via dart gun or hand injection. Blood samples were collected during immobilization, and the separated serum was archived at −20 °C.
Most tigers (26/28) were captured in and around the Chitwan–Parsa Complex (CPC; 27°46′ N, 84°53′ E) which includes CNP, PNP and the surrounding buffer zones. The remaining two tigers were captured in or around the Bardia–Banke Complex (BBC; 28°33′ N, 81°65′ E) which includes BNP, BaNP and the surrounding buffers zones. Meanwhile, most leopards (17/20) were captured in the central Mid-hill districts, two were captured in the CPC and one was captured in Koshi Thappu Wildlife Reserve (KTWR; 26°65′ N, 87°00′ E).
Prior to testing, all samples were heat inactivated at 56 °C for 30 min. A modified version of a previously described serum neutralization test [52 (link)] was used to screen the heat-inactivated samples. This protocol uses an engineered cell line, HEK293dogSLAM, which expresses the canine signaling lymphocytic activation molecule (SLAM-F1, the receptor used by CDV for cell entry) and a replication deficient vesicular stomatitis virus pseudotype expressing hemagglutinin and fusion surface glycoproteins from the Onderstepoort strain of CDV. The referenced protocol was adapted by substituting green fluorescent protein (GFP) for luciferase as the marker for infection [12 (link)]. Cell lines HEK293dogSLAM and HEK293T were supplied by the University of Glasgow. Tests were read by examining wells using an inverted fluorescent microscope under low power. Dilutions which achieved a 90% reduction in the number of infected cells compared to the mean count of four serum-free controls were considered to have effectively neutralized the virus.
All samples were tested in quadruplicate using a 1:16 serum dilution along with a panel of 20 dog sera as positive controls. Samples which tested positive on the initial assay were re-tested to determine the titer using four-fold serial serum dilutions from 1:16 to 1:16,384. The Spearman–Karber method [53 ] was then used to calculate the final titer. Samples with a titer of 1:16 or greater were considered positive. Seroprevalence was calculated as the number of animals testing positive, which was divided by the total number of animals tested and expressed with 95% binomial confidence intervals. Population comparisons were conducted using a two-proportions z-test in R [54 ]. Results were reviewed in conjunction with the clinical histories for the individuals concerned.
Mortality records from the Central Zoo in Lalitpur and in CNP were reviewed for the period September 2019 to September 2022 to identify additional tigers and leopards that had suffered fatal neurological disease, which was characterized by seizures.
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