Mycobacterium avium subsp. paratuberculosis in the catchment area and water of the River Taff in South Wales, United Kingdom, and its potential relationship to clustering of Crohn's disease cases in the city of Cardiff

Abstract

In South Wales, United Kingdom, a populated coastal region lies beneath hill pastures grazed by livestock in which Mycobacterium avium subsp. paratuberculosis is endemic. The Taff is a spate river running off the hills and through the principal city of Cardiff. We sampled Taff water above Cardiff twice weekly from November 2001 to November 2002. M. avium subsp. paratuberculosis was detected by IS900 PCR and culture. Thirty-one of 96 daily samples (32.3%) were IS900 PCR positive, and 12 grew M. avium subsp. paratuberculosis bovine strains. Amplicon sequences from colonies were identical to the sequence with GenBank accession no. X16293, whereas 16 of 19 sequences from river water DNA extracts had a single-nucleotide polymorphism at position 214. This is consistent with a different strain of M. avium subsp. paratuberculosis in the river, which is unculturable by the methods we used. Parallel studies showed that M. avium subsp. paratuberculosis remained culturable in lake water microcosms for 632 days and persisted to 841 days. Of four reservoirs controlling the catchment area of the Taff, M. avium subsp. paratuberculosis was present in surface sediments from three and in sediment cores from two, consistent with deposition over at least 50 years. Previous epidemiological research in Cardiff demonstrated a highly significant increase of Crohn's 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 southwesterly winds. This would influence the distribution of aerosols carrying M. avium subsp. paratuberculosis from the river.

FIG. 1.

Location of the study region in South Wales, United Kingdom. The enlargement shows the catchment area (enclosed by the dashed line) of the river Taff, which at the time of the study contained 30,435 beef and dairy cattle and 571,429 sheep, of which 304,443 were breeding ewes (see text). The river Taff itself is a spate river which runs down from the Brecon hills and through the city of Cardiff before entering the Bristol Channel. The prevailing winds are from the Atlantic to the southwest. 1, Upper Neuadd reservoir; 2, Pontsticill reservoir; 3, Taff Fechan River; 4, Brecons reservoir; 5, Llwyn-on reservoir; 6, Taff Faw River; 7, river Taff. MT, town of Merthyr Tydfil; C, city of Cardiff. *, sampling sites.

FIG. 2.

Relationship between presence or absence of M. avium subsp. paratuberculosis and flow and river height of the river Taff (November 2001 to November 2002). Data were obtained by using the Hydrolog data management system, version 2.61 (Hydro-Logic Ltd., Bromyard, United Kingdom), from Pontypridd logging station (grid reference ST07938973). Plotted data were recorded at 9:00 a.m. each day. The solid line represents the river height, while the dotted line represents the flow rate. Sample points are shown on the river height graph as grey diamonds, and samples that were PCR positive for M. avium subsp. paratuberculosis are shown as black diamonds.

FIG. 3.

Culture of M. avium subsp. paratuberculosis from decontaminated samples of Taff river water and catchment area sediment. Growth was first seen after 8 to 11 months of incubation on Herrold's egg yolk medium. (A and B) Typical crumbly colonies. (C, D, and E) Early colonies forming below the surface of the solid HEYM. All colonies were confirmed as M. avium subsp. paratuberculosis by IS900 PCR with amplicon sequencing and by MIRU typing.

FIG. 4.

Products obtained by amplification at MIRU locus 1 (upper panel), MIRU locus 2 (middle panel), and MIRU locus 4 (lower panel) of M. avium subsp. paratuberculosis isolates from reservoir sediment and Taff river water. Lanes: 1, 100-bp ladder; 2, isolate from sediment from the Upper Neuadd reservoir; 3 to 10, isolates from Taff river water; 11, contemporary isolate of a bovine strain of M. avium subsp. paratuberculosis from a cow with Johne's disease in the study region; 12, M. avium subsp. avium ATCC25291; 13, Mycobacterium sp. strain 2333 from Sweden, containing a single copy of an IS900-related element (25); 14, M. chelonae; 15, M. scrofulaceum; 16 to 18, three Mycobacterium strains with IS900-related elements from Australia (20); 19, negative control; 20, 100-bp ladder.

FIG. 5.

Persistence of M. avium subsp. paratuberculosis in sterile lake water microcosms as determined by plating, real-time PCR, and corresponding growth in MGIT tubes and PCR. (A) Persistence (expressed as CFU milliliter⁻¹) of M. avium subsp. paratuberculosis in sterile lake water microcosms A (▾), B (○), and C (•) and estimated count by real-time PCR in microcosm C (▿) over 400 days. Results for microcosm D (saline inoculated control) are not shown. Error bars indicate standard deviations. (B) Extended study of persistence of M. avium subsp. paratuberculosis in sterile freshwater microcosms A to D by using MGIT culture and PCR over 841 days (a further 434 days after the last sample was taken for culture). + and −, presence and absence of M. avium subsp. paratuberculosis culture or PCR signal, respectively; ND, not determined.

FIG. 6.

Relationship between disease clusters and prevailing wind in Cardiff, Wales, United Kingdom. (A) Distribution of the 11 electoral wards in the city of Cardiff that were shown previously (52, 53) to have a highly significant (P < 0.001) increase in the incidence of Crohn's disease. (B) Relief map of Cardiff, with the course of the river Taff through the city indicated in blue. The wards with the high incidence of Crohn's disease are seen to lie along the Taff, with the exception of a gap in the center stretch of the windward right bank of the river (facing downstream). This gap directly faces a valley between hills to the north and south, which is open to the prevailing southwesterly winds. (Map reproduced with permission from the Ordnance Survey, Crown copyright NC/2004/29668.)