Phylogenomic analysis supports Mycobacterium tuberculosis transmission between humans and elephants.

Introduction: Tuberculosis is an infectious disease caused by a group of acid-fast bacilli known as Mycobacterium tuberculosis complex (MTC), which has a major impact on humans. Transmission of MTC across the human-animal interface has been demonstrated by several studies. However, the reverse zoonotic transmission from humans to animals (zooanthroponosis) has often been neglected. Methods: In this study, we used Nanopore MinION and Illumina MiSeq approaches to sequence the whole genome of M. tuberculosis strains isolated from two deceased Asian elephants (Elephas maximus) and one human in Chitwan, Nepal. The evolutionary relationships and drug resistance capacity of these strains were assessed using the whole genome data generated by the stand-alone tool Tb-Profiler. Phylogenomic trees were also constructed using a non-synonymous SNP alignment of 2,596 bp, including 94 whole genome sequences representative of the previously described M. tuberculosis lineages from elephants worldwide (lineages 1 and 4) and from humans in Nepal (lineages 1, 2 and 3). Results and Discussion: The new genomes achieved an average coverage of 99.6%, with an average depth of 55.67x. These M. tuberculosis strains belong to lineage 1 (elephant DG), lineage 2 (elephant PK) and lineage 4 (human), and none of them were found to have drug-resistant variants. The elephant-derived isolates were evolutionarily closely related to human-derived isolates previously described in Nepal, both in lineages 1 and 2, providing additional support for zooanthroponosis or bidirectional transmission between humans and elephants. The human-derived isolate clustered together with other published human isolates from Argentina, Russia and the United Kingdom in the lineage 4 clade. This complex multi-pathogen, multi-host system is challenging and highlights the need for a One Health approach to tuberculosis prevention and control at human-animal interface, particularly in regions where human tuberculosis is highly endemic.

Canine Distemper Virus in Tigers (Panthera tigris) and Leopards (P. pardus) in Nepal.

From wild dogs (Lycaon pictus) in the Serengeti to tigers (Panthera tigris altaica) in the Russian Far East, canine distemper virus (CDV) has been repeatedly identified as a threat to wild carnivores. Between 2020 and 2022, six Indian leopards (P. pardus fusca) presented to Nepali authorities with fatal neurological disease, consistent with CDV. Here, we report the findings of a serosurvey of wild felids from Nepal. A total of 48 serum samples were tested, comprising 28 Bengal tigers (P. t. tigris) and 20 Indian leopards. Neutralizing antibodies were identified in three tigers and six leopards, equating to seroprevalences of 11% (CI: 2.8-29.3%, n = 28) and 30% (CI: 12.8-54.3%, n = 20), respectively. More than one-third of seropositive animals were symptomatic, and three died within a week of being sampled. The predation of domestic dogs (Canis lupus familiaris) has been posited as a potential route of infection. A comparison of existing diet studies revealed that while leopards in Nepal frequently predate on dogs, tigers do not, potentially supporting this hypothesis. However, further work, including molecular analyses, would be needed to confirm this.

Seroprevalence of canine distemper virus (CDV) in the free-roaming dog (Canis familiaris) population surrounding Chitwan National Park, Nepal.

Canine distemper virus (CDV) is a global multi-host pathogen that is capable of causing considerable mortality in a range of species and is important in the field of conservation medicine. Nepal's Chitwan National Park is a protected area providing habitat for 32% of the country's mammal species including endangered carnivores such as the Bengal tiger (Panthera tigris tigris) that are susceptible to CDV. The presence of free-roaming dogs around protected areas could represent a source of infectious disease for transmission to local wildlife. A cross-sectional demographic and canine distemper virus seroprevalence study of 100 free-roaming dogs from the Chitwan National Park buffer zone and surrounding area was conducted in November 2019. The overall seroprevalence indicating past exposure to canine distemper virus was 80.0% (95% CI: 70.8-87.3). Of the host variables assessed, sex and age were positively associated with seroprevalence at the univariable level, with male dogs demonstrating lower seroprevalence than females (OR = 0.32, 95% CI: 0.11-0.91) and adult dogs demonstrating higher seroprevalence than juveniles (OR = 13.94, 95% CI: 1.37-142.29). The effect of sex was no longer significant at the multivariable level, but the direction of the effect remained the same. The effect of age remained significant after multivariable analysis (OR = 9.00, 95% CI: 1.03-192.75). No spatial associations were demonstrated in relation to the buffer zone area or boundary of Chitwan National Park. Free-roaming dog neutering and vaccination programmes can provide a useful baseline for future CDV studies in the region, and a proxy to monitor disease threats to susceptible wildlife.

Differentially Represented Proteins in Response to Infection with Mycobacterium tuberculosis Identified by Quantitative Serum Proteomics in Asian Elephants.

Tuberculosis is a major global concern. Tuberculosis in wildlife is a risk for zoonotic transmission and becoming one of the challenges for conservation globally. In elephants, the number of cases is likely rising. The aim of this study was to identify proteins related to tuberculosis infection in elephants, which could then be used for the development of diagnostic tools and/or vaccines. A serum proteomics approach was used to characterize differentially represented proteins in response to Mycobacterium tuberculosis in Asian elephants (Elaphas maximus). Blood samples were collected from eight elephants, four of which were antibody positive for tuberculosis and four were antibody negative. Proteomics analysis identified 26 significantly dysregulated proteins in response to tuberculosis. Of these, 10 (38%) were identified as immunoglobulin and 16 (62%) as non-immunoglobulin proteins. The results provided new information on the antibody response to mycobacterial infection and biomarkers associated with tuberculosis and protective response to mycobacteria in Asian elephants. Protective mechanisms included defense against infection (Alpha-1-B glycoprotein A1BG, Serpin family A member 1 SERPINA1, Transthyretin TTR), neuroprotection (TTR), and reduced risks of inflammation, infections, and cancer (SERPINA1, Keratin 10 KRT10). Using a translational biotechnology approach, the results provided information for the identification of candidate diagnostic, prognostic, and protective antigens for monitoring and control of tuberculosis in Asian elephants.

Serologic Survey of Selected Pathogens in Free-Ranging Bengal Tigers (Panthera tigris tigris) in Nepal.

Serum samples of 11 Bengal tigers (Panthera tigris tigris) from Chitwan National Park in Nepal, collected between 2011-17, were evaluated for the presence of antibodies to eight diseases commonly investigated in large felids. This initial serologic survey was done to establish baseline information to understand the exposure of Nepal's free-ranging tiger population to these diseases. Tiger serum samples collected opportunistically during encounters such as translocation, human conflict, and injury were placed in cold storage for later use. Frozen serum samples were assessed for feline coronavirus (FCoV), feline immunodeficiency virus, feline leukemia virus, feline herpesvirus (FHV), canine distemper virus, canine parvovirus-2 (CPV-2), leptospirosis (LEP; seven serovars), and toxoplasmosis (TOX). Six tigers were found to be positive for LEP, eight for CPV-2, five for FHV, one for FCoV, and 10 for TOX. Tigers, like other wild felids, have been exposed to these common pathogens, but further research is needed to determine the significance of these pathogens to the Nepali population.

Hematological Study of Captive White-Rumped Vultures (Gyps bengalensis) to Assess Their Health Status.

A study was conducted to estimate the hematological values of captive white-rumped vultures (Gyps bengalensis) in Nepal. Blood samples were collected from 23 adults and 14 juvenile captive white-rumped vultures during their annual health examinations at the Vulture Conservation and Breeding Center, Kasara, Chitwan, Nepal. Of 23 adults, 12 (52%) were male and 11 (48%) were females, whereas the sex of the 14 juveniles was undifferentiated. The mean (± SD) values for the adult birds were estimated as red blood cell count (2.86 ± 1.01 × 106/µL), white blood cell count (14.75 ± 6.01 × 103/µL, hemoglobin concentration (12.86 ± 1.67 g/dL), and packed cell volume (44.69 ± 3.63%). The mean (± SD) values for the juvenile vultures were estimated as red blood cell count (1.98 ± 0.5 × 106/µL), white blood cell count (16.73 ± 7.11 × 103/µL), hemoglobin concentration (11.57 ± 0.39 g/dL), and packed cell volume (44.5 ± 2.67%). There were no significant differences between the mean values of the hematological parameters based on the age or sex of vultures.

Risks from disease caused by Mycobacterium orygis as a consequence of Greater one-horned Rhinoceros (Rhinoceros unicornis) translocation in Nepal.

The greater one-horned rhinoceros (Rhinoceros unicornis) is listed as vulnerable by the IUCN Red List. Mycobacterium orygis-associated disease was identified in a single greater one-horned rhino in Chitwan National Park in February 2015 prior to a planned translocation of five greater one-horned rhinoceros from Chitwan National Park to Bardia National Park for conservation purposes. This paper describes a qualitative disease risk analysis conducted retrospectively post-translocation for Mycobacterium orygis and this translocation, with the aim to improve the understanding of disease threats to the conservation of greater one-horned rhino. The disease risk analysis method used was devised by Sainsbury & Vaughan-Higgins (Conservation Biology, 26, 2017, 442) with modifications by Bobadilla Suarez et al (EcoHealth, 14, 2017, 1) and Rideout et al (EcoHealth, 14, 2017, 42) and included the use of a scenario tree and an analysis of uncertainty as recommended by Murray et al. (Handbook on import risk analysis for animals and animal products. Volume 1. Introduction and qualitative risk analysis, 2004), and the first time this combination of methods has been used to assess the risk from disease in a conservation translocation. The scenario tree and analysis of uncertainty increased the clarity and transparency of the analysis. Rideout et al.'s (EcoHealth, 14, 2017, 42) criteria were used to assess the source hazard and may be useful in comparative assessment of source hazards for future conservation translocations. The likelihood of release into the destination site of Mycobacterium orygis as a source hazard was estimated as of low risk, the risk of exposure of populations at the destination was of high risk and the likelihood of biological and environmental consequences was low. Overall, the risk from disease associated with Mycobacterium orygis as a result of this translocation was found to be low. Recommendations on disease risk management strategies could be improved with a better understanding of the epidemiology including the presence/absence of Mycobacterium orygis in greater one-horned rhino to develop effective disease risk management strategies.

First report of malaria parasites in water buffalo in Nepal.

We present the first molecular-based report on ungulate malaria parasites from water buffalo in Nepal. Fifty-six blood samples were collected from different groups of water buffalo (wild, feral, and domestic) and PCR assays were conducted using Plasmodium spp. cytb specific primers. Two positive cases were detected, one each from feral and domestic individuals. Complete mitochondrial genome sequence (5987 bp) was obtained and examined for nucleotide variations. Sequence analysis revealed identity with type II water buffalo malaria parasites, reported previously, with one A to T nucleotide difference at position 5344. Prevalence, as well as possible economic impacts of water buffalo malaria, should be determined on a wider set of samples from buffalo across Nepal.