1 2 Molecular isolation of pathogenic non-tuberculous mycobacteria in free ranging migratory wildebeests (Connochaetes taurinus) and cattle of Masai Mara, Kenya. 3 4 Domnic Mijele 1, Isaac Lekolool 1, Francis Gakuya 1, Irmgard Moser 4, Rudovick Kazwala5, Moses Otiende1, Manfred Tanner 2&3 5 6 7 1 8 9 2 10 3 Friedrich-Loeffler Institut, Südufer 10, 17493 Greifswald-Isle of Riems, Germany. 11 4 Friedrich-Loeffler Institut, Naumburger Strasse 96a, 07743 Jena, Germany. 12 5 Sokoine University of Agriculture P.O Box 3021, Morogoro, Tanzania. 13 Abstract 14 The genus Mycobacterium consists of the members of the Mycobacterium (M.) tuberculosis 15 complex (MTC), non-tuberculous mycobacteria (NTM) and M. leprae. MTC and some NTM 16 organisms cause tuberculosis or tuberculosis-like diseases in humans and other animal species 17 respectively. In Masai Mara conservation area in Kenya, a potential interface between wildlife, 18 livestock and humans is existing due to frequent wildlife and livestock movements and 19 interactions. This study proved the presence of various NTM species in free ranging migratory 20 wildebeests wildebeests (Connochaetes taurinus) and cattle (Bos indicus) through culture, 21 isolation, polymerase chain reaction (PCR) and DNA sequencing techniques using16S rRNA 22 genes followed by hsp65 gene to confirm the results. The study revealed the presence of M. 23 kansasii sub-species VI in 4/60 (6.7%) wildebeests and in 10/89 (11.2%) cattle. One of the 24 wildebeests and 3/89 (3.4%) cattle had M. lentiflavum. M. gordonae was isolated from only one 25 wildebeest while M. intracellulare was detected in 1/89 (1.1%) cattle. No MTC organisms were 26 isolated. The DNA extracts of the isolated mycobacteria were sequence-analysed and published 27 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. 28 migratory wildebeests and cattle may pose a great risk of human infection through consumption 29 of cattle and other livestock or bush-meat products. 30 31 Key words: Wildebeests, M. kansasii, M. lentiflavum, M. intracellulare, hsp65 gene, 16S rRNA gene. 32 33 Corresponding author: Domnic Mijele; email: firstname.lastname@example.org 34 Background 35 Mycobacterial organisms are the causative agents of tuberculosis and other tuberculosis-like 36 diseases in humans and animals. Mycobacterial infection has a negative impact on livestock 37 production, wildlife conservation and poses an increased risk to humans who may get infected 38 through consumption of uncooked meat or untreated milk from infected animals (Minja et al., 39 1998). Vice versa, animals may acquire the infection from tuberculosis-infected humans 40 (Alexander et al., 2002). Consumption of infected prey is considered to be the predominant route 41 by which lions in the Kruger National park became infected with M. bovis (Keet, et. al., 2000b). 42 Mycobacterial organisms belonging to the Mycobacterium (M.) tuberculosis complex (MTC) are 43 the causative agents of tuberculosis disease in humans and a broad range of mammalian species. 44 Mycobacterium tuberculosis is the major cause of tuberculosis in humans causing mainly lung 45 disease, while all mammals are susceptible to M. bovis infections, including humans causing 46 mainly extra-pulmonary pathology. 47 Although the causative agent of tuberculosis in birds is normally M. avium, under certain 48 conditions even birds may acquire mammalian tuberculosis. M. avium belongs to the numerous 49 groups of the non-tuberculous mycobacteria (NTM) which are of minor pathogenicity for 50 humans and mammals. Besides these, there are many other emerging NTM species which have 51 been reported to cause disease especially in immunocompromised humans and animals alike. 52 Currently there are over 125 mycobacterial species already identified; both pathogenic and non- 53 pathogenic (Shirin et al., 2010). 54 The Masai Mara ecosystem is characterized by intensive wildlife-livestock-human interactions 55 favourable for disease transmission among different species. The prevalence and distribution of 56 pathogenic and non-pathogenic mycobacterial organisms in wildlife and livestock has not been 57 documented in the ecosystem. This study investigated the presence of both pathogenic and non- 58 pathogenic mycobacteria organisms in wildebeests (Connochaetes taurinus) and cattle (Bos 59 indicus) in this area. The role of NTM in wildebeests and cattle in Masai Mara need to be 60 investigated if they have any impact on public health. 61 Masai Mara ecosystem is situated in Narok County on the south-western part of Kenya. The 62 ecosystem comprises the Masai Mara National Reserve (MMNR) surrounded by several adjacent 63 community-owned wildlife conservancies (figure 1.). 64 65 66 Figure 1. Masai Mara National Reserve in Narok county and wildebeests sampling sites along the Mara river. 67 MMNR is a government protected area for wildlife conservation which covers an area of 68 approximately 1510 square kilometers and is located on the South-Western part of Kenya along 69 the Kenyan – Tanzanian border between 1°13’ and 1°45’ South and 34°45’ and 35°25’ East. The 70 reserve extends to Serengeti National Park (SNP) in Tanzania where tuberculosis has been 71 reported in lions (figure 1.). 72 The ecosystem has dense populations of wildlife including large mammals such as African 73 elephants, lions, leopards, African buffaloes, black rhinoceros, wildebeests and several antelope 74 species (Mijele et al., 2013). The area is inhabited mainly by Masai pastoral communities 75 keeping large herds of cattle, sheep and goats. 76 There is regular interaction between wildlife, human and livestock within and outside the reserve 77 (Mijele et al., 2013) and frequent cross-border movements of wildlife and livestock between 78 Kenya and Tanzania. MMNR is also known for spectacular annual wildebeests migration 79 between SNP and MMNR in search of adequate pastures (Ogutu et al., 2011). Wildebeests 80 usually drown and die in large numbers while crossing the Mara River during the annual 81 migrations. Frequent animal movements in the area have the potential of enhancing disease 82 transmission among the wildlife-livestock-human populations across the border of Kenya and 83 Tanzania. 84 Methods 85 Wildebeests sampling 86 Postmortem examination was done on 60 wildebeests (Conochaetes taurinus) that drowned in 87 three sites along the Mara River in August 2010. The three sites included Mara bridge (30 88 wildebeests), Serena (20 wildebeests) and Little Governors (10 wildebeests) (figure 1.). The 89 sampling of wildebeests was opportunistic and any wildebeest carcass irrespective of age or sex 90 was retrieved from the river and a detailed postmortem examination conducted to detect 91 granulomas or any other lesions compatible with tuberculosis. Lymph node tissue samples were 92 then collected from retropharyngeal, mediastinal and mesenteric lymph nodes. The samples were 93 labeled and immediately preserved in liquid nitrogen before being transferred to 80°C freezer at 94 the Kenya Wildlife Service laboratory in Nairobi. 95 Cattle sampling 96 Detailed postmortem examination was performed on 89 cattle at Ewaso-Ngiro slaughterhouse 97 within the Masai Mara ecosystem. The selection of cattle for post-mortem was simple random 98 and animals above 2 years of age were targeted. Each carcass was examined for existence of any 99 lesions or pathology related to tuberculosis infection. Tissue samples were collected from 100 retropharyngeal, mediastinal and mesenteric lymph nodes irrespective of whether the animal had 101 lesions or not. The samples were immediately preserved in liquid nitrogen and later transferred 102 to a -80°C freezer at the Kenya Wildlife Service laboratory in Nairobi. All the samples collection 103 was done at the same time in August, 2010. 104 Laboratory analysis 105 Frozen lymph node tissue samples from wildebeest and cattle were transferred to the tuberculosis 106 laboratory at Sokoine University of Agriculture (SUA), Tanzania, where they were processed 107 according to standard methods for isolation of mycobacteria from tissue (Hosek et al., 2006) and 108 cultured on Loewenstein-Jensen (LJ) media for 8 weeks. Colonies that grew on LJ media were 109 stained according to Ziehl-Neelsen and examined for the presence of acid fast bacilli (AFB) 110 under light microscope. From positive cultures, material was taken for polymerase chain reaction 111 (PCR) analysis at SUA and DNA sequence analysis was performed at Friedrich-Loeffler- 112 Institute in Jena, Germany. 113 Colony material was suspended in sterile water, boiled for ten minutes to inactivate the 114 mycobacteria and centrifuged. The supernatant was taken as source of DNA and introduced into 115 mycogenous standard PCR (Kirschner & Boettger 1998). The PCR product was run on an 116 agarose gel with Ethidium Bromide and visualized under UV light. The PCR product band was 117 cut out of the gel and DNA was extracted from agarose gel using QIAquick Gel Extraction kit 118 (Qiagen, Hilden, Germany). The resultant DNA together with sequencing primers was submitted 119 to GATC Biotech Company (Konstanz, Germany) for sequencing. 120 DNA-Sequencing 121 Sequencing of the 16S rRNA gene to identify mycobacterial species was performed according to 122 Kirschner et al. (1998) using primer 271 as sequencing primer. The primers were obtained from 123 Jena Bioscience, Jena, Germany. The sequence data were analyzed at FLI, Jena, Germany, using 124 NCBI Blast software to identify the mycobacteria with the highest match to the sequences. 125 Sequencing of hsp65 gene was performed to differentiate M. kansasii and M. gastri using the 126 primers TB11 (Devallois et al., 1997). Subspecies identity of M. kansasii was determined 127 according to Richter et al., 1999. All the sequence data was published in Gen-bank and provided 128 with accession numbers (table 1a). BioEdit software was used to assess the similarities between 129 the new sequences and the existing sequences in the database. 130 Results 131 There were no lesions or pathology related to tuberculosis infection in all the 60 wildebeests and 132 89 cattle examined. Twenty PCR-positive mycobacterial isolates from 6 wildebeests and 14 133 cattle were sequence-analyzed. Sequencing the 16S rRNA gene yielded DNA stretches of 749 to 134 923 bp in all but one strain where only a length of 311bp was achieved. Sequencing of the hsp65 135 gene yielded DNA stretches of 297 to 421 bp. The maximal identity to the data base entries was 136 99% for the 16S rRNA gene and 99% - 100% for the hsp65 gene (table 2a). 137 Wildebeests results 138 From the 60 wildebeest lymph node samples examined from the Mara River, members of the 139 MTC or M. avium were not isolated. Six out of 60 (10%) wildebeests were found positive for 140 non-tuberculous mycobacteria. Sequences for 16S rRNA gene revealed Mycobacterium 141 kansasii/gastri in retropharyngeal lymph nodes of three (5%) wildebeests and in mediastinal 142 lymph node of one wildebeest (1.7%). One wildebeest (1.6%) had M. lentiflavum in mesenteric 143 lymph node and one (1.6%) had M. gordonae in mediastinal lymph node (table 1a). 144 Further sequencing of the same samples using hsp65 gene confirmed that the four samples which 145 could not be differentiated using 16S rRNA, were all M. kansasii sub-species VI. The results for 146 the other two wildebeests samples remained the same showing M. lentiflavum and M. gordonae 147 respectively (table 1a). 148 Cattle results 149 A variety of non-tuberculous mycobacteria species were isolated from 14 out of 89 (15.7%) 150 cattle lymph node samples examined at the Ewaso Nyiro slaughterhouse. Sequencing of the 16S 151 rRNA gene revealed Mycobacterium kansasii/gastri in ten (11.2%) cattle, Mycobacterium 152 lentiflavum was detected in three (3.4%) cattle and M. intracellulare in one cattle (1.1%) as 153 indicated in (table 1a). Members of the MTC or M. avium were not isolated. 154 Sequencing of the hsp65 gene confirmed that all the M. kansasii/gastri in 10 cattle were M. 155 kansasii sub-species VI. The results for the other four cattle samples remained the same showing 156 M. lentiflavum and M. intracellulare, respectively (table 1a). There was a mixed infection in one 157 cattle Talek01 in which M. lentiflavum was isolated from the mediastinum lymph node and M. 158 kansasii sub-species VI isolated from the retropharyngeal lymph node. 159 160 161 162 163 164 165 166 167 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. 168 169 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. 170 171 Discussion 172 The presence of NTM organisms in free-ranging wildebeests and cattle generates a risk for 173 human infection and may be responsible for the presence of human disease compatible with 174 tuberculosis in Masai Mara ecosystem of Kenya. 175 NTM organisms are ubiquitous and are usually recovered from the environment. They can cause 176 disease to humans or animals through respiratory, cutaneous, parenteral and gastrointestinal 177 exposure. The most pathogenic NTM species, M. avium causing tuberculosis in birds and 178 tuberculosis-like disease in humans, especially in immunocompromised persons (e. g. HIV, 179 elderly people) has not been detected. However, other NTM species which have been isolated in 180 the course of this study are also known to cause disease in humans and animals alike. 181 Mycobacterium kansasii is a slow-growing acid-fast bacillius (AFB) and belongs to the NTM 182 group of mycobacteria (Sang et al., 2010) also known as environmental mycobacteria or even 183 called “atypical mycobacteria”. Local water supplies are considered as the major reservoir for M. 184 kansasii, evidence of person-to-person transmission has not been reported. The most common 185 presentation of M. kansasii infection in humans is a chronic bronchopulmonary disease, which 186 manifests typically in adult patients with chronic obstructive pulmonary disease or cystic fibrosis 187 (Sang et al., 2010). In addition, M. kansasii can cause skeletal infections, skin and soft tissue 188 infection, cervical or other lymphadenitis, and disseminated infection (Brown-Elliot et al., 2004). 189 M. kansasii is pathogenic for humans and may cause severe tuberculosis-like disease (Sang et 190 al., 2010). M. kansasii isolation is more common in HIV-positive persons, patients with 191 hematologic malignancy, or patients receiving long-term steroid regimens (Wallace et al., 1997). 192 Infection with M. kansasii requires the same treatment as M. tuberculosis infection (Evans et al., 193 1996). 194 In wildlife, cattle and other livestock M. kansasii is not pathogenic but it may cause immune 195 reactions and thus interfere with tuberculin skin test or gamma interferon release assay tests. 196 There is a considerable risk of transmission from cattle or wildebeests to humans through 197 consumption of meat or even contact. This is the first time M. kansasii has been isolated from 198 wildebeests of Masai Mara ecosystem. 199 Even though M. kansasii infection has not been reported in Kenya or elsewhere in Africa, it is 200 the second most frequently recognized NTM pathogen and second most frequent cause of 201 disseminated NTM disease, after M. avium complex (MAC), in the Unites States and Japan 202 (Tsukamura et al., 1988; Lillo et al., 1990). In southeast England, M. kansasii is more common 203 than M. avium Complex (Yattes et al., 1997). In South Korea, M. kansasii is the fourth most 204 commonly isolated NTM pathogen, after MAC, M. abscessus-chelonae complex, and M. 205 fortuitum, but its incidence has increased, especially in highly industrialized areas (Yim et al., 206 2005). Most patients with M. kansasii infection show clinical and radiologic evidence of 207 infection regardless of HIV status. 208 Mycobacterium lentiflavum was recently described as non-tuberculous mycobacterial species 209 (Springer et al., 1996). It is described as a causative agent of cervical lymphadenitis in immuno- 210 compromised human patients and children. M. lentiflavum is resistant to most first line 211 antituberculous drugs. M. lentiflavum is a slow growing acid-fast bacillus (AFB) that has 212 biochemical characteristics identical to those of organisms belonging to the Mycobacterium 213 avium complex (MAC) and mycolic acid and fatty acid chromatography patterns very similar to 214 those of Mycobacterium simiae, so genetic analysis is necessary for conclusive identification 215 (Springer et al., 1996). This organism has been isolated from clinical samples in Italy, 216 Switzerland, Germany, France and Spain (Springer et al., 1996; Tortoli et al., 1997; Niobe et al., 217 2001; Ibanez et al., 2002) and from sputum samples in Brazil (da Silva Rocha et al., 1999) and 218 Italy (Molteni et al., 2005). Recently, cases of human disease have been reported, including 219 chronic pulmonary disease (Molteni et al., 2005), cervical lymphadenitis (Cabria et al., 2002), 220 liver abscess (Tortoli et al., 2002) and fatal disseminated infection (Ibanez et al., 2002). Our 221 findings are the first evidence of M. lentiflavum infection reported in Kenya. The main reservoir 222 in the environment has not been firmly established, but organisms with M. lentiflavum-like 16S 223 rRNA gene sequences were detected in soil samples from the UK and from France (Mendum et 224 al., 2000) and the species seem to be frequently present in drinking water distribution systems in 225 Finland (Torvinen et al., 2004). 226 Mycobacterium gordonae is the least pathogenic and its isolation is typically regarded as a 227 contaminant (Lessnau et al., 1993). The organism is ubiquitous and it is most commonly isolated 228 from soil and water (Caterina et al., 2009). Clinically significant disease caused by M. gordonae 229 has been reported (Weinberger et al., 1992). Its presence in wildebeest samples could have been 230 due to environmental contamination of the carcasses. 231 Mycobacterium intracellulare is closely related to Mycobacterium avium and similarly causes 232 pulmonary disease in humans. Environmental sources, especially water are the main reservoir for 233 most human infections. Wildebeests and cattle could have acquired M. intracellulare from the 234 environment. 235 The high similarities between mycobacterial organisms in wildebeests and cattle is an indication 236 that there could be cross-transmission of mycobacterial organisms between cattle and 237 wildebeests and possibly to humans or other animals thereby putting human beings at a higher 238 risk of infection. There is a possibility that the isolated mycobacteria exists in the environment 239 from where wildebeests and cattle acquired infection from water or pasture. 240 Authors Contributions 241 DM IL FG RK IM MT MO conceived and designed the experiments for the paper. DM FG IL 242 IM MT performed the experiments. DM IM RK IL analyzed the data. DM RK IL IM contributed 243 reagents, materials and analysis tools. DM IL FG RK IM MT MO wrote the paper. All authors 244 read and approved the final manuscript. 245 Acknowledgement 246 We are grateful for the financial support we received from the German Foundation for Research 247 (DFG). Thanks to all staff at Kenya Wildlife Service, Sokoine University of Agriculture (Faculty 248 of Veterinary Medicine), Friederich Loeffler Institute, Germany who supported this study in one 249 way or another and without whom this work would not have been possible. We acknowledge the 250 significant role of anonymous reviewers who have helped to improve the quality of this work. 251 References 252 253 254 1. Alexander K, Pleydell E, Williams M, Lane E, Nyange J, Michel A Mycobacterium tuberculosis: an emerging disease of free-ranging wildlife. Emerg Infect Dis. 2002;8:598–601. 255 256 257 2. Brown-Elliott BA, Wallacc RJ, 2004: Infections Caused by Non-tuberculosis Mycobacteria. In: Mandell GL, Bennett JE, Dolin R, eds. Principle and Practice of Infectious Disease. 6th ed. 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Mycobacterial species isolated from lymph node tissue samples of wildebeests and cattle of Masai Mara. 360 361 Table 2a. Percentage of similarities between mycobacterial species isolated from wildebeests and cattle of Masai Mara and sequences already published in the Genbank.