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We conducted a tuberculosis serosurveillance program of captive elephants in Nepal and compared hematology and biochemistry parameters between seropositive and seronegative elephants. A total of 153 elephants (male=20, female=133) from four national parks were tested for Tuberculosis (TB) using the ElephantTB STAT-PAK® Assay (ChemBio Diagnostic Systems, Inc., Medford, NY, USA). The mean reported age for 138 elephants was 38.5 years (range 2-71 years). Seroprevalence for TB was 21.56% (33/153). The majority of seropositive elephants were female (n=30) and from Chitwan National Park (n=29. The occurrence of TB seropositive cases in other more remote national parks suggests TB may be widespread among the captive elephant population of Nepal. Hematology and biochemistry analyses were performed on 13 and 22 seropositive elephants, respectively and, nine elephants from a seronegative TB herd for comparison. Hematology parameters (hemoglobin, packed cell volume, platelet, white blood cells, and erythrocyte sedimentation rate) were comparable between the two groups. Total protein, globulin, and lactate dehydrogenase were significantly higher in seronegative elephants, and bilirubin was significantly higher in seropositive elephants whereas blood urea nitrogen, creatinine, glutamic oxaloacetic transaminase/aspartate aminotransferase (GOT/AST), glutamic pyruvic transaminase/alanine aminotransferase (GPT/ALT), Gamma glutamyl transferase (GT), and albumin were not significantly different. The range of biochemical parameters that were significantly different between seropositive and seronegative elephants had narrow ranges. Thus, the potential of these parameters as a direct biomarker for TB diagnosis is limited based on the findings in this study. We recommend including blood parameters in future TB surveillance studies.

This report describes the genome sequences of two Mycobacterium tuberculosis isolates, S1 and S3, recovered from Asian elephants in Nepal. These genome sequences will enhance our understanding of the genomic epidemiology of Mycobacterium tuberculosis in Asian elephants.

Endocytosis mediates the internalization and ingestion of a variety of endogenous or exogenous substances, including virus particles, under the control of intracellular signaling pathways. We have previously reported that the complex formed between the small GTPase Ras and phosphoinositide 3-kinase (PI3K) translocates from the plasma membrane to endosomes, signaling from which thereby regulates clathrin-independent endocytosis, endosome maturation, influenza virus internalization, and infection. However, the molecular mechanism by which the Ras-PI3K complex is recruited to endosomes remains unclear. Here, we have identified the amino acid sequence responsible for endosomal localization of the Ras-PI3K complex. PI3K lacking this sequence failed to translocate to endosomes, and expression of the peptide comprising this PI3K-derived sequence inhibited clathrin-independent endocytosis, influenza virus internalization, and infection. Moreover, treatment of cells with this peptide in an arginine-rich, cell-penetrating form successfully suppressed influenza virus infection in vitro and ex vivo, making this peptide a potential therapeutic agent against influenza virus infection.Key words: signal transduction, endocytosis, endosome, imaging, influenza virus.

In 2015, an estimated 17-year-old female Bornean elephant (Elephas maximus borneensis) at Fukuyama Zoo in Japan exhibited anorexia and significant weight loss. Pan-susceptible Mycobacterium tuberculosis complex (MTBC) was isolated from vaginal discharge, oral mucus, urine, and fecal samples by culture. The isolate was identified as Mycobacterium caprae by genetic analysis. Isoniazid, pyrazinamide, and levofloxacin were administered rectally. Body weight increased to normal, but subsequently decreased again. Elevation of liver enzymes occurred, likely related to the increase in isoniazid dosage. After recovery from side effects, the elephant's weight increased further. However, isoniazid-resistant M. caprae was isolated from oral mucus after anti-tuberculosis drug treatment for 9 mo. The regimen was changed to rifampicin, pyrazinamide, ethambutol, and levofloxacin, administered orally or rectally. The 18-mo treatment was completed in October 2018. This elephant has shown no clinical sign since. No MTBC-positive sample had been obtained as of March 2020.

The discovery of fluorescent proteins (FPs) has revolutionized cell biology. The fusion of targeting sequences to FPs enables the investigation of cellular organelles and their dynamics; however, occasionally, such fluorescent fusion proteins (FFPs) exhibit behavior different from that of the native proteins. Here, we constructed a color pallet comprising different organelle markers and found that FFPs targeted to the mitochondria were mislocalized when fused to certain types of FPs. Such FPs included several variants of Aequorea victoria green FP (aqGFP) and a monomeric variant of the red FP. Because the FFPs that are mislocalized include FPs with faster maturing or folding mutations, the increase in the maturation rate is likely to prevent their expected localization. Indeed, when we reintroduced amino acid substitutions so that the FP sequences were equivalent to that of wild-type aqGFP, FFP localization to the mitochondria was significantly enhanced. Moreover, similar amino acid substitutions improved the localization of mitochondria-targeted pHluorin, which is a pH-sensitive variant of GFP, and its capability to monitor pH changes in the mitochondrial matrix. Our findings demonstrate the importance of selecting FPs that maximize FFP function.

We developed an interferon-γ release assay (IGRA) specific for Asian elephants (Elephas maximus). Whole blood collected from forty captive Asian elephants was stimulated with three different mitogens i.e., phytohemagglutinin (PHA), pokweed mitogen (PWM) and phorbol myristate aceteate/ionomycin (PMA/I). A sandwich ELISA that was able to recognize the recombinant elephant interferon-γ (rEIFN-γ) as well as native interferon-γ from the Asian elephants was performed using anti-elephant IFN-γ rabbit polyclonal antibodies as capture antibodies and biotinylated anti-elephant IFN-γ rabbit polyclonal antibodies as detection antibodies. PMA/I was the best mitogen to use as a positive control for an Asian elephant IGRA. The development of an Asian elephant-specific IGRA that detects native IFN-γ in elephant whole blood provides promising results for its application as a potential diagnostic tool for diseases, such as tuberculosis (TB) in Asian elephants.

We compared cortisol and thyroid hormone (T3 and T4) concentrations between tuberculosis (TB)-suspected (n=10) and healthy (n=10) elephants of Nepal. Whole blood was collected from captive elephants throughout Nepal, and TB testing was performed using the ElephantTB STAT-PAK® and DPP VetTB® serological assays that detect antibodies against Mycobacterium tuberculosis and M. bovis in elephant serum. Cortisol, T3 and T4 were quantified by competitive enzyme immunoassays, and the results showed no significant differences in hormone concentrations between TB-suspect and healthy elephants. These preliminary data suggest neither adrenal nor thyroid function is altered by TB disease status. However, more elephants, including those positively diagnosed for TB by trunk wash cultures, need to be evaluated over time to confirm results.

Tuberculosis (TB) is a devastating disease in elephants caused by either Mycobacterium tuberculosis or M. bovis. It is an ancient disease, and TB in elephants was first reported over two millennia ago in Sri Lanka. Outbreaks of TB worldwide, in captive and free-ranging elephant populations, have been recorded. Interspecies transmission of TB among elephants and humans has been confirmed in several geographic localities using spoligotyping, MIRU-VNTR analysis, and/or comparative genomics. Active surveillance of TB in wild and captive elephants and their handlers is necessary to prevent TB transmission at the elephant-human interface and to aid in the conservation of Asian and African elephants. In this review, we present an overview of diagnosis, reports of TB outbreaks in the past 25 years, TB in wild elephants, its transmission, and possible prevention and control strategies that can be applied at the elephant-human interface.

Influenza A virus (IAV) infection is initiated by the attachment of the viral glycoprotein hemagglutinin (HA) to sialic acid on the host cell surface. However, the sialic acid-containing receptor crucial for IAV infection has remained unidentified. Here, we show that HA binds to the voltage-dependent Ca

A comprehensive elephant tuberculosis (TB) survey using culture and four serological screening tests was conducted in Nepal in response to concern raised by wildlife officials that TB could threaten wild populations of elephants, rhinos, and other susceptible species. Captive elephants come into close contact with wild animals during conservation and tourism activities inside Nepal’s national parks. Private and government-owned male and female captive Asian elephants (Elephas maximus) were included in the study. The mean reported age was 38 years (range 5–60 years). A total of 289 samples from 120 elephants were collected for mycobacterial culture. Culture samples were processed at the National Tuberculosis Centre (NTC) in Nepal and the National Veterinary Services Laboratories (NVSL) in Ames, IA. Acid-fast organisms were observed in 11 and 21 samples processed at NTC and NVSL, respectively, and nontuberculous mycobacteria (NTMs) were isolated from six elephants. There were no isolations of Mycobacterium tuberculosis or Mycobacterium bovis. Blood samples were also collected from 115 of the elephants for serological testing using the Chembio ElephantTB STAT-PAK®, the Chembio MultiAntigen Print Immunoassay test, a multi-antigen ELISA, and an immunoblot assay. Culture and serological results were variable and required careful interpretation to develop criteria to assess TB risk. Elephants were assigned to one of four disease risk groups (high, moderate, low, and undetermined), and management recommendations for each group were made to government authorities. Serological results were prioritized in developing recommendations because of culture limitations and inconclusive culture results. This strategy was based on evidence for the early predictive value of serological tests and the urgent need expressed by wildlife authorities in Nepal to protect their captive elephants, mitigate TB at the captive-wild interface, and safeguard tourism.

To the Editor: Mycobacterium orygis, previously described as oryx bacilli, has recently been categorized as a member of M. tuberculosis complex and has been reported to cause tuberculosis (TB) in a variety of animals and in humans. Most reported isolates were of South Asian origin (1). In a previous study (2), we isolated and molecularly characterized M. orygis isolates from wild animals living in a captive facility in Kathmandu, Nepal. The greater one-horned rhinoceros (Rhinoceros unicornis), or Indian rhinoceros, is the largest species of rhinoceros. It is listed in Appendix I of the Convention on International Trade in Endangered Species (https://cites.org/eng/app/appendices.php), designated as vulnerable by the International Union for Conservation of Nature Red List (http://www.iucnredlist.org/search), and designated as a protected species by the Government of Nepal (3). Because of successful conservation efforts, the current wild population of greater one-horned rhinoceros in Nepal and India has increased from 600 in 1975 to 3,555 in mid-2015 (4). As of 2015, the population of these rhinoceros in Nepal was 645, including 605 animals living in Chitwan National Park (CNP) (5). On February 16, 2015, CNP officials observed a sick female rhinoceros in the buffer zone of the western sector of the park near Amaltari. The rhinoceros was dull, depressed, and not feeding. The following day, the animal was found dead in the same area (Technical Appendix Figure 1). Superficial maggot-infested wounds were on both sides of the vulva, indicating that the rhinoceros was not able to naturally remove the maggots and suggesting that the animal was sick for some time. During the necropsy, several granulomatous lesions were observed in the lungs and considered to be compatible with TB infection. The lesions were extensively distributed and well encapsulated and contained caseous necrotic material (Technical Appendix Figure 2). No other pathologic changes were observed in any of the organs examined, leading to the conclusion that the rhinoceros died from TB. A lung tissue sample positive for TB by acid-fast staining was cultured on Lowenstein-Jensen media. We performed spoligotyping and mycobacterial interspersed repetitive units–variable-number tandem-repeat (MIRU-VNTR) procedure on the isolate as previously described (6,7). Spoligotyping analysis, performed as previously described (2), showed that the isolate had a spoligo–international type 587 pattern, indicating it was M. orygis. We also performed multilocus sequence typing on various genes (2), and confirmed that the isolate was M. orygis. We then constructed a dendrogram by comparing the MIRU-VNTR result from rhinoceros isolate with published M. orygis MIRU-VNTR types (Figure) (1,2,8). The rhinoceros M. orygis isolate fell in a unique position in the dendrogram; we identified a difference in only 1 locus (MIRU 424) when we compared the isolate with the largest cluster of reported M. orygis isolates, including those previously reported from Nepal. Figure Phylogeny of Mycobacterium orygis isolates as determine on the basis of mycobacterial interspersed repetitive units–variable-number tandem-repeat (MIRU-VNTR) results of 22 loci. The unweighted pair group method with arithmetic mean dendrogram ... In our earlier study (2), we isolated M. orygis from chital deer (Axis axis) and blue bull (Boselaphus tragocamelus) from a captive wild-animal facility and postulated that the origin of the infection might be from infected animals in CNP, where the deer and blue bull originated. This new finding of a different strain type of M. orygis in a free-ranging rhinoceros in CNP provides evidence for our hypothesis. Other reports of M. orygis in captive wild animals in Nepal (2), cattle and a rhesus monkey in Bangladesh (1), humans in South Asia (1), and an immigrant from India in New Zealand (9) further support this bacterium’s potential widespread distribution in South Asia and attests to the One Health significance of this organism. In a demographic study of rhinoceros in Nepal (10), the animals were found to be living in a narrow area of riverine grassland in CNP. A chronic and devastating disease like TB in this vulnerable and isolated population, which is already threatened from habitat destruction and poaching, is a matter of great conservation concern for the animal’s long-term survivability. Also, CNP is listed by the United Nations Educational, Scientific and Cultural Organization as a World Heritage site because of its rich biodiversity and as an important habitat for endangered animals, including Bengal tigers (Panthera tigris) and Asian elephants (Elephas maximus). Thus, M. orygis–associated TB in rhinoceros in CNP may also indicate a threat to other animals, including some that are endangered. There is a strong possibility of unknown maintenance hosts of M. orygis in and around the national park. Our findings support the need for further investigation to understand the ecology and epidemiology of M. orygis and provide justification for active surveillance of this bacterium in animals in the national park and in livestock and humans in the buffer-zone areas. Furthermore, the increasing evidence for widespread distribution of M. orygis in South Asia provides a new picture of TB and may lead to a new understanding of M. tuberculosis complex. Technical Appendix: Location in Nepal where Mycobacterium orygis–infected rhinoceros was found dead and image of granulomatous tuberculosis lesion. Click here to view.(559K, pdf)

Tuberculosis (TB) is an emerging disease in elephants primarily caused by Mycobacterium tuberculosis (M. tb) and in some occassion by M. bovis. We performed culture and three serological teststhe Elephant TB STAT-PAK,(R) DPP VetTB (R) Assay, and MAPIA (multi-antigen print immunoassay)prospectively on samples from eight elephants in Nepal that died of suspected or confirmed tuberculosis (TB) between 2007 and 2013. Among them, all elephants were reactive to DPP VetTB (R) Assay, five to Elephant TB STAT-PAK,(R) and two were reactive to MAPIA. Similarly, six elephants were positive on culture on samples collected antemortem or postmortem. We observed antibody responses months to years before culture confirmation of TB which shows that serological tests can be highly useful for the early diagnosis of TB in elephants. Validated point-of-care serological tests are easily performed in the field and hold promise for improved TB surveillance in other non-domestic species.

We developed an interferon-γ release assay (IGRA) specific for Asian elephants (Elephas maximus). Whole blood collected from forty captive Asian elephants was stimulated with three different mitogens i.e., phytohemagglutinin (PHA), pokweed mitogen (PWM) and phorbol myristate aceteate/ionomycin (PMA/I). A sandwich ELISA that was able to recognize the recombinant elephant interferon-γ (rEIFN-γ) as well as native interferon-γ from the Asian elephants was performed using anti-elephant IFN-γ rabbit polyclonal antibodies as capture antibodies and biotinylated anti-elephant IFN-γ rabbit polyclonal antibodies as detection antibodies. PMA/I was the best mitogen to use as a positive control for an Asian elephant IGRA. The development of an Asian elephant-specific IGRA that detects native IFN-γ in elephant whole blood provides promising results for its application as a potential diagnostic tool for diseases, such as tuberculosis (TB) in Asian elephants.

Born and raised in western Nepal, Sarad Paudel saw his first elephant at Chitwan National Park, southern Nepal, and resolved to build his career around them.

Influenza A virus (IAV) infection is initiated by the attachment of the viral glycoprotein hemagglutinin (HA) to sialic acid on the host cell surface. However, the sialic acid-containing receptor crucial for IAV infection has remained unidentified. Here, we show that HA binds to the voltage-dependent Ca2+ channel Cav1.2 to trigger intracellular Ca2+ oscillations and subsequent IAV entry and replication. IAV entry was inhibited by Ca2+ channel blockers (CCBs) or by knockdown of Cav1.2. The CCB diltiazem also inhibited virus replication in vivo. Reintroduction of wild-type but not the glycosylation-deficient mutants of Cav1.2 restored Ca2+ oscillations and virus infection in Cav1.2-depleted cells, demonstrating the significance of Cav1.2 sialylation. Taken together, we identify Cav1.2 as a sialylated host cell surface receptor that binds HA and is critical for IAV entry.

Mycobacterium tuberculosis was cultured from the lung tissues of 3 captive elephants in Nepal that died with extensive lung lesions. Spoligotyping, TbD1 detection and multi-locus variable number of tandem repeat analysis (MLVA) results suggested 3 isolates belonged to a specific lineage of Indo-Oceanic clade, EAI5 SIT 138. One of the elephant isolates had a new synonymous single nucleotide polymorphism (SNP) T231C in the gyrA sequence, and the same SNP was also found in human isolates in Nepal. MLVA results and transfer history of the elephants suggested that 2 of them might be infected with M. tuberculosis from the same source. These findings indicated the source of M. tuberculosis infection of those elephants were local residents, presumably their handlers. Further investigation including detailed genotyping of elephant and human isolates is needed to clarify the infection route and eventually prevent the transmission of tuberculosis to susceptible hosts.