Epidemiology and molecular characterization of Mycobacterium tuberculosis including a drug-resistant strain associated with mortality of Asian elephants in Nepal 2019-2022.

Tuberculosis (TB) is an emerging threat to the survival of elephants in Nepal. We investigated the lung tissue samples from nine elephants that died from 2019 to 2022 in Nepal using culture, conventional PCR, and loop-mediated isothermal amplification (LAMP) and then performed genotyping of five PCR-positive isolates to understand the possible transmission dynamics of Mycobacterium tuberculosis (Mtb). Results showed that two-thirds (6/9) of elephants were confirmed to be infected from Mtb by LAMP, 5/9 by PCR, and 4/9 by culture. Genotyping of Mtb isolates showed that elephants were infected with the Indo-Oceanic and Beijing lineages including an isoniazid-resistant Beijing lineage. MIRU-VNTR-based phylogeny, gyrA, and katG sequencing showed the possibility of ongoing transmission of Indo-Oceanic lineages and likely transmission of the drug-resistant Beijing lineage from human to elephant. Implementation of comprehensive surveillance and preventive measures are urgently needed to address this zoonotic disease and protect elephants from TB in Nepal.

Tuberculosis seroprevalence and comparison of hematology and biochemistry parameters between seropositive and seronegative captive Asian elephants of Nepal.

We conducted a tuberculosis (TB) 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 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.

Severe fever and thrombocytopenia syndrome virus infection: Considerations for vaccine evaluation of a rare disease.

Infection caused by the severe fever and thrombocytopenia syndrome virus (SFTSV) causes a hemorrhagic illness with a mortality between 20% and 40%. Initially recognized in 2009 in China, cases have additionally been documented in Japan and Korea although retrospective studies have documented seroprevalence since 1996. Although case rates have increased due to increased awareness and more widely available diagnostics, SFTSV infection remains rare with the highest rates documented in Korea for Jeju Province (3.5 cases per 100,000 population) and the Inje-gun region (66.2 cases per 100,000). Because of the very low incidence of infection, a placebo-controlled study with 1:1 randomization to evaluate an SFTSV vaccine would require a sample size that is 25% greater than the region of study. We discuss alternatives to licensure. Vaccine effectiveness may be assessed through a registry, comparing rates of infection over time between vaccine recipients versus regional populations. Modeled data can be updated based on actual case rates and population changes over the years of follow-up. Using one model, statistically significant differences are seen after 10 years in Inje-gun and 15 years of follow-up in Jeju. This approach may be applicable to other uncommon infectious diseases for which a standard study design is difficult.

Mixed Mycobacterium tuberculosis Lineage Infection in 2 Elephants, Nepal.

Tuberculosis in elephants is primarily caused by Mycobacterium tuberculosis. We identified mixed M. tuberculosis lineage infection in 2 captive elephants in Nepal by using spoligotyping and large sequence polymorphism. One elephant was infected with Indo-Oceanic and East African-Indian (CAS-Delhi) lineages; the other was infected with Indo-Oceanic and East Asian (Beijing) lineages.

Mycobacterium tuberculosis in Wild Asian Elephants, Southern India.

We tested 3 ild Asian elephants (Elephas maximus) in southern India and confirmed infection in 3 animals with Mycobacterium tuberculosis, an obligate human pathogen, by PCR and genetic sequencing. Our results indicate that tuberculosis may be spilling over from humans (reverse zoonosis) and emerging in wild elephants.

Comparison of cortisol and thyroid hormones between tuberculosis-suspect and healthy elephants of Nepal.

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.

Development and evaluation of an interferon-γ release assay in Asian elephants (Elephas maximus).

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.

Molecular characterization of Mycobacterium tuberculosis isolates from elephants of Nepal.

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.

Tuberculosis at the human-animal interface: an emerging disease of elephants.

Over the past 15 years, cases of infection with organisms of the Mycobacterium tuberculosis complex have been diagnosed among captive elephants in the United States and worldwide. Outbreak investigations have documented that among staff employed at facilities housing infected animals, skin test conversion to purified protein derivative have been documented. Clonal spread among animals in close contact and even inter-species spread between elephant and human has been documented. Detection of actively infected animals relies on samples obtained by trunk wash. Diagnosis has been augmented by the development of a multi-antigen serologic assay with excellent specificity and sensitivity. Treatment regimens are still in development with efficacy largely unknown due to a paucity of both premortem follow-up and necropsy data of treated animals. The epidemiology, diagnosis and treatment of tuberculosis in elephants require additional careful study of clinical data.

Comparison of systemic cytokine levels in Mycobacterium spp. seropositive and seronegative Asian elephants (Elephas maximus).

Mycobacterium spp. infection is an important health concern for Asian elephant (Elephas maximus) populations worldwide. The disease is of particular concern considering its potential to affect not only the individual animal but also herd and public health. Although elephant tuberculosis susceptibility is poorly understood, immune function alterations are central to disease pathogenesis in other species and probably affect outcome of mycobacterial infections in elephants. Measurement of immune mediator (cytokine) levels within blood samples can provide information regarding immune function that may elucidate disease susceptibility. For this study, mRNA levels of interleukin (IL)-2, IL-4, IL-10, and IL-12; interferon (IFN)-gamma; tumor necrosis factor (TNF)-alpha; and transforming growth factor (TGF)-beta were measured using elephant-specific, real-time reverse transcription-polymerase chain reaction (RT-PCR) assays in RNA-preserved whole blood samples from 106 Asian elephants, 15% of which were Mycobacterium tuberculosis complex seropositive. The Elephant TB STAT-PAK (Chembio Diagnostics, Inc., Medford, New York 11763, USA), a novel lateral flow antibody detection assay developed for specific use in elephants, was used to determine serologic status for the study. Seropositive animals had higher levels of TNF-alpha and lower levels of TGF-beta than seronegative animals; these differences between groups were statistically significant when levels were analyzed as categorical variables. Trends toward higher levels of IFN-gamma and IL-4 and slightly lower levels of IL-10 and IL-12 were noted in the seropositive group, although differences between groups were not statistically significant. Presence of other inflammatory conditions was found to be a significant confounding variable in the analysis of the relationship between tuberculosis status and TNF-alpha levels, necessitating its inclusion in statistical models. Age and sex were not found to significantly affect the relationship between tuberculosis status and any of the cytokines measured. Interleukin-2 levels were below the sensitivity of the real-time RT-PCR assay irrespective of tuberculosis status. These findings provide a foundation for future research into the immunopathogenesis of elephant tuberculosis.

Development and validation of cytokine quantitative, real time RT-PCR assays for characterization of Asian elephant immune responses.

Infectious disease is an important factor in Asian elephant health and long-term species survival. In studying disease pathogenesis, it is important to consider not only the pathogen, but also the effectiveness of the host immune response. Currently, there is a paucity of information available on elephant immune function. Measurement of cytokine levels within clinical samples can provide valuable information regarding immune function during health and disease that may elucidate disease susceptibility. To develop tools for assessment of elephant immune function, Asian elephant partial mRNA sequences for interleukin (IL)-2, IL-4, IL-10, IL-12, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and beta-actin were determined. Sequence information was then utilized to design elephant-specific primers and probes for quantitative, real time, RT-PCR assays for the measurement of cytokine mRNA. Greater than 300bps of Asian elephant mRNA sequence were determined for each cytokine of interest. Consistent and reproducible, real time, RT-PCR assays with efficiencies of greater than 93% were also developed. Assay sensitivities ranged from less than 1 to 5000 DNA copies with the exception of IL-12, which had a sensitivity of 42,200 copies. Employment of molecular techniques utilizing mRNA-based detection systems, such as real time, RT-PCR, facilitate sensitive and specific cytokine detection and measurement in samples from species for which commercial reagents are not available. Future studies utilizing these techniques to compare elephant immune function during health and in the face of infection will be useful for characterizing the contribution of the elephant immune system to disease.

Population pharmacokinetics of antituberculous drugs and treatment of Mycobacterium bovis infection in bongo antelope (Tragelaphus eurycerus isaaci).

After clinical illness, treatment, and death of a captive male bongo antelope (Tragelaphus eurycerus isaaci) caused by tuberculosis involving Mycobacterium bovis, four tuberculin test reactive captive bongos were treated for 6 mo with isoniazid (INH) and rifampin (RIF) and intermittent single doses of other medications before being euthanized. In all cases, postmortem examination indicated no evidence of active disease and cultures of multiple organs were negative. We present detailed pharmacokinetic (PK) data for amikacin (AMK), ethambutol (EMB), INH, pyrazinamide (PZA), RIF, and levofloxacin in four female bongos. Adequate absorption and serum levels were obtained after parenteral administration of AMK, EMB, and INH and after oral administration of INH and PZA. Parenterally administered drugs were well described by a one-compartment PK model with first-order absorption and elimination processes. Treatment with INH and RIF over a 6-mo period did not result in demonstrable adverse effects. Starting doses of 10-15 mg/kg, i.m., or 30 mg/kg, p.o., of INH, 50 mg/kg, p.o., of EMB, and 25 mg/kg, i.m., s.i.d., of AMK are recommended. The treatment is continued with at least two drugs to which the organism is susceptible for a total treatment length of 6-12 mo. Treatment may be an option to eradicate M. bovis from suspect animals, with carefully administered and monitored drug treatment.