Summary:
Bovine tuberculosis (bTB), caused by Mycobacterium bovis(M. bovis ) infection, is a multi-host disease which negatively
affects the wildlife industry, with adverse consequences for
conservation, ecotourism, and game/wildlife sales. Although interspecies
transmission has been reported between some wildlife hosts, the risk of
spread in complex ecosystems is largely unknown. As a controlled
disease, tools for accurate detection of M. bovis infection is
crucial for effective surveillance and management, especially in
wildlife populations. There are however, limited species-specific
diagnostic tests available for wildlife. Hippopotamuses are rarely
tested for M. bovis infection and infection has not previously
been confirmed in these species. In this study, blood and tissue samples
collected from common hippopotamus (Hippopotamus amphibius )
residing in a bTB endemic area, the Greater Kruger Protected area
(GKPA), were retrospectively screened to determine whether there was
evidence for interspecies transmission of M. bovis, and to
identify tools for M. bovis detection in this species. Using the
multi-species DPP® VetTB serological assay, a bTB
seroprevelance of 8% was found in hippopotamus from GKPA. In addition,
the first confirmed case of M. bovis infection in a free-ranging
common hippopotamus is reported, based on the isolation in mycobacterial
culture, genetic speciation, and detection of DNA in tissue samples.
Importantly, the M. bovis spoligotype (SB0121) isolated from this
common hippopotamus is shared with other M. bovis- infected hosts
in GKPA, suggesting interspecies transmission. These results support the
hypothesis that M. bovis infection may be under recognized in
hippopotamus. Further investigation is needed to determine the risk of
interspecies transmission of M. bovis to common hippopotamus in
bTB endemic ecosystems and evaluate serological and other diagnostic
tools in this species.
Keywords: Common hippopotamus, DPP® Vet TB
Assay, GeneXpert® MTB/RIF Ultra qPCR Assay, Greater
Kruger Protected Area, interspecies transmission, Mycobacterium
bovis
INTRODUCTION
Bovine tuberculosis (bTB), caused by Mycobacterium bovis(M. bovis ) infection, is a multi-host disease which negatively
affects the wildlife industry, with adverse consequences for
conservation, ecotourism, and game/wildlife sales (Renwick, White, &
Bengis, 2007). Importantly, the presence of wildlife maintenance hosts
further complicates the control of M. bovis infections and
increases the risk of interspecies transmission, especially in
multi-host systems (Brites et al ., 2018). In South Africa, this
is demonstrated by ongoing outbreaks of bTB caused by spread of M.
bovis from African buffaloes (Syncerus caffer ) to livestock and
other wildlife species (Hlokwe, van Helden, & Michel, 2014).
Interspecies transmission of M. bovis is recognized as a risk for
spreading disease within and between populations of susceptible hosts,
therefore, accurate tools for detection in multiple species are needed
for effective management of bTB.
Although M. bovis is a controlled disease, surveillance and
development of diagnostic tools are often limited to livestock, with a
paucity of species-specific diagnostic tests available for wildlife. For
this reason, several direct detection methods are used for surveillance
purposes including mycobacterial culture and Xpert®MTB/RIF Ultra qPCR Assay (GXU), as well as multi-species serological
assays, such as the Chembio Dual Path Platform (DPP®)
Vet TB Assay (Hlokwe & Mogano, 2020; Kerr et al ., 2020; Miller,
Buss, Roos, et al ., 2019). These tests may prove useful for
screening susceptible hosts from bTB endemic areas, such as the Greater
Kruger Protected Area (GKPA), South Africa, to investigate interspecies
transmission and risk of disease.
Common hippopotamuses (Hippopotamus amphibius ) have a wide
distribution in sub-Saharan Africa but are considered vulnerable by the
International Union for the Conservation of Nature (IUCN) (Lewison &
Pluháček, 2017). As a result of the difficulty and danger associated
with handling this species, few studies have been undertaken to evaluate
disease status, especially in free-ranging populations. For example, it
is unknown whether M. bovis infection can be transmitted to
hippopotamuses in the bTB-endemic GKPA (over 2 million hectares in
size), which has an estimated 3100 hippopotamuses (Lewison & Pluháček,
2017).
M. bovis infection has not previously been confirmed in any
hippopotamus species, and these animals are rarely tested for bTB
(Bouts, Vordermeier, Flach, & Routh, 2009; Mann, Bush, Janssen, Frank,
& Montali, 1981; Miller & Lyashchenko, 2015). However, in order to
assess the infection risk prior to translocating animals from bTB
endemic areas, it is necessary to develop tools for surveillance and
diagnosis. Therefore, the aim of this project was to perform
retrospective screening of blood (serum) and tissue samples (lung and
lymph node) collected from common hippopotamuses in GKPA as part of a
bTB surveillance project and determine the potential risk of
interspecies transmission. These findings will inform management
decisions and policy development for bTB in
wildlife.
- MATERIALS AND METHODS
- Animals and samples:
Between 2016 and 2019, opportunistic samples were collected from 60
randomly culled free-ranging common hippopotamuses in the Kruger
National Park, South Africa and adjacent private reserves included
within the GKPA (> 2 million hectares). These culls were
part of a species management plan undertaken during periods of extreme
drought. The sex (male = 29; female = 30; not recorded = 1) and age
category of each hippopotamus were recorded at the time of sampling.
Individuals were categorized as adult (n= 48); young/sub-adult (n=7); or
juvenile (n=4), based on body size. The age of one individual was not
recorded. Serum (n = 49 animals) and post-mortem tracheobronchial lymph
node and lung samples (n = 50 animals) were collected by state
veterinary personnel and stored at -20°C prior to testing. Not all
sample types were available from every individual. Ethical clearance to
retrospectively screen these samples was obtained from the Stellenbosch
University Research Ethics Committee (Protocol #: SU-ACU-9086;
SU-ACU-10933), and Section 20 approval was granted by the South African
Department of Agriculture, Forestry, and Fisheries (DAFF Section 20:
12/11/1/7/2). All testing was performed as part of this research project
and not in official diagnostic laboratories for controlled animal
diseases.
Dual Path Platform
(DPP®) Vet TB Assay
All sera were screened for antibodies to mycobacterial antigens using a
multi-species rapid serological test, the DPP® Vet TB
Assay (Chembio Diagnostic Systems, Inc., Medford, NY, USA), according to
the manufacturer’s instructions. Antibody binding was measured using a
DPP® optical reader (Chembio) and reflectance reported
in relative light units (RLU). Reactivity of serum to either test line 1
(antigen MBP83) and/or test line 2 (antigen CFP-10/ESAT-6) above the
visual cut-off value (RLU ≥ 5) was considered test-positive (Greenwaldet al ., 2009). Test results were considered valid only if the
control line could be visualized.
- Mycobacterial culture
- Tissue homogenisation
Tissue homogenisation and mycobacterial culture was performed under
Biosafety Level 3 (BSL-3) conditions. Briefly, approximately 10 grams of
tissue (from either tracheobronchial lymph nodes or lung samples
depending on each individual hippopotamus sample availability) were
homogenized in 50 mL skirted falcon tubes (Becton Dickinson, Franklin
Lakes, NJ, USA) containing eight 4.8 mm metal beads and 4 mL sterile
BBL™ MycoPrep™ Phosphate Buffer (PO4 buffer) (Becton
Dickinson) using a Bullet Blender® 50 (Next Advance,
Averill Park, NY, USA) for 15 min at maximum speed. Following
homogenisation, remaining intact tissue was discarded and tissue
homogenates split in equal volumes for downstream testing with
mycobacterial culture and the GXU assay (Cepheid, Sunnyvale, CA, USA).
Culture and genomic speciation
Following homogenisation, 2 mL BD BBL™ MycoPrep™ (Becton Dickinson) was
added to 2 mL of each tissue homogenate sample and incubated for
15 minutes at room temperature. Thereafter, samples were neutralized
with 15 mL PO4 buffer. All samples were centrifuged for
20 min at 3000 rcf and the supernatant decanted. Each pellet was
resuspended in 1 mL PO4 buffer and 500 μl of this
suspension was transferred to a Mycobacteria Growth Indicator Tube
(MGIT™) containing 800 μl of BD BACTEC™ MGIT™ 960 Supplement and
incubated in a BACTEC™ MGIT™ 960 Mycobacterial Detection System for at
least 56 days (all Becton Dickinson). Cultured bacterial isolates were
genetically speciated by genomic regions of difference PCR (Warrenet al ., 2006), 16S rRNA sequencing (Leclerc, Haddad, Moreau, &
Thorel, 2000) and spoligotyping (Kamerbeek et al ., 1997). The
spoligotype of the isolate from this study was authenticated by
comparing the observed spoligotype pattern to known spoligotypes in theMycobacterium bovis Spoligotype Database
(https://www.mbovis.org/database.php; Smith & Upton, 2012).
Xpert® MTB/RIF Ultra Assay (GXU)
As a rapid ancillary method to mycobacterial culture, the GXU assay was
used to detect Mycobacterium tuberculosis complex (MTBC) DNA
within tissue homogenates (Chakravorty et al., 2017). Briefly 1.3 mL of
the Xpert® Sample Lysis Reagent (Cepheid) was added to
each 700 µl tissue homogenate aliquot under BSL-3 conditions. Samples
were vortexed for 10 seconds, incubated for 10 minutes at room
temperature, mixed again by vortexing for another 10 seconds, and
incubated for a final 5 minutes for optimal microbial DNA release and
bacilli inactivation. The total volume of 2 mL was transferred to a GXU
Cartridge (Cepheid) and analysed per manufacturer’s instruction
(Cepheid, Alternative Specimen Testing).
RESULTS
Of the 49 serum samples tested in this study, sera from four
hippopotamuses were positive using the DPP® Vet TB
Assay (Table 1), resulting in a bTB seroprevalence of 8% (95% CI 3 –
19%) in the tested population. The RLU values for the four
DPP® -positive hippopotamuses were between 0 and 14.3
RLU for test line 1 (MBP83) and between 0 and 18.1 RLU for test line 2
(CFP-10/ESAT-6), with only one individual (SU ID: 19/142) reactive to
both test lines (Table 1). Serum collected from the M. bovisculture-positive hippopotamus (SU ID: 19/169) had antibodies to
CFP-10/ESAT-6 only (Table 1).
Using the GXU assay, MTBC DNA was detected (LOW) in the tissue
homogenates from two hippopotamuses (SU IDs: 19/169 and 19/170), with
TRACE amounts in an additional three animals (Table 2). M. boviswas cultured from both the lung and thoracic lymph node samples
collected from a single hippopotamus (SU ID: 19/169; Table 2). Further
PCR-based speciation confirmed the strain present in both the lung and
thoracic lymph node samples was spoligotype SB0121 (Table 3). The raw
data that support the findings of this study are available from the
corresponding author upon reasonable request.
DISCUSSION:
The findings of this retrospective study confirm that common
hippopotamus can be infected with M. bovis. This was verified by
the isolation of bacilli from lung and thoracic lymph node tissue
collected from a single free-ranging adult male hippopotamus.
Significantly, the M. bovis strain (SB0121) isolated from the
hippopotamus in the GKPA is commonly found in this region and has been
previously isolated in several wildlife species including African
buffalo (Syncerus caffer ), Chacma baboon (Papio ursinus ),
greater kudu (Tragelaphus strepsiceros ), leopard (Panthera
pardus ) and African lion (Dippenaar et al ., 2017; Michelet al ., 2009). African buffaloes are considered a reservoir host
for bTB in this ecosystem and interspecies spillover to livestock and
other wildlife has been reported (Hlokwe et al ., 2014; Musoke,
Hlokwe, Marcotty, du Plessis, & Michel, 2015; Renwick etal ., 2007). Therefore, buffaloes are considered the most likely
source of infection. Since studies of wild boar populations have
demonstrated that M. bovis can be spread through environmental
contamination around waterholes to other species (Naranjo, Gortazar,
Vicente, & de la Fuente, 2008), it is speculated that the hippopotamus
was indirectly infected.
Although mycobacterial culture of post-mortem tissues and speciation of
bacterial isolates remain the gold standard method for the confirmation
of M. bovis infection in wildlife, this method is limited by the
ability to acquire tissue samples as well as suboptimal sensitivity of
culture (de la Rua-Domenech et al ., 2006). Therefore, other
methods are needed for accurate detection of infection. In the case of
the M. bovis culture-confirmed hippopotamus in this study,
mycobacterial specific antibodies were detected, suggesting that the
DPP® Vet TB assay may be useful for indirect detection
of MTBC infection in this species. The mycobacterial culture results
were also consistent with the positive GXU results, which were obtained
directly from tissue homogenates. Although culture confirmed infection
in only one hippopotamus, there were three additional animals with
seropositive results, suggesting that these animals may have been
previously infected. Overall, using mycobacterial culture, GXU, and the
serological test, there were 7 out of 60 hippopotamuses that had
evidence of M. bovis infection, suggesting prevalence could be
higher in GKPA than previously expected.
A positive antibody response to bTB specific antigens MPB83 and/or
CFP-10/ESAT-6 was observed in four hippopotamuses, including one
culture-positive individual screened using the DPP®Vet TB Assay, resulting in an estimated bTB seroprevalence of 8%. The
DPP® Vet TB Assay is not a host species-specific
assay, and several species including African and Asian elephants
(Loxodonta africana; Elephas maximus ), African lions
(Panthera leo ), cheetah (Acinonyx jubatus ) and common
warthogs (Phacochoerus africanus ) with culture-confirmed TB have
previously been shown to produce antibody responses to MPB83 and/or
CFP-10/ESAT-6 (Greenwald et al ., 2009; Kerr et al ., 2019,
2020; Lyashchenko et al ., 2006; Miller, Buss, Sylvester, et
al ., 2019; Miller et al ., 2016; Roos et al ., 2016). The
association between seropositivity and culture-confirmed MTBC infection
suggests that rapid antibody assays might be useful as an ante-mortem
screening test for M. bovis infection in hippopotamuses.
Furthermore, low concentrations of MTBC DNA were detected in the tissue
homogenates from two hippopotamuses using GXU. While the GXU assay
cannot distinguish between members of the MTBC, it is still highly
specific to MTBC, based on the simultaneous detection of MTBC-specific
multicopy genes IS6110 and IS1081 (Chakravorty et
al ., 2017). However, like most qPCR assays, it also cannot distinguish
between live or dead mycobacteria bacilli (Theron et al ., 2014).
However, as an ancillary method to culture, this DNA test is a strong
indicator that if live mycobacteria are present, it is at the same
quantity of DNA detected in the sample (Chakravorty et al .,
2017). Usually, a positive mycobacterial culture result can be
anticipated from a positive GXU sample, as previously observed in
post-mortem tissue samples collected from an M. bovis -infected
cheetah (Kerr et al ., 2020). Thus, a positive PCR test
result strongly supports the subsequent diagnosis of M. bovisinfection based on culture.
Limitations associated with this study include sample acquisition and
the nature of the samples themselves. Firstly, all samples used in this
study were acquired opportunistically through culling. Culling usually
targets specific age groups, sexes or family groups resulting in a
sampling approach biased towards certain demographics. In this study,
family groups were targeted to reduce the number of individuals within a
pod. Secondly, long term storage of frozen tissue samples (since 2016)
may have affected the number of viable mycobacteria present in some of
these tissues. Finally, unlike tissue samples acquired from other
species, the tissue samples from hippopotamuses have a very high fat
content which affected tissue homogenisation and therefore, downstream
application of GXU and mycobacterial culture. During initial sample
processing, we observed several invalid GXU results. For subsequent
samples, we increased the initial volume of PO4 buffer
(for tissue homogenization) from 2 to 4 mL. Therefore, additional
optimisations of mycobacterial culture for use in this species may
further improve the utility of this assay.
Hippopotamus sampling occurred during a prolonged drought in GKPA,
therefore, the presence of artificial waterholes and increased density
of hippopotamus as well as use by other M. bovis hosts such as
buffaloes in this area may have contributed to risk of infection and
should be investigated further. Based on a single culture confirmed
case, the susceptibility to infection, potential progression to disease,
and route of infection in hippopotamus remain unknown. However, since no
obvious clinical signs or macroscopic pathology were observed in this
cohort of hippopotamuses, there is no evidence that this species
develops active disease. Further
research should therefore be performed to investigate infected
hippopotamus, especially in endemic populations, and continued abattoir
surveillance should also be conducted when the opportunities arise.
In conclusion, the results reported here demonstrate the utility of
DPP® VetTB assay, GXU, and mycobacterial culture for
detecting M. bovis infection in common hippopotamus. Our findings
suggest potential tools for screening this species for bTB, which will
aid in diagnosis in zoo hippopotamus and surveillance in free-ranging
populations.