Research interests

1
Research interests

• Freshwater microbial ecology
• Community structure/biogeochemical cycling
• Natural attenuation in deep aquifers
• Distribution of antibiotic resistance, genetic
  elements (plasmids, transposons, IS elements)
• Survival and behaviour of bacterial pathogens in
  large catchments

2
Bacterial pathogens

• Survival of pathogens in freshwater and deep
  sediments (Windermere/coliforms)
• culture 37 years/Molecular signal 6000 years
• Source tracking E. coli in river Tamar
• Distinguish human from ovine/bovine from birds
• Distribution of
• antibiotic resistance in lakes and upland tarns
• Tetr determinants and plasmids in fish farm and
  hospital environments across Europe
• Mycobacterium avium subspecies paratuberculosis
  in large catchments

3
Today

• Background on bacterial pathogens in the
  environment
• Example of a animal pathogen with a potential
  human link
• Using it as an example of how bacterial pathogen
  can be studied in large scale catchments

4
Why are bacterial pathogens difficult to study?

• Variable numbers
• Sampling issues (sample heterogeneity)
• Natural environments are invariably nutrient
  limited
• Populations of micro-organisms are largely black
  boxes
• Source tracking/identification
• Culturability

5
The culturability problem

• Cfu underestimate numbers of prokaryotes - only
  0.1 to 10 organisms present can be cultured
• Viable but non-culturable cells (mainly
  pathogens)
• Possible reasons
• Substrate accelerated death
• Oxidative killing
• Laboratory media are selective
• In vitro conditions different to in vivo
• Culture methods detect microbial weeds?

6
From yesterday

• Noted that the environment is a transport
  mechanism for disease
• from farm to fork
• from crap to tap (this presentation)
• Noted that disease places an economic burden
  (e.g. on NHS)

7
Mycobacterium avium subspecies paratuberculosis
(MAP) and its distribution in catchments
MAP causes Johnes Disease MAP is excreted into
the environment by diseased animals in very large
numbers MAP implicated as cause of Crohns
Disease and IBS (humans) - controversial
8
Broad species pathogenicity of MAP

• Domestic wild ruminants
• Deer, Alpaca, Horses
• Pigs Dogs
• Rabbits their predators
• Chickens carrion birds
• Primates (recognised in 4 out of 5.but not
  humans)

9
Link between Map and Crohns disease

• No consensus on aetiological link
• MAP reported in Crohns tissue- 0 to 93
• Johnes disease incidence increasing in the UK
• CD rising (particularly in children
  Europe-wide)
• RECENT DEFRA STUDY NO LINK

10
DEFRA study interpretation

• Two alternative explanations
• No causal link
• Causal link but study design unable to detect
  effect
• Possible reasons
• MAP ubiquitous in the environment
• other exposure routes not taken into account
• Direct contact with animals
• Milk and dairy products
• Water (river, potable, aerosols)

11
Live MAP in retail pasteurised milk

• Milk and dairy products

• Irish Republic - IS900 PCR 9.8 culture no
• OReilly et al. Appl Env Microbiol
  2004705138-44
• USA- culture 2.8
  yes
• Ellingson et al. J Food Protect 2005 in press
• Czech Republic - culture 2
  yes
• Ayele et al. Appl Env Microbiol 2005711210-14
• Argentina - culture 5.7
  yes
• Cirone et al. abstr 2004

12

• Water (river, potable, aerosols)

Environmental studies on waterborne transmission
of Mycobacterium avium subspecies
paratuberculosis (MAP) from animals to humans in
South Wales
Centre for Ecology and Hydrology St
Georges Hospital Medical School
13
Basis of the project

• MAP is implicated in Crohns disease
• We can define some of the environmental sources
  of MAP
• Infection may have an environmental route
• Bacterial pathogens survive in the environment
• Possible to track difficult-to- culture MAP in
  the environment using PCR (IS900) and culture

14
Hypotheses

• MAP is present in the rivers Taff and Towy in
  planktonic form and within biofilm communities
  and protozoa
• MAP is present in the holding reservoir for
  domestic water supplies abstracted from the Towy
• The profile of MAP detection in sedimentary cores
  will demonstrate co-emergence with
  industrialisation and intensive farming

15
Study areas

• River Taff and catchment (South Wales, UK)
• River Towy and catchment (South Wales, UK)
• Several Lake District catchments (N. England, UK)
• Windermere catchment (in detail)

16
Catchment 1

• River Taff, South Wales, UK

17
R. Taff catchment

• The Taff is a spate river running off the hills
  and through the principal city of Cardiff
• Cardiff
• a highly populated coastal region
• lies beneath hill pastures
• grazed by livestock
• Mycobacterium avium subspecies paratuberculosis
  is endemic
• published distribution of CD patients
  (association with river)
• Sampled
• Taff water above Cardiff twice weekly from
  November 2001 to November 2002 (urban area)
• higher catchment and reservoirs (bottom sediment
  and cores)

18
River Taff and catchment
Applied and Environmental Microbiology (2005) 71
2130-2139
19
Sampling water
20
Interpretation of PCR data

• Positive PCR
• Unequivocally IS900
• Product identified
• by size then sequencing and defined against IS900
  sequence
• Negative PCR
• Not necessarily negative
• Inhibition of PCR (humic acid etc)
• Patchiness of sampling
• PCR may underestimate the presence of MAP in the
  environment

21
Presence of IS900 in R. Taff from Nov 01 to Nov
02
22
Sediment (upper Taff catchment)
23
Vertical of MAP distribution in sediments

• Several cores showed MAP below the surface
  showing that it has a historical deposition
  record
• Pontiscillin- gt20 years
• Llwyn-on - gt 40 years

30 cm
24
Distribution of Crohns patients in Cardiff
(Wales) associated with river MAP present in R.
Taff consistently (32 samples in 1
year) Distributed throughout the
catchment historically deposited
(gt40years) Presence significantly associated
with rainfall Presence could not be predicted
from rainfall, river flow or river height (not
significant but close) Parallel studies showed
that MAP culturable in lake water microcosms
for 632 days and detectable gt841 days
25
R. Taff study

• Previous epidemiological research in Cardiff
  demonstrated a highly significant increase of
  Crohns disease in 11 districts
• These bordered the river except for a gap on the
  windward side
• A topographical relief map shows that this gap is
  directly opposite a valley open to the prevailing
  south-westerly winds
• This would influence the distribution of aerosols
  carrying M. avium subspecies paratuberculosis
  from the river

26
(No Transcript)
27
Catchment 2

• River Towy, South Wales, UK

28
River Towy catchment

• The Towy is a spate river running off the hills
  and through Carmarthen
• Towy provides water for abstraction and public
  supply
• Catchment
• comprises hill pastures, grazed by livestock
• MAP is endemic
• Sampled
• Towy water above Nantgaredig twice weekly from
  August 2002 to April 2003 (rural area)
• Higher catchment and reservoirs (bottom sediment
  and cores)

29
River Towy and catchment
30
R. Towy/water distribution river (site 9 twice
weekly sampling)
?
31
(No Transcript)
32
R. Towy study

• River Towy (S. Wales, UK) showed
• Of the 70 sample dates (all data included),
  IS900 was present on 48 occasions (69 cf Taff
  32)
• A significant association between MAP and river
  hydrography
• Its presence was entirely predictable based on
  rainfall or river flow data

33
R. Towy study

• River height/flow higher average heights and
  flows were significantly associated with the
  presence of IS900 compared to lower values when
  IS900 was absent
• Using a threshold flow of 3.4 m3s-1, river flow
  in the Towy could predict the presence of IS900
  (cf Cardiff)
• Presence of IS900 (MAP) is associated with rain
  falling up to 7 days before sample date

Applied and Environmental Microbiology 72, 4067-
4077
34
R. Towy study

• Clustering in time (significant at the 1 level)
  was demonstrated
• Furthermore, clustering showed MAP was present
  continuously as prolonged pulses (sometimes
  several weeks) influenced by rainfall up to 8
  days prior to sampling
• The presence of MAP is predictable in the River
  Towy

Applied and Environmental Microbiology 72, 40677-
4077
35
R. Towy water abstraction/purification (Cocodaf)
plant
36
R. Towy/water distribution/abstraction point
?
37
Pipeline (27km)
?
11
12b?
12a
12c
13 ?
12d?
38
Pipeline (27km)
11
Towy Pumping station
12b?
?
12a
12c
13 ?
12d?
39
Pipeline (27km)
11
Towy Pumping station
12b?
12a
?
12c
13 ?
12d?
40
(No Transcript)
41
Catchment 3

• English Lake District, Cumbria, UK

42
English Lake District 14 major lakes gt500 tarns
43
Applied and Environmental Microbiology
(submitted)
44
Lake District

• MAP was shown to be present
• in the tarns and lakes in the high and low fells
  
• in quaternary treated sewage effluent discharged
  into rivers
• within river and lake sediments (core data),
  deposited continuously since 1950 or before
  (possible)

Applied and Environmental Microbiology
(submitted)
45
Summary MAP in the freshwater catchments

• MAP is widely distributed in 3 large catchment
  areas
• Presence in rivers influenced by hydrography and
  rainfall
• Possible exit via aerosols
• High survival potential in freshwater
• (gt3 years in lab system)
• Historical deposition record in sediments
• Reduced numbers in water treatment but not
  eliminated
• found in water tanks supplied with
  potable/treated water

46
Summary MAP in the freshwater catchments

• Implied association with sediment
• requires sediment attachment for persistence/not
  found in non-sediment areas
• Biofilm association
• found in environmental biofilm (internal
  association not determined)
• long-term association with Acanthamoeba polyphage
• Mura, Bull, Evans, Sidi-Boumedine, McMinn,
  Rhodes, Pickup, Hermon-Taylor. AEM submitted

47
(No Transcript)
48
Countryside Survey of Great Britain

• Major audit of the British countryside
• Complete land cover census for Great Britain and
  Northern Ireland
• Partnership between NERC and Government
  Departments
• CS2007 8M over 4 years
• moving to continuous programme?

49

• CS2007
• Habitats, landscape features, land use
• Plant community structure
• Freshwater biology
• Soils chemical, physical and biological
• Land Cover Map
• Integrated Ecosystem Assessments

50
CS field survey
Mapping habitats and landscape features in each
1km square
Surveying/sampling of vegetation, birds,
freshwater biota, soils and soil biodiversity
51
CS field survey

• 1 km squares across GB
• wide range of survey sampling plots

52
CS soil sampling
Inner 2 m2 quadrat of 200 m2 X-plot

• 5 X plots in each 1 km square
• 1978 and CS2000 256 squares ? 1256 samples
• CS2007 max. 600 1 km squares ? 3000 samples

53

• New Methods for Looking at Change
• CS2007 and Molecular Ecology
• first country-level survey of microbial diversity
  in terrestrial ecosystems and bacterial pathogen
  distribution (1300 samples)
• establish baseline measurements for future
  surveys
• UK wide genomic archive of our microbial
  biodiversity
• key drivers of change in microbial communities

high density microarray technologies to assess
multiple taxa and relationships to soil quality
(ca 650 determinative probes to most major
bacterial soil taxa)
Soil cores
54
Isolates cultured from R. Taff
Colonies Amplicon sequences from colonies were
identical to GenBank X16293 (bovine MAP) Water
(PCR amplicon) 16 of 19 from river water DNA
extract had a single nucleotide polymorphism at
position 214 This is consistent with a different
strain of M. avium subspecies paratuberculosis in
the river, unculturable by the methods we
used----now associated with ovine sources
55
River Taff
Fig1. Distribution of MAP isolates and
corresponding RFLP types.
RFLP-type A
RFLP-type D
RFLP-type B-C1
RFLP-type B
RFLP-type E
RFLP-type B-C13
RFLP-type B-C17
RFLP-type C