MOLECULAR PROFILE OF MYCOBACTERIUM SPP. ISOLATES FROM CATTLE AND OTHER ANIMAL SPECIES

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CHAPTER 3: NOVEL MYCOBACTERIUM AVIUM SPECIES ISOLATED FROM BLACK WILDEBEEST (CONNOCHAETES GNOU) IN SOUTH AFRICA

ABSTRACT

A study was undertaken to isolate and characterize Mycobacterium species from black wildebeest suspected of being infected with tuberculosis in South Africa. This led to the discovery of a new Mycobacterium avium species, provisionally referred to as “Gnou isolate” from black wildebeest (Connochaetus gnou). Sixteen samples from nine black wildebeest were processed for Mycobacterium isolation. Following decontamination; samples were incubated in an ordinary incubator at 37°C on Löwenstein-Jensen slants and in liquid medium tubes using the BACTECTM MGITTM 960 system respectively. Identification of the isolate was done by standard biochemical tests and using the line probe assay from the GenoType® CM/AS kit (Hain Life Science GmbH, Nehren, Germany). The DNA extract was also analysed using gene sequencing. Partial gene sequencing and analysis of 16S rRNA gene, 16S-23S rRNA (ITS), rpoB and hsp65 and phylogenetic analyses by searching GenBank using the BLAST algorithm were conducted. Phylogenetic trees were constructed using four methods, namely Bayesian inference, maximum likelihood, maximum parsimony and neighbor-joining methods. The isolate was identified as Mycobacterium intracellulare using the GenoType® CM/AS kit and as Mycobacterium avium complex (MAC) by gene sequencing. The gene sequence targeting all the genes, ITS, 16S rRNA, rpoB and hsp65 and phylogenetic analyses indicated that this isolate presented a nucleotide sequence different from all currently published sequences, and its position was far enough from other MAC species to suggest that it might be a new species.

BACKGROUND

In late 2006, animals from a commercial game farm reserve in Mpumalanga Province in South Africa were harvested for game meat exportation. During meat inspection, the animal carcasses showed lesions suspicious of tuberculosis which was supported by histopathological results. The exact cause of the disease was not determined and the farm was put under quarantine for suspected bovine tuberculosis.
In February 2009, 158 animals were harvested. A high number of animals (n = 135) showed gross-visible tuberculosis-like lesions and lesions from 6 animals processed for mycobacterial cultures yielded non-tuberculous mycobacteria.
Samples (n = 16) from 9 animals were submitted to the National Health Laboratory Service (NHLS, Pretoria, South Africa) for mycobacterial isolation and a non-tuberculous mycobacterium (NTM) was isolated and identified as Mycobacterium intracellulare using a commercial kit and as Mycobacterium avium complex (MAC) by gene sequencing. The results were submitted to the Department of Agriculture, Forestry and Fisheries (DAFF, South Africa) as M. intracellulare, and characterization of the isolate was then done in Japan.
The complete history of the herd including the pathology report described well-developed encapsulated granulomatous lesions observed on the different samples of organs as very suspicious for bovine tuberculosis (BTB). Other lesions observed which were not typical of M. bovis (pseudotuberculosis) comprised lack of caseous necrosis and liquefaction in the granulomas. The inspissated material from within the capsules could almost be squeezed out in total, leaving behind an empty “shell”. There were also several smaller granulomas with a typical onion ring appearance, but absence of calcification and liquefaction with no gritty sensation on cut section of these capsules. .
The genus Mycobacterium contains more than 170 species (http://www.bacterio.net/mycobacterium.html), most of which are classified as NTM or potentially pathogenic mycobacteria (PPM) (Chege et al., 2008; Kim et al., 2014; Malama et al., 2014; Tortoli, 2014) and mycobacteria belonging to the Mycobacterium tuberculosis complex (MTC). MTC comprises M. tuberculosis, M. bovis, M. africanum, M. canetti, M. pinnipedii, M. caprae, M. microti, M. mungi, Dassie bacillus, M. orygis (Oryx bacillus), M. surricatae and the attenuated M. bovis Bacille-Calmette-Guerin (BCG) vaccine strain. With the exception of BCG, these species are pathogenic and can cause tuberculosis (TB) in mammalian hosts (Alexander et al., 2010; Helden et al., 2009; Pittius et al., 2012; Vos et al., 2001).
The M. avium-intracellulare complex is the most commonly encountered group of NTM, and the clinically most important members are M. intracellulare and M. avium (Biet et al., 2005). Mycobacterium intracellulare has not been subdivided into subspecies whereas M. avium consists of four subspecies, namely M. avium subsp. avium, M. avium subsp. hominissuis, M. avium subsp. silvaticum and M. avium subsp. paratuberculosis. Mycobacterium Avium Complex (MAC) includes 10 different species, namely M. avium, M. intracellulare, M. colombiense, M. bouchedurhonense, M. timonense, M. arosiense, M. chimaera, M. vulneris, M. yongonense and M. marseillense (Tortoli, 2014).
The importance of NTM has received attention during the past decade, especially in humans. NTM are found in environmental systems (such as various soil and water systems) near human settlements and can be associated with colonization, serious infection or pseudo-outbreaks with a wide variety of presentations (Biet et al., 2005; Kankya et al., 2011; Katale et al., 2014). Indeed in humans, the isolation of NTM from clinical samples of patients presenting with pulmonary symptoms as suspected cases of tuberculosis has increased over the years and has been observed in different countries in Africa, America and Europe (Kankya et al., 2011; Katale et al., 2014; Mirsaeidi et al., 2014a, 2014b; Moore et al., 2010) whereas in animals the clinical significance of NTM has yet to be elucidated in the disease causing process (Chege et al., 2008; Kankya et al., 2011; Katale et al., 2014). The members of the genus Mycobacterium are genetically closer to each other than the microorganisms belonging to other genera, making identification a difficult and challenging task. The management, treatment and infection control measures differ significantly between M. tuberculosis and NTM infections.
More than one hundred and fifty species of NTM have been reported worldwide, of which more than 60% are pathogenic to animals or humans (Kim et al., 2014; Tortoli, 2014). In South Africa, reports on the isolation of NTM in animals, humans and environment and their effects in disease-causing processes are limited (Gcebe et al., 2013; Kabongo-Kayoka et al., 2015; Michel et al., 2007; Müller et al., 2011).
Black wildebeest (Connochaetus gnou), known in Afrikaans as “Swartwildebees” and in German as “Weisschwanzgnu”, have been hunted in South Africa for meat and hides. The overall research project was mainly on tuberculosis, so samples from black wildebeest suspected of being infected with tuberculosis were processed as part of phase I of the project focusing on prevalence and molecular studies of mycobacteria. The emergence of multi-drug and extremely drug resistant Mycobacterium tuberculosis strains was one of the main justifications of the project. This work resulted in the reporting of a novel Mycobacterium avium complex species from wildebeest in South Africa, which is expected to add to the corpus of knowledge and extend the frontiers of research on NTM.

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MATERIALS AND METHODS

Study area

Mpumalanga Province was selected as the area of study based on previous publications reporting TB in wildlife and livestock (Bengis et al., 1996; Michel et al., 2007; Vos et al., 2001) and availability of veterinary staff members experienced in conducting the comparative tuberculin skin test. Mpumalanga is one of the nine provinces in South Africa which is located in the north-eastern part of the country, bordering Swaziland and Mozambique to the East. It embraces the southern half of the Kruger National Park, a vast nature reserve with abundant wildlife including big game. It has a subtropical foliage supporting about 1 439 000 cattle according to the Trends in the Agricultural Sector (2013).

Study design and sampling

The study was designed as a cross-sectional study sampling animals in the designated area from January 2009 to January 2011. The local municipalities were selected based on the number of commercial farms, proximity to abattoirs, and location at the human-wildlife interface and the movement of animals as well. The municipalities selected comprised Malelane, Nelspruit, Lydenburg, Ermelo, Witbank and Standerton. The target population comprised cattle carcasses showing gross tuberculous-like lesions at meat inspection from positive reactors to tuberculin skin test at the municipality abattoirs during the study period. Samples from any other animal species showing gross tuberculous-like lesions were also included as convenience samples. The sampling was purposive to increase the chances of isolating mycobacteria and animals were selected based on positive reaction to the tuberculin skin test and suggestive lesions at the abattoir upon meat inspection.

Sources of samples

Samples from black wildebeest were received as part of phase I of a research project related to “Prevalence and molecular studies of Mycobacteria”. The samples were processed at the National Health Laboratory Service (NHLS) as part of a joint collaboration between University of Pretoria and NHLS. The history of the case was provided by the state veterinarian in charge. During a hunting period in February 2009 on a commercial game reserve located in Mpumalanga (South Africa), game animals (n=158) were randomly harvested and processed in the local abattoir according to standard operating procedures. These animals comprised black wildebeest (Connochaetes gnou) (n=137), blesbok (Damaliscus dorcas phillipsi) (n=15), blue wildebeest (Connochaetes taurinus) (n=2), red hartebeest (Alcelaphus buselaphus caama) (n=2) and springbok (Antidorcas marsupialis) (n=2). The animals lagging behind were the main target as well as females. During routine meat inspection of these animals, a high number of black wildebeest (n=135) showed granulomatous lesions in one or more lymph nodes or organs and this is the reason why samples were selected from this antelope species. Sixteen samples randomly selected from nine black wildebeest showing fresh lesions suggestive of tuberculosis infection were submitted to NHLS for isolation and identification of Mycobacterium spp. The samples included different organs and lymph nodes transported on ice (Table 3.1).

Mycobacterial isolation

Samples were frozen at -20°C until processing at NHLS. Direct impression smears were made from lesions and smears were stained using the Ziehl-Neelsen method. Tissue samples taken in a sterile manner from the inside of granulomatous lesions at the border between healthy and pathological tissues were finely cut using a sterile scalpel blade and homogenized using sterile glass beads by vortexing as described by Bengis et al. (1996) and Warren et al. (2006) with some modifications. To maximize the mycobacterial yield, specimens were subjected to a digestion and decontamination procedure using N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) solution with NaOH at a final concentration of 2% (Chatterjee et al., 2013). The specimens were left at room temperature for 15 min during the decontamination process and thereafter neutralized with phosphate buffer, centrifuged (Beckman Coulter) at 3000 x g for 15 min at 4°C, and the supernatant decanted and pellet suspended into 1 mL of phosphate buffer. The sediment was inoculated onto two LJ slants supplemented with pyruvate and glycerol and an antibiotic mixture of polymyxin B, amphotericin B, carbenicillin and Trimethoprim (PACT) (National Health Laboratory Service, South Africa, and Becton Dickinson, Germany) using a 0.01 mL calibrated loop. A further 0.5 mL of the sediment was inoculated with a graduated Pasteur pipette into a prepared liquid medium tube (BBLTM MGITTM Mycobacterium Growth indicator tubes) enriched with OADC and containing 800 µL of PANTATM antibiotic mixture (BDTM). This was incubated in the BACTECTM MGITTM 960 mycobacterial detection system at 37°C (Warren et al., 2006). The system was monitored for a maximum period of 42 days for bacterial growth whereas LJ slants were observed for colony growth and any other contaminant at two week intervals for 10 weeks. Tubes detected as positive within that period were further processed using Ziehl-Neelsen staining and examined microscopically for the presence of acid fast organisms and morphology. Thereafter, they were subcultured on LJ slant supplemented with glycerol and pyruvate. For identification purposes, a single colony was subcultured on a fresh LJ slant to obtain pure colonies. The same was repeated with two other colonies on different LJ slants to rule out the possibility of missing a different organism. Reference cultures of M. avium (ATCC 25291), M. bovis BCG and M. tuberculosis (ATCC 25177) were used as positive controls.

CHAPTER 1:GENERAL INTRODUCTION AND LITERATURE REVIEW 
1.1 General introduction
1.2 Literature review
1.3 Problem statement
1.4 Aims and objectives
1.5 Hypothesis
1.6 Scope of the thesis
1.7 Structure of the thesis
CHAPTER 2:MOLECULAR PROFILE OF MYCOBACTERIUM SPP. ISOLATES FROM CATTLE AND OTHER ANIMAL SPECIES
Abstract
2.1 Introduction
2.2 Materials and methods
2.3 Results and discussion
2.4 Conclusion
CHAPTER 3:NOVEL MYCOBACTERIUM AVIUM SPECIES ISOLATED FROM BLACK WILDEBEEST (CONNOCHAETES GNOU) IN SOUTH AFRICA
Abstract
3.1 Background
3.2 Materials and methods
3.3 Results
3.4 Discussion and conclusion
CHAPTER 4:ANTIMYCOBACTERIAL ACTIVITY AND CYTOTOXICITY OF LEAF EXTRACTS OF SOME AFRICAN ANACARDIACEAE TREE SPECIES
Abstract
4.1 INTRODUCTION
4.2 Materials and methods
4.3 Results and discussion
4.4 Conclusion
CHAPTER 5:BIOASSAY-GUIDED ISOLATION OF FRACTIONS AND COMPOUNDS FROM SEARSIA UNDULATA 
ABSTRACT
5.1 Background
5.2 Materials and methods
5.3 Results and discussion
5.4 Conclusion
CHAPTER 6:SUMMARY AND CONCLUSIONS
6.1 Isolation and characterization of mycobacterium species
6.2 Antimycobacterial activity of acetone leaf extracts of plant species from anacardiaceae family
6.2.1 Antimycobacterial activity of fractions and compounds of S. undulata
6.3 Cytotoxicity of acetone leaf extracts of plant species from anacardiaceae family
6.4 Isolation of bioactive fractions and compounds from the leaf of searsia undulata
6.5 Structure elucidation and identification of compounds
6.6 Research challenges
6.7 General conclusion, recommendations and future perspectives
REFERENCES
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CHARACTERIZATION OF MYCOBACTERIA SPP. AND ANTIMYCOBACTERIAL ACTIVITIES OF PLANT DERIVED COMPOUNDS FROM ANACARDIACEAE FAMILY

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