Phylogenetic characterization of canine distemper virus from stray dogs in Kathmandu Valley.

Canine distemper is a highly contagious, often fatal disease caused by canine distemper virus (CDV) in domestic dogs and wild carnivores. The virus has caused mass epidemics in both wild and captive carnivores of high conservation value such as tigers, lions and leopards. Hence, understanding and managing CDV outbreaks is particularly important in Nepal, which is home to many species of threatened wild carnivores including tigers, leopards, snow leopards, dholes and wolves, and also contains a large population of stray dogs. Previous studies have suggested that CDV may pose a threat to wild carnivores, but there have not been any studies characterizing the genetic strains of the virus circulating in Nepal's carnivores. We collected invasive and non-invasive biological samples from stray dogs in Kathmandu Valley and genetically characterized the strains of CDV in the dogs to belong to the Asia-5 lineage by using phylogenetic analysis. The same lineage also contained CDV strains sequenced from dogs, civets, red panda and lions in India. Based on our phylogenetic analysis, we think it is likely that CDV is maintained through sylvatic cycle among sympatric carnivores allowing the recurring spillovers and outbreaks. It is crucial to prevent the virus transmission from reservoir hosts to other species, especially threatened populations of large carnivores in Nepal. Hence, we recommend for regular surveillance of CDV targeting wild carnivores in addition to the domestic dogs.

Natural Clerodendrum-derived tick repellent: learning from Nepali culture.

Ticks attaching to ear canals of humans and animals are the cause of otoacariasis, common in rural areas of Nepal. The plant Clerodendrum viscosum is used in multiple indigenous systems of medicine by ethnic communities in the Indo-Nepali-Malaysian region. Visiting the Chitwan National Park, we learned that in indigenous medicine, flower extract of C. viscosum is utilized to treat digestive disorders and extracts from leaves as tick repellent to prevent ticks from invading or to remove them from the ear canal. The objective of our study was to provide support to indigenous medicine by characterizing the in vivo effect of leave extracts on ticks under laboratory conditions and its phytochemical composition. We collected plant parts of C. viscosum (leaves and flowers) and mango (Mangifera indica) leaves at the Chitwan National Park, previously associated with repellent activity to characterize their effect on Ixodes ricinus ticks by in vivo bioassays. A Q-ToF high-resolution analysis (HPLC-ESI-QToF) was conducted to elucidate phenolic compounds with potential repellent activity. Clerodendrum viscosum and M. indica leaf extracts had the highest tick repellent efficacy (%E = 80-100%) with significant differences when compared to C. viscosum flowers extracts (%E = 20-60%) and phosphate-buffered saline. Phytochemicals with tick repellent function as caffeic acid, fumaric acid and p-coumaric acid glucoside were identified in C. viscosum leaf extracts by HPLC-ESI-QToF, but not in non-repellent flower extracts. These results support the Nepali indigenous medicine application of C. viscosum leaf extracts to repel ticks. Additional research is needed for the development of natural and green repellent formulations to reduce the risks associated with ticks resistant to acaricides.

Novel strains of Campylobacter cause diarrheal outbreak in Rhesus macaques (Macaca mulatta) of Kathmandu Valley.

Campylobacter spp. is often underreported and underrated bacteria that present real health risks to both humans and animals, including non-human primates. It is a commensal microorganism of gastrointestinal tract known to cause gastroenteritis in humans. Commonly found in many wild animals including non-human primates (monkeys- Rhesus macaques) these pathogens are known to be a common cause of diarrhea in humans in many parts of developing and under developed countries. Rhesus macaques from the two holy sites in Kathmandu (Pashupati and Swoyambhu) were included in this cross-sectional study. Diarrheal samples of monkeys were analyzed to detect and characterize the pathogen using 16S rRNA-based PCR screening, followed by DNA sequencing and phylogenetic analysis. Out of a total 67 collected diarrheal samples, Campylobacter spp. were detected in the majority of the samples (n = 64; 96%). DNA sequences of the amplified PCR products were successfully obtained from 13 samples. Phylogenetic analysis identified Candidatus Campylobacter infans (n = 10, Kimura-2 parameter (K2P) pairwise distance values of 0.002287). Remaining three sequences might potentially belong to a novel Campylobacter species/sub-species- closely relating to known species of C. helviticus (K2P pairwise distance of 0.0267). Both Candidatus Campylobacter infans and C. helvitucus are known to infect humans and animals. Additionally, we also detected the bacteria in water and soil samples from the sites. Campylobacter spp. caused the 2018 diarrhea outbreak in Rhesus macaques in the Kathmandu valley. Campylobacter might be one of the important contributing pathogens in diarrheal outbreaks-both in humans and animals (monkeys) in Nepal. Due to close interactions of these animals with humans and other animals, One Health approach might be the most effective way to prevent and mitigate the threat posed by this pathogen.

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.

Newcastle disease burden in Nepal and efficacy of Tablet I2 vaccine in commercial and backyard poultry production.

Poultry (Gallus domesticus) farming plays an important role as an income generating enterprise in a developing country like Nepal, contributing more than 4% to the national Gross Domestic Product (GDP). Newcastle Disease (ND) is a major poultry disease affecting both commercial and backyard poultry production worldwide. There were more than 90 reported ND outbreaks in Nepal in 2018 with over 74,986 birds being affected. ND is responsible for over 7% of total poultry mortality in the country. Recent outbreaks of ND in 2021 affected many farms throughout Nepal and caused massive loss in poultry production. ND is caused by a single-stranded ribonucleic acid (RNA) virus that presents very similar clinical symptoms as Influenza A (commonly known as bird flu) adding much complexity to clinical disease identification and intervention. We conducted a nationwide ND and Influenza A (IA) prevalence study, collecting samples from representative commercial and backyard poultry farms from across the major poultry production hubs of Nepal. We used both serological and molecular assessments to determine disease exposure history and identification of strains of ND Virus (NDV). Of the 40 commercial farms tested, both NDV (n = 28, 70%) and IAV (n = 11, 27.5%) antibodies were detected in majority of the samples. In the backyard farms (n = 36), sero-prevalence of NDV and IAV were 17.5% (n = 7) and 7.5% (n = 3) respectively. Genotype II NDV was present in most of the commercial farms, which was likely due to live vaccine usage. We detected never reported Genotype I NDV in two backyard farm samples. Our investigation into 2021 ND outbreak implicated Genotype VII.2 NDV strain as the causative pathogen. Additionally, we developed a Tablet formulation of the thermostable I2-NDV vaccine (Ranigoldunga™) and assessed its efficacy on various (mixed) breeds of chicken (Gallus domesticus). Ranigoldunga™ demonstrated an overall efficacy >85% with a stability of 30 days at room temperature (25°C). The intraocularly administered vaccine was highly effective in preventing ND, including Genotype VII.2 NDV strain.

Identification and characterization of probiotics isolated from indigenous chicken (Gallus domesticus) of Nepal.

BACKGROUND: Excessive and irrational use of antibiotics as growth promoters in poultry has been one of key factors contributing to increased emergence of antibiotics resistant bacteria. Several alternatives for antibiotic growth promoters are being sought, and the search for effective probiotics to be used as feed additives is amongst the promising ones. Our study aimed to isolate and test potential probiotics bacteria from cloacal swabs of various indigenous chicken (Gallus domesticus) breeds from rural outskirts of the Kathmandu valley (Nepal). METHODS: Selective isolation of probiotics was conducted by micro-aerophilic enrichment of sample in MRS Broth at 37°C, followed by culturing on MRS agar supplemented with 5 g/L of CaCO3. Isolated bacterial colonies producing transparent halo were selected as potential lactic acid bacteria (LAB), and tested for their antibacterial activity, phenotypic and biochemical characteristics, acidic yield, and tolerance to acid and bile. RESULTS: A total of 90 potential LAB were isolated from cloacal samples collected from 41 free-ranging chickens of indigenous breeds. Of these, 52 LAB isolates (57%) showed variable antibacterial activity to at least one bacterial pathogen. Of 52 LAB, 46 isolates fulfilled phenotypic and biochemical criteria of Lactobacillus spp. Of these, 37 isolates produced varying percentage yields of lactic acid, 27 isolates showed survival at pH 3.0, and 17 isolates showed survival tolerances in the presence of 0.3% and 0.5% bile salts for 24 hours. Phylogenetic analysis of 16S rDNA sequencing of LAB isolates fulfilling in vitro probiotics properties showed that 3 isolates had genetic identity of 99.38% with Lactobacillus plantarum, while one isolate was genetically similar (99.85%) with the clade of L. reuteri, L. antri and L. panis. CONCLUSION: Our study identified four Lactobacillus spp. strains having potential probiotics properties. Further investigations are needed to evaluate these isolates to be used as poultry probiotics feed supplement.

Rapid genomic surveillance of SARS-CoV-2 in a dense urban community of Kathmandu Valley using sewage samples.

Understanding disease burden and transmission dynamics in resource-limited, low-income countries like Nepal are often challenging due to inadequate surveillance systems. These issues are exacerbated by limited access to diagnostic and research facilities throughout the country. Nepal has one of the highest COVID-19 case rates (915 cases per 100,000 people) in South Asia, with densely-populated Kathmandu experiencing the highest number of cases. Swiftly identifying case clusters (hotspots) and introducing effective intervention programs is crucial to mounting an effective containment strategy. The rapid identification of circulating SARS-CoV-2 variants can also provide important information on viral evolution and epidemiology. Genomic-based environmental surveillance can help in the early detection of outbreaks before clinical cases are recognized and identify viral micro-diversity that can be used for designing real-time risk-based interventions. This research aimed to develop a genomic-based environmental surveillance system by detecting and characterizing SARS-CoV-2 in sewage samples of Kathmandu using portable next-generation DNA sequencing devices. Out of 22 sites in the Kathmandu Valley from June to August 2020, sewage samples from 16 (80%) sites had detectable SARS-CoV-2. A heatmap was created to visualize the presence of SARS-CoV-2 infection in the community based on viral load intensity and corresponding geospatial data. Further, 47 mutations were observed in the SARS-CoV-2 genome. Some detected mutations (n = 9, 22%) were novel at the time of data analysis and yet to be reported in the global database, with one indicating a frameshift deletion in the spike gene. SNP analysis revealed possibility of assessing circulating major/minor variant diversity on environmental samples based on key mutations. Our study demonstrated the feasibility of rapidly obtaining vital information on community transmission and disease dynamics of SARS-CoV-2 using genomic-based environmental surveillance.

Molecular and morphological identification of frog species collected at Rara Lake in Rara National Park, Nepal.

This study seeks to clarify the taxonomic identity of three adult frogs and two tadpoles from Rara Lake situated in Rara National Park, Mugu district, Western Nepal, using both phenotypic and mitochondrial sequence data (16S rRNA gene). Based on the molecular data, we determined our specimens belonged to two species; Paa cf. ercepeae (Dubois, 1974) and Paa rarica (Dubois, Matsui, and Ohler, 2001), and this assignment is also supported by morphology. Principal component analysis of phenotypic data clustered species into large-bodied (SVL > 55 mm) and small-bodied (SVL < 55 mm) groups. The molecular data suggest that Paa rarica belonged to a group that contains several undescribed lineages and that Paa liebigii (Gnther, 1860) hosts extensive cryptic diversity. Our study also documents that Paa species in the Himalayan region are more widely distributed than previously believed.

Phylogeographical analysis shows the need to protect the wild yaks' last refuge in Nepal.

The wild yak Bos mutus was believed to be regionally extinct in Nepal for decades until our team documented two individuals from Upper Humla, north-western Nepal, in 2014. The International Union for Conservation of Nature (IUCN) seeks further evidence for the conclusive confirmation of that sighting. We conducted line transects and opportunistic sign surveys in the potential wild yak habitats of Humla, Dolpa, and Mustang districts between 2015 and 2017 and collected genetic samples (present and historic) of wild and domestic yaks Bos grunniens. We also sighted another wild yak in Upper Humla in 2015. Phylogenetic and haplotype network analyses based on mitochondrial D-loop sequences (~450 bp) revealed that wild yaks in Humla share the haplotype with wild yaks from the north-western region of the Qinghai-Tibetan Plateau in China. While hybridization with domestic yaks is a major long-term threat, illegal hunting for meat and trophy put the very small populations of wild yaks in Nepal at risk. Our study indicates that the unprotected habitat of Upper Humla is the last refuge for wild yaks in Nepal. We recommend wild yak conservation efforts in the country to focus on Upper Humla by (i) assigning a formal status of protected area to the region, (ii) raising awareness in the local communities for wild yak conservation, and (iii) providing support for adaptation of herding practice and pastureland use to ensure the viability of the population.

Gut microbiota and their putative metabolic functions in fragmented Bengal tiger population of Nepal.

Bengal tigers (Panthera tigris tigris) serve a pivotal role as an apex predator in forest ecosystems. To increase our knowledge on factors impacting the viability and health of this endangered species, we studied the gut microbiota in 32 individual Bengal tigers from three geographically separated areas (Chitwan National Park (CNP), Bardia National Park (BNP) and Suklaphanta Wildlife Reserve (SWR)) in Nepal, using noninvasive genetic sampling methods. Gut microbiota influence the immune system, impact various physiological functions, and modulates metabolic reactions, that ultimately impact the host health, behavior and development. Across the tiger populations in Nepal, we found significant differences in the composition of microbial communities based on their geographic locations. Specifically, we detected significant differences between CNP and the other two protected areas (CNP vs BNP: pseudo t = 1.944, P = 0.006; CNP vs SWR: pseudo t = 1.9942, P = 0.0071), but no differences between BNP and SWR. This mirrors what has been found for tiger gene flow in the same populations, suggesting gut microbiota composition and host gene flow may be linked. Furthermore, predictive metagenome functional content analysis (PICRUSt) revealed a higher functional enrichment and diversity for significant gut microbiota in the Chitwan tiger population and the lowest enrichment and diversity in Suklaphanta. The CNP tiger population contained higher proportions of microbiota that are associated with predicted functions relevant for metabolism of amino acid, lipid, xenobiotics biodegradation, terpenoides and polyketides than the SWR population. We conclude the tiger population structure, gut microbiota profile and associated functional metabolic categories are correlated, with geographically most separated CNP and SWR tiger population having the most distinct and different host genotype and microbiota profiles. Our work dramatically expands the understanding of tiger microbiota in wild populations and provides a valuable case study on how to investigate genetic diversity at different hierarchical levels, including hosts as well as their microbial communities.

Species, sex and geo-location identification of seized tiger (Panthera tigris tigris) parts in Nepal-A molecular forensic approach.

Tiger (Panthera tigris) populations are in danger across their entire range due to habitat loss, poaching and the demand for tiger parts. The Bengal tiger (Panthera tigris tigris) is an endangered apex predator with a population size estimated to be less than 200 in Nepal. In spite of strict wildlife protection laws, illegal trade of tiger parts is increasing; and Nepal has become one of the major sources and transit routes for poached wildlife parts. Identification of wildlife parts is often challenging for law enforcement officials due to inadequate training and lack of available tools. Here, we describe a molecular forensic approach to gain insight into illegally trafficked tiger parts seized across Nepal. We created Nepal's first comprehensive reference genetic database of wild tigers through the Nepal Tiger Genome Project (2011-2013). This database has nuclear DNA microsatellite genotype and sex profiles, including geo-spatial information, of over 60% (n = 120) of the wild tigers of Nepal. We analyzed 15 putative cases of confiscated poached tiger parts and all were confirmed to be of tiger. Ten samples were identified as male and five were female. We determined probable geo-source location for 9 of the 14 samples with 6-8 nuclear DNA microsatellite loci using inferences from four different statistical assignment methods. Six samples were assigned to Bardia National Park and one of these was an exact match to a female tiger previously profiled in our fecal DNA reference database. Two tiger samples were assigned to Shuklaphanta Wildlife Reserve and one to Chitwan National Park. We are unable to definitively assign five tiger samples which could be offspring dispersers or might have come from tiger population outside of Nepal. Our study revealed that the western region, particularly Bardia National Park, is a poaching hotspot for illegal tiger trade in Nepal. We present feasibility of using molecular forensic based evidence to incriminate criminals in a court of law in the fight against wildlife crime.

Identification and characterization of a novel zebrafish (Danio rerio) pentraxin-carbonic anhydrase.

BACKGROUND: Carbonic anhydrases (CAs) are ubiquitous, essential enzymes which catalyze the conversion of carbon dioxide and water to bicarbonate and H+ ions. Vertebrate genomes generally contain gene loci for 15-21 different CA isoforms, three of which are enzymatically inactive. CA VI is the only secretory protein of the enzymatically active isoforms. We discovered that non-mammalian CA VI contains a C-terminal pentraxin (PTX) domain, a novel combination for both CAs and PTXs. METHODS: We isolated and sequenced zebrafish (Danio rerio) CA VI cDNA, complete with the sequence coding for the PTX domain, and produced the recombinant CA VI-PTX protein. Enzymatic activity and kinetic parameters were measured with a stopped-flow instrument. Mass spectrometry, analytical gel filtration and dynamic light scattering were used for biophysical characterization. Sequence analyses and Bayesian phylogenetics were used in generating hypotheses of protein structure and CA VI gene evolution. A CA VI-PTX antiserum was produced, and the expression of CA VI protein was studied by immunohistochemistry. A knock-down zebrafish model was constructed, and larvae were observed up to five days post-fertilization (dpf). The expression of ca6 mRNA was quantitated by qRT-PCR in different developmental times in morphant and wild-type larvae and in different adult fish tissues. Finally, the swimming behavior of the morphant fish was compared to that of wild-type fish. RESULTS: The recombinant enzyme has a very high carbonate dehydratase activity. Sequencing confirms a 530-residue protein identical to one of the predicted proteins in the Ensembl database (ensembl.org). The protein is pentameric in solution, as studied by gel filtration and light scattering, presumably joined by the PTX domains. Mass spectrometry confirms the predicted signal peptide cleavage and disulfides, and N-glycosylation in two of the four observed glycosylation motifs. Molecular modeling of the pentamer is consistent with the modifications observed in mass spectrometry. Phylogenetics and sequence analyses provide a consistent hypothesis of the evolutionary history of domains associated with CA VI in mammals and non-mammals. Briefly, the evidence suggests that ancestral CA VI was a transmembrane protein, the exon coding for the cytoplasmic domain was replaced by one coding for PTX domain, and finally, in the therian lineage, the PTX-coding exon was lost. We knocked down CA VI expression in zebrafish embryos with antisense morpholino oligonucleotides, resulting in phenotype features of decreased buoyancy and swim bladder deflation in 4 dpf larvae. DISCUSSION: These findings provide novel insights into the evolution, structure, and function of this unique CA form.