Download Molecular isolation of pathogenic non

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Molecular isolation of pathogenic non-tuberculous mycobacteria in free ranging migratory
wildebeests (Connochaetes taurinus) and cattle of Masai Mara, Kenya.
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Domnic Mijele 1, Isaac Lekolool 1, Francis Gakuya 1, Irmgard Moser 4, Rudovick Kazwala5,
Moses Otiende1, Manfred Tanner 2&3
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Friedrich-Loeffler Institut, Südufer 10, 17493 Greifswald-Isle of Riems, Germany.
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Friedrich-Loeffler Institut, Naumburger Strasse 96a, 07743 Jena, Germany.
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Sokoine University of Agriculture P.O Box 3021, Morogoro, Tanzania.
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Abstract
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The genus Mycobacterium consists of the members of the Mycobacterium (M.) tuberculosis
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complex (MTC), non-tuberculous mycobacteria (NTM) and M. leprae. MTC and some NTM
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organisms cause tuberculosis or tuberculosis-like diseases in humans and other animal species
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respectively. In Masai Mara conservation area in Kenya, a potential interface between wildlife,
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livestock and humans is existing due to frequent wildlife and livestock movements and
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interactions. This study proved the presence of various NTM species in free ranging migratory
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wildebeests wildebeests (Connochaetes taurinus) and cattle (Bos indicus) through culture,
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isolation, polymerase chain reaction (PCR) and DNA sequencing techniques using16S rRNA
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genes followed by hsp65 gene to confirm the results. The study revealed the presence of M.
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kansasii sub-species VI in 4/60 (6.7%) wildebeests and in 10/89 (11.2%) cattle. One of the
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wildebeests and 3/89 (3.4%) cattle had M. lentiflavum. M. gordonae was isolated from only one
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wildebeest while M. intracellulare was detected in 1/89 (1.1%) cattle. No MTC organisms were
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isolated. The DNA extracts of the isolated mycobacteria were sequence-analysed and published
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in Genbank with accession numbers assigned. The occurrence of NTM organisms in free ranging
Kenya Wildlife Service, Veterinary Services Department, P.O Box 40241-00100, Nairobi,
Kenya.
Institute for Infectious Diseases and Zoonoses, Department of Veterinary Science, LudwigMaximilians-Universität, Veterinärstr. 13, 80539 München, Germany.
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migratory wildebeests and cattle may pose a great risk of human infection through consumption
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of cattle and other livestock or bush-meat products.
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Key words: Wildebeests, M. kansasii, M. lentiflavum, M. intracellulare, hsp65 gene, 16S rRNA
gene.
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Corresponding author: Domnic Mijele; email: [email protected]
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Background
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Mycobacterial organisms are the causative agents of tuberculosis and other tuberculosis-like
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diseases in humans and animals. Mycobacterial infection has a negative impact on livestock
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production, wildlife conservation and poses an increased risk to humans who may get infected
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through consumption of uncooked meat or untreated milk from infected animals (Minja et al.,
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1998). Vice versa, animals may acquire the infection from tuberculosis-infected humans
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(Alexander et al., 2002). Consumption of infected prey is considered to be the predominant route
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by which lions in the Kruger National park became infected with M. bovis (Keet, et. al., 2000b).
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Mycobacterial organisms belonging to the Mycobacterium (M.) tuberculosis complex (MTC) are
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the causative agents of tuberculosis disease in humans and a broad range of mammalian species.
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Mycobacterium tuberculosis is the major cause of tuberculosis in humans causing mainly lung
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disease, while all mammals are susceptible to M. bovis infections, including humans causing
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mainly extra-pulmonary pathology.
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Although the causative agent of tuberculosis in birds is normally M. avium, under certain
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conditions even birds may acquire mammalian tuberculosis. M. avium belongs to the numerous
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groups of the non-tuberculous mycobacteria (NTM) which are of minor pathogenicity for
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humans and mammals. Besides these, there are many other emerging NTM species which have
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been reported to cause disease especially in immunocompromised humans and animals alike.
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Currently there are over 125 mycobacterial species already identified; both pathogenic and non-
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pathogenic (Shirin et al., 2010).
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The Masai Mara ecosystem is characterized by intensive wildlife-livestock-human interactions
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favourable for disease transmission among different species. The prevalence and distribution of
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pathogenic and non-pathogenic mycobacterial organisms in wildlife and livestock has not been
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documented in the ecosystem. This study investigated the presence of both pathogenic and non-
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pathogenic mycobacteria organisms in wildebeests (Connochaetes taurinus) and cattle (Bos
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indicus) in this area. The role of NTM in wildebeests and cattle in Masai Mara need to be
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investigated if they have any impact on public health.
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Masai Mara ecosystem is situated in Narok County on the south-western part of Kenya. The
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ecosystem comprises the Masai Mara National Reserve (MMNR) surrounded by several adjacent
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community-owned wildlife conservancies (figure 1.).
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Figure 1. Masai Mara National Reserve in Narok county and wildebeests sampling sites along
the Mara river.
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MMNR is a government protected area for wildlife conservation which covers an area of
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approximately 1510 square kilometers and is located on the South-Western part of Kenya along
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the Kenyan – Tanzanian border between 1°13’ and 1°45’ South and 34°45’ and 35°25’ East. The
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reserve extends to Serengeti National Park (SNP) in Tanzania where tuberculosis has been
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reported in lions (figure 1.).
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The ecosystem has dense populations of wildlife including large mammals such as African
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elephants, lions, leopards, African buffaloes, black rhinoceros, wildebeests and several antelope
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species (Mijele et al., 2013). The area is inhabited mainly by Masai pastoral communities
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keeping large herds of cattle, sheep and goats.
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There is regular interaction between wildlife, human and livestock within and outside the reserve
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(Mijele et al., 2013) and frequent cross-border movements of wildlife and livestock between
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Kenya and Tanzania. MMNR is also known for spectacular annual wildebeests migration
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between SNP and MMNR in search of adequate pastures (Ogutu et al., 2011). Wildebeests
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usually drown and die in large numbers while crossing the Mara River during the annual
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migrations. Frequent animal movements in the area have the potential of enhancing disease
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transmission among the wildlife-livestock-human populations across the border of Kenya and
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Tanzania.
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Methods
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Wildebeests sampling
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Postmortem examination was done on 60 wildebeests (Conochaetes taurinus) that drowned in
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three sites along the Mara River in August 2010. The three sites included Mara bridge (30
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wildebeests), Serena (20 wildebeests) and Little Governors (10 wildebeests) (figure 1.). The
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sampling of wildebeests was opportunistic and any wildebeest carcass irrespective of age or sex
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was retrieved from the river and a detailed postmortem examination conducted to detect
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granulomas or any other lesions compatible with tuberculosis. Lymph node tissue samples were
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then collected from retropharyngeal, mediastinal and mesenteric lymph nodes. The samples were
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labeled and immediately preserved in liquid nitrogen before being transferred to 80°C freezer at
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the Kenya Wildlife Service laboratory in Nairobi.
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Cattle sampling
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Detailed postmortem examination was performed on 89 cattle at Ewaso-Ngiro slaughterhouse
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within the Masai Mara ecosystem. The selection of cattle for post-mortem was simple random
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and animals above 2 years of age were targeted. Each carcass was examined for existence of any
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lesions or pathology related to tuberculosis infection. Tissue samples were collected from
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retropharyngeal, mediastinal and mesenteric lymph nodes irrespective of whether the animal had
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lesions or not. The samples were immediately preserved in liquid nitrogen and later transferred
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to a -80°C freezer at the Kenya Wildlife Service laboratory in Nairobi. All the samples collection
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was done at the same time in August, 2010.
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Laboratory analysis
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Frozen lymph node tissue samples from wildebeest and cattle were transferred to the tuberculosis
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laboratory at Sokoine University of Agriculture (SUA), Tanzania, where they were processed
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according to standard methods for isolation of mycobacteria from tissue (Hosek et al., 2006) and
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cultured on Loewenstein-Jensen (LJ) media for 8 weeks. Colonies that grew on LJ media were
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stained according to Ziehl-Neelsen and examined for the presence of acid fast bacilli (AFB)
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under light microscope. From positive cultures, material was taken for polymerase chain reaction
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(PCR) analysis at SUA and DNA sequence analysis was performed at Friedrich-Loeffler-
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Institute in Jena, Germany.
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Colony material was suspended in sterile water, boiled for ten minutes to inactivate the
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mycobacteria and centrifuged. The supernatant was taken as source of DNA and introduced into
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mycogenous standard PCR (Kirschner & Boettger 1998). The PCR product was run on an
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agarose gel with Ethidium Bromide and visualized under UV light. The PCR product band was
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cut out of the gel and DNA was extracted from agarose gel using QIAquick Gel Extraction kit
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(Qiagen, Hilden, Germany). The resultant DNA together with sequencing primers was submitted
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to GATC Biotech Company (Konstanz, Germany) for sequencing.
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DNA-Sequencing
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Sequencing of the 16S rRNA gene to identify mycobacterial species was performed according to
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Kirschner et al. (1998) using primer 271 as sequencing primer. The primers were obtained from
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Jena Bioscience, Jena, Germany. The sequence data were analyzed at FLI, Jena, Germany, using
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NCBI Blast software to identify the mycobacteria with the highest match to the sequences.
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Sequencing of hsp65 gene was performed to differentiate M. kansasii and M. gastri using the
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primers TB11 (Devallois et al., 1997). Subspecies identity of M. kansasii was determined
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according to Richter et al., 1999. All the sequence data was published in Gen-bank and provided
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with accession numbers (table 1a). BioEdit software was used to assess the similarities between
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the new sequences and the existing sequences in the database.
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Results
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There were no lesions or pathology related to tuberculosis infection in all the 60 wildebeests and
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89 cattle examined. Twenty PCR-positive mycobacterial isolates from 6 wildebeests and 14
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cattle were sequence-analyzed. Sequencing the 16S rRNA gene yielded DNA stretches of 749 to
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923 bp in all but one strain where only a length of 311bp was achieved. Sequencing of the hsp65
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gene yielded DNA stretches of 297 to 421 bp. The maximal identity to the data base entries was
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99% for the 16S rRNA gene and 99% - 100% for the hsp65 gene (table 2a).
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Wildebeests results
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From the 60 wildebeest lymph node samples examined from the Mara River, members of the
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MTC or M. avium were not isolated. Six out of 60 (10%) wildebeests were found positive for
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non-tuberculous mycobacteria. Sequences for 16S rRNA gene revealed Mycobacterium
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kansasii/gastri in retropharyngeal lymph nodes of three (5%) wildebeests and in mediastinal
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lymph node of one wildebeest (1.7%). One wildebeest (1.6%) had M. lentiflavum in mesenteric
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lymph node and one (1.6%) had M. gordonae in mediastinal lymph node (table 1a).
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Further sequencing of the same samples using hsp65 gene confirmed that the four samples which
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could not be differentiated using 16S rRNA, were all M. kansasii sub-species VI. The results for
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the other two wildebeests samples remained the same showing M. lentiflavum and M. gordonae
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respectively (table 1a).
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Cattle results
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A variety of non-tuberculous mycobacteria species were isolated from 14 out of 89 (15.7%)
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cattle lymph node samples examined at the Ewaso Nyiro slaughterhouse. Sequencing of the 16S
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rRNA gene revealed Mycobacterium kansasii/gastri in ten (11.2%) cattle, Mycobacterium
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lentiflavum was detected in three (3.4%) cattle and M. intracellulare in one cattle (1.1%) as
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indicated in (table 1a). Members of the MTC or M. avium were not isolated.
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Sequencing of the hsp65 gene confirmed that all the M. kansasii/gastri in 10 cattle were M.
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kansasii sub-species VI. The results for the other four cattle samples remained the same showing
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M. lentiflavum and M. intracellulare, respectively (table 1a). There was a mixed infection in one
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cattle Talek01 in which M. lentiflavum was isolated from the mediastinum lymph node and M.
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kansasii sub-species VI isolated from the retropharyngeal lymph node.
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No.
Sample number
Animal
species
FLI
number
Lymph node
Mycobacterial
species (hsp65
gene)
M. kansasii
Accession
numbers
Retropharyngeal
Mycobacterial
species (16S
rRNA gene)
M. kansasii/gastri
1
WB32RETRO
*W/beest
12MA1363
2
WB46MES
W/beest
12MA1366
Mesenteric
M. lentiflavum
M. lentiflavum
KF687953
3
WB48RETRO
4
WB44RETRO
W/beest
12MA1368
Retropharyngeal
M. kansasii/gastri
M. kansasii
KF687952
W/beest
12MA1373
Retropharyngeal
M. kansasii/gastri
M. kansasii
KF687952
5
WB23MED
W/beest
12MA1384
Mediastinal
M. gordonae
M. gordonae
-
6
WB49MED
W/beest
12MA1397
Mediastinal
M. kansasii/gastri
M. kansaii
KF687949
7
M276MES
Cattle
12MA1367
Mesenteric
M. kansasii/gastri
M. kansasii
KF687949
8
M144RETRO
Cattle
12MA1370
Retropharyngeal
M. kansasii/gastri
M. kansasii
KF687952
9
EWB52RETRO
Cattle
12MA1372
Retropharyngeal
M. lentiflavum
M. lentiflavum
KF687953
10
M294RETRO
Cattle
12MA1374
Retropharyngeal
M. kansasii/gastri
M. kansasii
KF687949
11
M309MES
Cattle
12MA1377
Mesenteric
M. lentiflavum
M. lentiflavum
KF687953
12
Talek01MED
Cattle
12MA1391
Mediastinal
M. lentiflavum
M. lentiflavum
KF687953
13
Talek01RETRO
Cattle
12MA1394
Retropharyngeal
M. kansasii
KF687952
14
Sek06 RETRO
Cattle
12MA1395
Retropharyngeal
M. kansasii/
gastri
M. kansasii/gastri
M. kansasii
KF687952
15
M286 RETRO
Cattle
12MA1399
Retropharyngeal
M. kansasii/gastri
M. kansasii
KF687952
16
Sek08 RETRO
Cattle
12MA1400
Retropharyngeal
M. kansasii/gastri
M. kansasii
KF687952
17
M309MES
Cattle
12MA1377
Mesenteric
M. kansasii/gastri
M. kansasii
KF687952
18
VNK01RETRO
Cattle
12MA1403
Retropharyngeal
M. intracellulare
M. intracellulare
KF687950
19
EWB76MES
Cattle
12MA1405
Mesenteric
M. kansasii/gastri
M. kansasii
KF687949
20
M151MED
Cattle
12MA1406
Mediastinal
M. kansasii/gastri
M. kansasii
KF687949
KF687951
Table 1a. Mycobacterial species isolated from lymph node tissue samples of wildebeests and
cattle of Masai Mara. *Wildebeest.
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No.
Animal
species
FLI
number
Mycobacterial
species (16S
rRNA gene)
Mycobacterial species
(hsp65 gene)
New
Genbank
Accession
numbers
Percentage
similarity
KF687951
Closest
existing
accession
numbers in
Genbank
HE575963.1
1
*W/beest
12MA1363
M. kansasii/gastri
M. kansasii ssp VI
2
W/beest
12MA1366
M. lentiflavum
M. lentiflavum
KF687953
KF019694.1
100%
3
W/beest
12MA1368
M. kansasii/gastri
M. kansasii ssp VI
KF687952
HE575963.1
99%
4
W/beest
5
W/beest
12MA1373
M. kansasii/gastri
M. kansasii ssp VI
KF687952
HE575963.1
99%
12MA1384
M. gordonae
M. gordonae
-
-
-
6
W/beest
12MA1397
M. kansasii/gastri
M. kansasii ssp VI
KF687949
AY550233.1
100%
7
Cattle
12MA1367
M. kansasii/gastri
M. kansasii ssp VI
KF687949
AY550233.1
100%
8
Cattle
12MA1370
M. kansasii/gastri
M. kansasii ssp VI
KF687952
HE575963.1
99%
9
Cattle
12MA1372
M. lentiflavum
M. lentiflavum
KF687953
KF019694.1
100%
10
Cattle
12MA1374
M. kansasii/gastri
M. kansasii ssp VI
KF687949
AY550233.1
100%
11
Cattle
12MA1377
M. lentiflavum
M. lentiflavum
KF687953
KF019694.1
100%
12
Cattle
12MA1391
M. lentiflavum
M. lentiflavum
KF687953
KF019694.1
100%
13
Cattle
12MA1394
M. kansasii/ gastri
M. kansasii ssp VI
KF687952
HE575963.1
99%
14
Cattle
12MA1395
M. kansasii/gastri
M. kansasii ssp VI
KF687952
HE575963.1
99%
15
Cattle
12MA1399
M. kansasii/gastri
M. kansasii ssp VI
KF687952
HE575963.1
99%
16
Cattle
12MA1400
M. kansasii/gastri
M. kansasii ssp VI
KF687952
HE575963.1
99%
17
Cattle
12MA1397
M. kansasii/gastri
M. kansasii ssp VI
KF687952
AY550233.1
100%
18
Cattle
12MA1403
M. intracellulare
M. intracellulare
KF687950
NR_074661.1
99%
19
Cattle
12MA1405
M. kansasii/gastri
M. kansasii ssp VI
KF687949
HE575963.1
99%
20
Cattle
12MA1406
M. kansasii/gastri
M. kansasii ssp VI
KF687949
HE575963.1
99%
99%
Table 2a. Percentage of similarities between mycobacterial species isolated from wildebeests
and cattle of Masai Mara and sequences already published in the Genbank. *Wildebeest.
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Discussion
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The presence of NTM organisms in free-ranging wildebeests and cattle generates a risk for
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human infection and may be responsible for the presence of human disease compatible with
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tuberculosis in Masai Mara ecosystem of Kenya.
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NTM organisms are ubiquitous and are usually recovered from the environment. They can cause
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disease to humans or animals through respiratory, cutaneous, parenteral and gastrointestinal
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exposure. The most pathogenic NTM species, M. avium causing tuberculosis in birds and
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tuberculosis-like disease in humans, especially in immunocompromised persons (e. g. HIV,
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elderly people) has not been detected. However, other NTM species which have been isolated in
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the course of this study are also known to cause disease in humans and animals alike.
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Mycobacterium kansasii is a slow-growing acid-fast bacillius (AFB) and belongs to the NTM
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group of mycobacteria (Sang et al., 2010) also known as environmental mycobacteria or even
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called “atypical mycobacteria”. Local water supplies are considered as the major reservoir for M.
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kansasii, evidence of person-to-person transmission has not been reported. The most common
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presentation of M. kansasii infection in humans is a chronic bronchopulmonary disease, which
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manifests typically in adult patients with chronic obstructive pulmonary disease or cystic fibrosis
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(Sang et al., 2010). In addition, M. kansasii can cause skeletal infections, skin and soft tissue
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infection, cervical or other lymphadenitis, and disseminated infection (Brown-Elliot et al., 2004).
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M. kansasii is pathogenic for humans and may cause severe tuberculosis-like disease (Sang et
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al., 2010). M. kansasii isolation is more common in HIV-positive persons, patients with
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hematologic malignancy, or patients receiving long-term steroid regimens (Wallace et al., 1997).
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Infection with M. kansasii requires the same treatment as M. tuberculosis infection (Evans et al.,
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1996).
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In wildlife, cattle and other livestock M. kansasii is not pathogenic but it may cause immune
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reactions and thus interfere with tuberculin skin test or gamma interferon release assay tests.
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There is a considerable risk of transmission from cattle or wildebeests to humans through
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consumption of meat or even contact. This is the first time M. kansasii has been isolated from
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wildebeests of Masai Mara ecosystem.
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Even though M. kansasii infection has not been reported in Kenya or elsewhere in Africa, it is
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the second most frequently recognized NTM pathogen and second most frequent cause of
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disseminated NTM disease, after M. avium complex (MAC), in the Unites States and Japan
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(Tsukamura et al., 1988; Lillo et al., 1990). In southeast England, M. kansasii is more common
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than M. avium Complex (Yattes et al., 1997). In South Korea, M. kansasii is the fourth most
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commonly isolated NTM pathogen, after MAC, M. abscessus-chelonae complex, and M.
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fortuitum, but its incidence has increased, especially in highly industrialized areas (Yim et al.,
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2005). Most patients with M. kansasii infection show clinical and radiologic evidence of
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infection regardless of HIV status.
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Mycobacterium lentiflavum was recently described as non-tuberculous mycobacterial species
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(Springer et al., 1996). It is described as a causative agent of cervical lymphadenitis in immuno-
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compromised human patients and children. M. lentiflavum is resistant to most first line
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antituberculous drugs. M. lentiflavum is a slow growing acid-fast bacillus (AFB) that has
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biochemical characteristics identical to those of organisms belonging to the Mycobacterium
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avium complex (MAC) and mycolic acid and fatty acid chromatography patterns very similar to
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those of Mycobacterium simiae, so genetic analysis is necessary for conclusive identification
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(Springer et al., 1996). This organism has been isolated from clinical samples in Italy,
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Switzerland, Germany, France and Spain (Springer et al., 1996; Tortoli et al., 1997; Niobe et al.,
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2001; Ibanez et al., 2002) and from sputum samples in Brazil (da Silva Rocha et al., 1999) and
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Italy (Molteni et al., 2005). Recently, cases of human disease have been reported, including
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chronic pulmonary disease (Molteni et al., 2005), cervical lymphadenitis (Cabria et al., 2002),
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liver abscess (Tortoli et al., 2002) and fatal disseminated infection (Ibanez et al., 2002). Our
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findings are the first evidence of M. lentiflavum infection reported in Kenya. The main reservoir
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in the environment has not been firmly established, but organisms with M. lentiflavum-like 16S
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rRNA gene sequences were detected in soil samples from the UK and from France (Mendum et
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al., 2000) and the species seem to be frequently present in drinking water distribution systems in
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Finland (Torvinen et al., 2004).
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Mycobacterium gordonae is the least pathogenic and its isolation is typically regarded as a
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contaminant (Lessnau et al., 1993). The organism is ubiquitous and it is most commonly isolated
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from soil and water (Caterina et al., 2009). Clinically significant disease caused by M. gordonae
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has been reported (Weinberger et al., 1992). Its presence in wildebeest samples could have been
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due to environmental contamination of the carcasses.
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Mycobacterium intracellulare is closely related to Mycobacterium avium and similarly causes
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pulmonary disease in humans. Environmental sources, especially water are the main reservoir for
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most human infections. Wildebeests and cattle could have acquired M. intracellulare from the
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environment.
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The high similarities between mycobacterial organisms in wildebeests and cattle is an indication
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that there could be cross-transmission of mycobacterial organisms between cattle and
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wildebeests and possibly to humans or other animals thereby putting human beings at a higher
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risk of infection. There is a possibility that the isolated mycobacteria exists in the environment
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from where wildebeests and cattle acquired infection from water or pasture.
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Authors Contributions
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DM IL FG RK IM MT MO conceived and designed the experiments for the paper. DM FG IL
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IM MT performed the experiments. DM IM RK IL analyzed the data. DM RK IL IM contributed
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reagents, materials and analysis tools. DM IL FG RK IM MT MO wrote the paper. All authors
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read and approved the final manuscript.
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Acknowledgement
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We are grateful for the financial support we received from the German Foundation for Research
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(DFG). Thanks to all staff at Kenya Wildlife Service, Sokoine University of Agriculture (Faculty
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of Veterinary Medicine), Friederich Loeffler Institute, Germany who supported this study in one
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way or another and without whom this work would not have been possible. We acknowledge the
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significant role of anonymous reviewers who have helped to improve the quality of this work.
251
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Figure Legends
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Figure 1. Map of Masai Mara National Reserve in Narok county and wildebeests sampling
sites along the Mara river.
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Tables
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Table 1a. Mycobacterial species isolated from lymph node tissue samples of wildebeests
and cattle of Masai Mara.
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Table 2a. Percentage of similarities between mycobacterial species isolated from
wildebeests and cattle of Masai Mara and sequences already published in the Genbank.