Molecular Detection of Mycobacterium bovis and Other Mycobacteria in Soil

Transcription

1 Part 2 Molecular detection techniques and problems of quantification Molecular Detection of Mycobacterium bovis and Other Mycobacteria in Soil

2 Pathogen reservoirs Case study 1: Detection of enteric bacteria. Monitoring salmonellae in the environment in soil and water Case study 2: Detection of Mycobacterium species in soil, faeces, potable waters/ animal drinking toughs Exploiting bacterial diversity Case study 3: New approaches to detection of novel Pharmaceuticals and enzymes

3 Mycobacterium bovis High GC, Gram positive actinomycete Member of the Slow-Growing group of mycobateria. Causative agent of tuberculosis in many warm blooded animals i.e. humans, cattle badgers, seals etc.

4 Bovine Tuberculosis Major problem in UK, Ireland, US and New Zealand Thought to be affected by animal reservoir Cases may have been affected by foot and mouth outbreak

5 Bovine Tuberculosis Testing and slaughter policy introduced in 1960s From

6 Bovine Tuberculosis Testing and slaughter policy introduced in 1960s Badger protection act introduced in With full policy in 1975 From

7 Bovine Tuberculosis Testing and slaughter policy introduced in 1960s Badger protection act introduced in With full policy in 1975 From

8 Detect survival and distribution of M. bovis Determine survival of M. bovis BCG in soil Determine diversity amongst the total mycobacterial population Determine DNA turnover rates in soil to better understand the longevity of DNA in soil

9 Survival and Distribution of M. bovis

10 Mycobacterium bovis in Soil Previous research has shown other pathogenic mycobacteria to be highly prevalent in soil e.g M avium and M. paratuberculosis M. bovis has evolved from a soil saprophyte and retains many genes involved in a saprophytic lifestyle Traditional cultivation uses harsh decontamination techniques to remove the majority of other organisms from the soil

11 Decontamination 1 g soil added to Ringer s solution Shaken for 10 min on Griffin shaker 5% NaOH added for 30 min Plates incubated in gas permeable bags for 8 weeks. Checked every 4 days Samples mixed, then plated onto Middlebrook 7H10 agar containing Cycloheximide Optional steps: Hyperchlorite, oxalic acid addition

12 2/8 weeks Decontamination Extraction of M. bovis BCG from Soil No decontamination decomtamination initial inoculum ln cfu/g soil

13 Molecular Detection Used molecular techniques as opposed to traditional culture methods Soil samples came from a farm in Southern Ireland with a history of bovine TB Used PCR, directed at three antigen genes, MPB70, MPB64 and Esat-6 Exact function of these genes is unknown, but are specific to M. bovis group

14 DNA was extracted from soils from the Irish farm, also Warwick, Greek, Italian, and Cotswold soils were tested. Samples were taken in triplicate from random locations in each field. Samples were mixed and sub sampling done in triplicate

15 MPB , 11) Molecular Markers. 2) Negative Control.3) 1A. 4) 1c. 5) 2a. 6) 2c) 7) 3C. 8) Infected Pasture. 9) Badger set. 10) Positive Control

16 MPB , 11) Molecular Markers. 2) Negative Control.3,4,5) Positive Control. 6) Badger Set. 7) Infected Pasture. 8) 3C 9) 1A. 10) 1C

17 PCR using Esat6 targeted primers , 2) M. bovis DNA 3, 4) DNA from badger set soil 5, 6, 7, 8) infected pasture soil 9) DNA from Warwick soil 10) Negative

18 Quantification of PCR products PCR was carried out using MPB64 primers, to determine when the reaction remains linear Increasing cycle number

19 Standards were then created, using a one in ten dilution series of M. bovis BCG. Each dilution was inoculated into soil, DNA extracted and PCR carried out 10 9 cells.g soil ln pixel intensity 10 2 cells/g soil ln pixel intensity

20 1C Quantification of PCR Badger set Pasture 3C 3A 2A 2C 1A Gene copy number/g soil

21 Number of individual Irish soil cores that gave PCR positive results for both mpb64 and mpb70. Levels of gene copies of both mpb64 and mpb70 in total community DNA extracted from Irish soil cores % Positives 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% BS1 BS2 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 Apr-00 Nov Number of gene copies per g soil Apr-00 Nov-02 0 BS1 BS2 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10

22 M.bovis DNA dilutions

23 Standard Curve

24 Environmental samples Positive control Neg control

25 Characteristic melting curve M.Bovis DNA RD4 flanking primers

26 Environmental samples

27 QUANTIFICATION

28 QUANTIFICATION

29 Conclusions Genes associated with M. bovis were detected in the infected pasture, entrances to badger sets and one other field. Highest level of targets was found in soil from badger set entrances.

30 Survival of M. bovis BCG in Soil

31 BCG Survival Set up 1g sterile and non-sterile soil microcosms. Inoculated with BCG and monitored survival over time using PCR and Plate count methods

32 Effect of Temperature on M. bovis BCG Survival C 15 0C 25 0C 37 0C Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 10 Day 13 Day 16 Day 20 Day 25 Day 30 Day 40 Day 60 ln cfu-g soil

33 Effect of Water Content on M. bovis BCG Survival 5% 10% 15% 20% ln cfu/g soil 30% 40% Day 40 Day 25 Day 16 Day 10 Day 6 Day 4 Day 2 Day 0

34 Survival of BCG in Non-sterile Soil C 15 0C 25 0C 37 0C ln gene copy/g soil Day 0 Day 2 Day 4 Day 6 Day 10 Day 16 Day 25 Day 40 In this case gene copy equates in 1:1 ratio with cell number

35 Summary BCG seems to survive for longer and at a higher rate the higher the temperature. Also with a high water content in the soil DNA was still present at a higher level than culturable cell equivalents at 60 days. And signal was present over 1 year later

36 Diversity Amongst the Total Mycobacterial Population

37 16S rrna Analysis Designed primers to target region of 16S rrna gene, encoding for the hyper-variable gamma region 2 Primer sets designed. One to be genus specific, and one to target slow growers Tested on a variety of Mycobacterium spp. and members of closely related genera, e.g. Nocardia spp. and Rhodococcus spp

38 Amplification of Product from Selected Soils Using PCR Primers Targeted Specifically to the Mycobacterium Genus 16S rrna Genes , 16) Molecular weight markers 2, 3) M. bovis DNA 4, 5) M. bovis BCG DNA 6, 7, 8, 9, 10) Badger set soil DNA 11, 12) DNA from soil at the University of Warwick 13, 14, 15, 17) 2A Soil 18, 19, 20, 21) Infected Pasture soil

39 Nocardia asteroides M.gilvum M. farcinogenes M.peregrinum M.fortuitum IP3JSY W7JSY M. smegmatis IP7JSY B14JSY IP6JSY B12JSY IP19JSY B10JSY B20JSY B13JSY B15JSY 76 M. fallax B21JSY M. hiberniae B16JSY IP5JSY B30JSY M.interjectum M. Murphy IP2JSY W3JSY W11JSY W12JSY M. gordonae M. asiaticum IWGMT90018 M. pinnipedii M. bovis M. africanum M.tuberculosis M. kansasii M. malmoense M. haemophilum W20JSY M. paratuberculosis M. avium M. intracellulare M. bohemicum W1JSY W5JSY W14JSY 99 B11JSY B35JSY W13JSY3 M. bovis group Neighbour-Joining boot-strapped Phylogenetic Tree Infected pasture clones Badger set clones Warwick soil clones IP4JSY W4JSY IP10JSY Genus specific primers Slow growing groups-pathogens

40 Targeting slow growing mycobacteria 16S rrna insertion

41 16S rrna Analysis ) Molecular Markers. 2) Positive Control. 3) Warwick Soil.4) Badger Set Soil. 5) Infected Pasture Soil.6) 2A Soil7) 2C Soil. 8) Negative Control

42 M. fortuitum M. peregrinum M. farcinogenes M. gilvum M. smegmatis M. fallax M. hiberniae W4JSY IP29JSY W14JSY W1JSY M. asiaticum IWGMT90018 M. gordonae W18JSY IP4JSY W2JSY W12JSY W6JSY M. kansasii M. tuberculosis M. pinnipedii M. africanum M. bovis IP25JSY IP21JSY BS4JSY BS6JSY M. paratuberculosis WS35JSY 85 Nocardia asteroides W16JSY BS2JSY W8JSY BS3JSY M. bovis group M. avium Neighbour-Joining boot-strapped Phylogenetic Tree Infected pasture clones Badger set clones Warwick soil clones 16S rrna primers for slow growing groups

43 Conclusions Sequences were cloned with high identity to M. fallax and M. hiberniae, both implicated in hypersensitivity reactions. Also sequences with identity to the pathogenic species M. avium were detected Second primer set revealed M. bovis 16S rrna gene sequences

44

45 Persistence of M. bovis BCG DNA from four sources in soil microcosms held at a) 10 C and b) 37 C. = DNA, = DNA from dead lysed cells, = DNA from dead intact cells and = 120 DNA from live intact cells Day 0 Day1 Day2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15 Day 16 Day 17 Day 18 Day 19 Day 20 % Of Original DNA 10 o C Dead lysed cells Free DNA Dead intact cells Live intact cells o C Day 0 Day1 Day2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15 Day 16 Day 17 Day 18 Day 19 Day 20 % Of Original DNA

46 Decrease of DNA per day rates from four different sources in soil microcosms incubated at 10 ºC and 30 ºC %Decrease/Day Live Cells Dead Intact Cells Lysed Cells Free DNA

47 Summary M. bovis can survive in soil for over 4 months, and DNA persist for over 1 year 16S rrna in RNA extracted from soil allowed specific detection indicating probably viable, active cells M. bovis BCG can survive for 60 days at high temperatures and high moisture content. DNA and rrna detected >1 year Presence of diverse mycobacteria species in soil, M. hiberniae, M.fallax and M. avium may cause immune reaction

48 Summary M. bovis can survive in soil for over 4 months, and DNA persist for over 1 year 16S rrna in RNA extracted from soil allowed specific detection indicating probably viable, active cells M. bovis BCG can survive for 60 days at high temperatures and high moisture content. DNA and rrna detected >1 year Presence of diverse mycobacteria species in soil, M. hiberniae, M.fallax and M. avium may cause immune reaction

49 Soil Location Altitude (m) Date of Collection 1108 Cattle Market, Mehal Meda (town) /10/ House yard, near Mehal Meda /10/ Ploughed Field, on road to Addis from Mehal Meda /10/ House Yard, on road to Addis from Debre Birham /10/08 Cryfield Field, farm land

50 Methods: Deep Sequencing 2007 Roche 454 FLX TITANIUM Genome Sequencer Research & Testing Laboratories, Texas, USA

51 454 Deep sequencing Data: Universal bacteria primers Species Cryfield Mycobacterium aubagnense Mycobacterium avium Mycobacterium duvalii Mycobacterium elephantis Mycobacterium heckeshornense Mycobacterium hodleri Mycobacterium holsaticum Mycobacterium obuense Mycobacterium psychrotolerans Mycobacterium pyrenivorans Mycobacterium sp Total Data expressed as a percentage of the original 484 species identified. Able to detect Mycobacteria so must be relatively abundant in the environment.

52 454 Deep sequencing Data: Mycobacterium genus specific Species Sample Type Soil Soil 1 Soil Cryfield Total Mycobacterium agri 1 1 Mycobacterium aichiense Mycobacterium alvei Mycobacterium arupense 4 4 Mycobacterium aubagnense 1 1 Mycobacterium aurum Mycobacterium avium complex Mycobacterium brumae Mycobacterium canariasense 1 1 Mycobacterium chelonae 3 3 Mycobacterium chlorophenolicum Mycobacterium confluentis Mycobacterium cosmeticum Mycobacterium duvalii Mycobacterium fallax Mycobacterium fluoranthenivorans Mycobacterium fortuitum Mycobacterium frederiksbergense Mycobacterium hodleri Mycobacterium holsaticum Mycobacterium mageritense 2 2 Mycobacterium moriokaense Mycobacterium novocastrense 1 1 Mycobacterium parafortuitum Mycobacterium peregrinum Mycobacterium poriferae Mycobacterium psychrotolerans 7 7 Mycobacterium pyrenivorans Mycobacterium rhodesiae Mycobacterium smegmatis Mycobacterium sp Mycobacterium tokaiense Mycobacterium tusciae Mycobacterium vanbaalenii Total

53 454 Deep sequencing Data: Mycobacterium genus specific 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Cryfield Diversity snapshot: distribution of clusters Similarity cut off of 97% similarity β diversity: The numbers in parenthesis next to the sample type refer to the number of representative sequences/otu in each sample

54 454 Deep sequencing Data Slow growing Mycobacteria specific primers Species Soil Soil Sample Type Soil Soil Soil 5 Cryfield Total Mycobacterium asiaticum Mycobacterium avium complex Mycobacterium bohemicum Mycobacterium conspicuum 1 Mycobacterium gastri Mycobacterium gordonae Mycobacterium lacus Mycobacterium marinum 37 8 Mycobacterium moriokaense 1 Mycobacterium nebraskense Mycobacterium pseudoshottsii 1 Mycobacterium sp Mycobacterium szulgai Mycobacterium tuberculosis complex Total

55 454 Deep sequencing Data Slow growing Mycobacteria specific primers

56 454 Deep sequencing Data Slow growing Mycobacteria specific primers

57 Environmental Stratification It is of interest to sample areas which are very different from each other in terms of 1)Climate/Season 2)Rainfall/Humidity 3)Altitude 4)Land Use To see how this effects the diversity and prevalence of Mycobacteria.

58 Environmental Stratification Sampling Methods A total of 40 villages 200 soil sample sites 80 water sample sites Equipment GPS SM200 Moisture meter Soilstick ph and temperature meter ST5 Tensiometer

59 Potential Sites Addis Ababa Butajira Jimma Jinka Hosiana Sellae Woldiya Future work To measure human exposure IFN y responses to EM PPDs