Title: Evaluation and validation of pathogen-specific biomarkers for the diagnosis of

tuberculosis in Asian elephants (Elephas maximus)

Submitted by
Sarad Paudel PhD
Research Associate
Department of Pathobiology & Diagnostic Investigation
College of Veterinary Medicine
Michigan State University
East Lansing, Michigan, USA
Email: paudelsa@msu.edu

Submitted to
Department of National Parks and Wildlife Conservation
Ministry of Forests and Environment,
Government of Nepal
Babarmahal, Kathmandu, Nepal

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INTRODUCTION
Asian elephants are classified as an endangered species (IUCN, 2008) with only about
45,000 remaining across the globe (Fernando and Pastorini, 2011). The survival of
elephants is threatened by habitat loss and fragmentation, conflict with humans,
poaching and more recently with the outbreak of infectious disease like tuberculosis
and elephant endothelio-herpesvirus (EEHV) infection.

Tuberculosis (TB) in elephants is primarily caused by Mycobacterium tuberculosis

(M.tb); however, infection by M. bovis has also been reported in some cases (Mikota,
2008). Culture of trunk wash is considered gold standard for the diagnosis of TB in
elephants; however, this technique has severe limitation (Lyaschenko et al., 2012).
Elephants use their trunks for varieties of purposes and growth of Mycobacteria other
than MTC may occur which will compromise the results. Elephants are known to be
intermittent shedders (Mikota et al., 2015). Elephants should be well trained for this
technique. Trunk wash samples should be collected at least for three alternative days
within a week that is also a time consuming. On the other hand, it takes long time to
get the culture results, its costly and requires a BSL 3 laboratory, which may not be
available in the elephant range countries. Tuberculin test, widely used in cattle, had
demonstrated poor sensitivity and specificity. Radiography is not possible in
pachyderms like elephants (Mikota et al., 2001).

ElephantTB STAT-PAK ® (STAT-PAK) and DPP VetTB ® assays (DPP) (Chembio

Diagnostic Systems, Inc., NY, USA) have been used for sero-surveillance. However,
the production of STAT-PAK was discontinued some years ago and only DPP is
available for TB screening in elephants currently. These assays detect antibodies
against putative immune-dominant TB antigens in serum/plasma samples of elephants
(Lyashchenko et al., 2012). The animals start producing antibodies in later stage of
the TB infection during adaptive immunity. Although few studies have developed and
evaluated interferon gamma release assays in elephants (Angkawanish et al., 2013;
Paudel et al., 2016), however, these assays are yet to be validated in larger
populations of TB confirmed and healthy elephants.

M.tb was also isolated from 5 government-owned captive elephants in Nepal that died
from 2009 to 2013 with suspected TB lesions in the lungs (Paudel et al., 2014; Paudel
et al., 2019). The genotyping of 5 TB isolates from Asian elephants of Nepal using

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spoligtotyping, MLVA and LSP revealed that all the isolates belonged to Indo-
oceanic lineage. Interestingly, 2 isolates had mixed M.tb lineages infection. One
elephant was infected with East African-Indian (CAS-Delhi) and the other was
infected with East Asian (Beijing) lineages in addition to the infection with Indo-
Oceanic lineage in both elephants (Paudel et al., 2014; Paudel et al., 2019). Indo-
oceanic lineage is present in 11.5% of TB infected human patients in Nepal (Malla et
al., 2012). This lineage is likely common among the TB patients dwelling near the
elephant facilities in Nepal. It can be assumed that Indo-oceanic lineage might be one
of the lineages that is well adapted among the elephants in Nepal.

TB is a chronic disease in mammals. During the infection with TB bacteria,

Mycobacterium-specific proteins, peptides, DNA and lipids are released into
circulation (Pollock et al., 2013). They can be used as the biomarkers for the early
detection of TB in elephants before the elephants develop the active TB in later stage.
The cost-effective control measures can be implemented so that this devastating
disease is not transmitted to other susceptible hosts including humans, other elephants
and endangered species.

Our laboratory here at Michigan State University has been actively involved in animal
TB research and we have identified 16 Mycobacterium tuberculosis complex (MTC)
specific proteins including MB2515c (transcriptional regulator [LuxR family]),
MB1895c (cell wall biosynthesis), and MB 1554c or pks5 (Lament et al., 2014). So,
we will use these pathogen-specific biomarkers for the diagnosis of TB in Asian
elephants (Elephas maximus) from Nepal.

MATERIALS AND METHODS

Serum samples
The applicant had collected the elephant TB samples from 117 captive Asian
elephants as a first comprehensive elephant TB surveillance in Nepal in 2005/2006.
CITES Export Permit was issued by DNPWC on February 10, 2006 to export serum
samples from these elephants for the diagnostic research to USA. The applicant will
utilize the serum samples that were imported to USA in 2006 for the proposed
biomarker studies. These serum samples are at one of the diagnostic laboratory here in
USA and the applicant will collaborate with this lab for the proposed study. Similarly,

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blood samples from 6 unexposed TB negative elephants will be collected from
zoological garden here in USA as a control.

TB testing
Serum samples (N=117) collected in Nepal during 2005/2006 were subjected to
STAT-PAK. The samples positive on this assay will be considered as TB reactive.
The samples negative on this assay will be regarded as TB non-reactive.

Biomarkers
An indirect ELISA using monoclonal antibodies synthesized against MB2515c,
MB1895c and MB 1554c or pks5 peptides to detect these biomarkers in elephant
serum will be used. These monoclonal antibodies have already been validated in
samples obtained from cattle and white tailed deer in USA (Wanzala et al., 2017).
Indirect ELISA will be performed as described (Wanzala et al., 2017).

Data analysis
The OD data obtained from indirect ELISA will be uploaded into a spreadsheet and
S/N values will be calculated for each biomarker. S/N ratio is defined as the ratio of
OD for TB positive elephants to mean OD of all unexposed TB negative deer. These
ratios will be uploaded to a excel sheet and Box-and-whisker plots will be generated
for each biomarker. As Nepal is a country with high prevalence of TB in human
population and the elephants in Nepal are diagnosed with M. tuberculosis (Paudel et
al., 2019), So, low S/N cutoff values will be applied for maximum accurate detection
of TB in elephants of Nepal and eventually identify all infected elephants.

EXPECTED OUTPUTS
This study will determine the presence or absence of three MTC biomarkers in the
STAT-PAK reactive and non-reactive elephants from Nepal. This study will also
validate these MTC biomarkers in Asian elephants. Our findings from proposed study
will definitely be helpful in the early diagnosis of TB in Asian elephants and will
eventually help in the conservation of this endangered species in the in the range
countries as well as in the zoological setting across the globe.

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