Article Figures & Data
Figures
- FIG. 1.
Flow diagram of drinking water systems in the broiler houses of intensively reared poultry. The broilers drink the water, obtained from main water supplies, at the nipples.
- FIG. 2.
(A to C) Detection and analysis of C. jejuni DNA from the drinking water and feces/bedding and of isolates from cloacal swabs from five broiler farms. The primer system was based on that of the Winters et al. (78) seminested primer system for the Cj0343c gene in C. jejuni, with positive detection resulting in the presence of 122-bp amplicons. (A) Agarose gel (1.5%) of C. jejuni PCR amplicons detected from the drinking water and feces/bedding and C. jejuni isolated from cloacal swabs from five broiler farms. Positive results are indicated by 122-bp amplicons. (Block A) Lanes: 1, 100-bp DNA ladder; 2, negative control (no DNA); 3, positive control (C. jejuni NCTC 11351); 4, feces/bedding (farm 4); 5, nipples; 6, end of line; 7, tube to line; 8, tank water; 9, stop-cock water (in tank); 10, feces/bedding (farm 5); 11, nipples; 12, end of line; 13, tube to line; 14, 100-bp DNA ladder. (Block B) Lanes: 1, 100-bp DNA ladder; 2, tank water; 3, stop-cock water (in tank); 4, 100-bp DNA ladder; 5, swab (farm 1); 6, swab (farm 4); 7, 5 swab (farm 5); 8, 100-bp DNA ladder. (B) Phylogenetic tree of C. jejuni PCR amplicons detected from the drinking water, feces/bedding, and C. jejuni isolated from cloacal swabs from five broiler farms. The tree was constructed by using the neighbor-joining method based on all nucleotide sites, with corrections for multiple substitutions by the Jukes-Cantor method in MEGA version 2.1. The letter “S” represents cloacal swab isolates, and the percent values indicate the percent similarity to gene Cj0343c, coordinates 66984 to 67087, in the C. jejuni subsp. jejuni NCTC 11168 genome. (C) EMBL-EBI CLUSTAL W alignment of 13 DNA sequences from the Cj0343c gene in C. jejuni; 10 of the sequences were detected from broiler drinking water and feces/bedding, and 3 of the sequences were amplified from C. jejuni isolated from cloacal swabs, again represented by the letter “S.” The asterisk represents conserved sequences in all C. jejuni seminested PCR amplicons.
- FIG. 3.
(A and B) Microscopy of T. pyriformis (CCAP 1630/14A) after 24 h of coculture with C. jejuni NCTC 11351 in PAS at 25°C. C. jejuni was stained with Baclight viability dye before coculture (1:1) with T. pyriformis. Magnification, ×100. The arrows indicate T. pyriformis vacuoles containing viable and intact C. jejuni. (A) Bright-field image of an intact T. pyriformis cell cocultured with C. jejuni. (B) Fluorescent image of viable (green) C. jejuni inside T. pyriformis.
- FIG. 4.
(A and B) Microscopy of A. castellanii (CCAP 1501/10) after 3 days of coculture with C. jejuni NCTC 11351 in PAS at 25°C. C. jejuni was stained with Baclight viability dye before coculture (1:1) with A. castellanii. magnification, ×40. The arrows indicate A. castellanii vacuoles containing dead C. jejuni. (A) Bright-field image of an intact A. castellanii cell cocultured with C. jejuni. (B) Fluorescent image of dead (red) C. jejuni inside A. castellanii.
- FIG. 5.
(A through C) Microscopy of T. pyriformis (CCAP 1630/14A) after 3 h of coculture with C. jejuni NCTC 11351 at 25°C. C. jejuni was stained with FITC-labeled rabbit antibody before coculture (1:1) with T. pyriformis. magnification, ×100. (A) Bright-field image of an intact T. pyriformis cell cocultured with C. jejuni. (B) Fluorescent image of C. jejuni inside T. pyriformis. The arrow indicates T. pyriformis vacuoles containing C. jejuni. (C) Intact T. pyriformis stained with DAPI after 3 h. DNA (especially in the nucleus) was stained blue with DAPI, and the arrow indicates a T. pyriformis nucleus. C. jejuni, also stained blue, can be seen outside around the intact T. pyriformis cell.
- FIG. 6.
(A through C) Survival of Campylobacter jejuni NCTC 11351, C. coli NCTC 11366, and C. jejuni subsp. jejuni (poultry isolate) when cocultured with A. castellanii (CCAP 1501/10) and T. pyriformis (CCAP 1630/14A). Cocultures (1:1 ratio of Campylobacter to protozoa) and planktonic strains of Campylobacter were prepared in PAS solution and incubated at 25°C for up to 10 days, when viable Campylobacter were no longer obtained. The results are presented as average (performed in quadruplicate) viable Campylobacter recovered per ml from PAS, and error bars indicate the standard deviations. (A) Viablecounts of recovered Campylobacter during the first 24 h of incubation from cocultures with A. castellanii and T. pyriformis. (B) Viable counts of recovered Campylobacter during 10 days of incubation from cocultures containing T. pyriformis. (C) Viable counts of recovered Campylobacter during 10 days of incubation from cocultures containing A. castellanii.
- FIG. 7.
Effect of coculture time on the survival of C. jejuni NCTC 11351 during disinfection. Cocultures with C. jejuni and A. castellanii (CCAP 1501/10) and T. pyriformis (CCAP 1630/14A) (1:1 ratio of Campylobacter to protozoa) were prepared in PAS solution and incubated for up to 24 h at 25°C. After 3, 6, 9, 12, and 24 h, a 1:1,000 dilution of Virudine (1-min contact time) was used to kill planktonic C. jejuni, followed by neutralization with STS, gravity filtration (0.8 μm), rinsing and resuspension in PAS, sonication (10 s at 40 W), and viable Campylobacter counts. The results are presented as average (performed in quadruplicate) viable Campylobacter recovered per ml of PAS, and error bars indicate the standard deviations.
Tables
- TABLE 1.
Oligonucleotide primers used in this study
Primer Sequence (5′-3′) Annealing position Reference WIN1 AAA TAA AGT TAG AGG TAG AAT GT C. jejuni NCTC 11168, coordinates 66984 to 67006 in genome; Cj0343c gene 78 WIN2 GGA TAA GCA CTA GCT AGC TGA T C. jejuni NCTC 11168, coordinates 67140 to 67121 in genome; Cj0343c gene 78 WIN3 GCA CGC CTA AAC CTA TAG C C. jejuni NCTC 11168, coordinates 67105 to 67087 in genome; Cj0343c gene 78 EIN1 ATT GGA GGG CAA GTC TGG TG T. pyriformis, coordinates 537 to 557 in small subunit of rRNA 18 EIN2 CCG ATC CCT AGT CGG AT AG T. pyriformis, coordinates 998 to 1024 in small subunit of rRNA 18 - TABLE 2.
Library of C. jejuni clones with sample collection data
Clone name Sample data GenBank no. 1A Farm 1, feces/bedding AY830861 1B Farm 1, feces/bedding AY830862 2A Farm 1, tank water AY830863 2B Farm 1, tank water AY830864 3A Farm 4, feces/bedding AY830865 3B Farm 4, feces/bedding AY830866 4A Farm 4, end of line AY830867 4B Farm 4, end of line AY830868 5A Farm 4, tube to line AY830869 5B Farm 4, tube to line AY830870 6A Farm 4, tank water AY830871 6B Farm 4, tank water AY830872 7A Farm 5, feces/bedding AY830873 7B Farm 5, feces/bedding AY830874 8A Farm 5, end of line AY830875 8B Farm 5, end of line AY830876 9A Farm 5, tube to line AY830877 9B Farm 5, tube to line AY830878 10A Farm 5, stop-cock water (in tank) AY830879 10B Farm 5, stop-cock water (in tank) AY830880 11S Farm 1, cloacal swab AY830881 12S Farm 4, cloacal swab AY830882 13S Farm 5, cloacal swab AY830883 - TABLE 3.
Library of eukaryotic clones with sample collection data
Clone Sample data GenBank no. Most closely related microbe, % similarity Clone Sample data GenBank no. Most closely related microbe, % similarity 1A Farm 1, external tap AY837467 Trichosporon montevideense, 99% small subunit rRNA 1B Farm 1, external tap AY837468 Trichosporon montevideense, 99% small subunit rRNA 1C Farm 1, external tap AY837469 Trichosporon montevideense, 97% small subunit rRNA 2A Farm 4, external tap AY837470 Phialocephala fortinii, 91% small subunit rRNA 2B Farm 4, external tap AY837471 Botryotinia fuckeliana, 18S 99% rRNA 2C Farm 4, external tap AY837472 Botryotinia fuckeliana, 99% 18S rRNA 3A Farm 5, external tap AY837473 Scopulariopsis brevicaulis, 99% 18S rRNA 3B Farm 5, external tap AY837474 Paecilomyces lilacinus, 98% 18S rRNA 3C Farm 5, external tap AY837475 Debaryomyces hansenii var. fabryi, 98% 18S rRNA 4A Farm 1, tank AY837476 Paraurostyla weissei 17S, 90% rRNA gene, 4B Farm 1, tank AY837477 Uncultured alveolate clone LEMD251, 93% small subunit rRNA 4C Farm 1, tank AY837478 Trichosporon montevideense, 99% small subunit rRNA 5A Farm 2, tank AY837479 Paraphysomonas butcheri, 96% 18S rRNA 5B Farm 2, tank AY837480 Eurotium herbariorum, 98% 18S rRNA gene 5C Farm 2, tank AY837481 Eurotium herbariorum, 97% 18S rRNA gene 6A Farm 3, tank AY837482 Hydnum rufescens 18S, 96% rRNA gene 6B Farm 3, tank AY837483 Spumella oblique, 96% 18S rDNA gene 6C Farm 3, tank AY837484 Scopulariopsis brevicaulis, 97% 18S rRNA gene, 7A Farm 4, tank AY837485 Phialophora verrucosa, 99% 18S rRNA 7B Farm 4, tank AY837486 Phialophora verrucosa, 98% 18S rRNA 7C Farm 4, tank AY837487 Unidentified eukaryote clone LKM67, 97% 18S rRNA 8A Farm 5, tank AY837488 Scopulariopsis brevicaulis, 97% 18S rRNA gene 8B Farm 5, tank AY837489 Uncultured alveolate clone LEMD251, 95% small subunit rRNA 8C Farm 5, tank AY837490 Uncultured alveolate clone LEMD251, 95% small subunit rRNA 9A Farm 1, nipples AY837491 Trichosporon montevideense, 98% small subunit rRNA 9B Farm 1, nipples AY837492 Aspidisca steini, 92% small subunit rRNA 10A Farm 2, nipples AY837493 Eurotium herbariorum, 99% 18S rRNA gene 10B Farm 2, nipples AY837494 Gibberella pulicaris, 99% 18S rRNA 10B Farm 2, nipples AY837494 Gibberella pulicaris, 99% 18S rRNA 11A Farm 3, nipples AY837495 Uncultured alveolate clone LEMD251, 94% small subunit rRNA 12A Farm 4, nipples AY837497 Capronia coronata, 97% 18S rRNA 12B Farm 4, nipples AY837498 Penicillium italicum, 94% 18S rRNA 13A Farm 5, nipples AY837499 Cladosporium cladosporioides, 99% 18S rRNA 13B Farm 5, nipples AY837500 Gibberella pulicaris, 96% 18S rRNA - TABLE 4.
Effect of age of protozoa during coculture on Campylobacter disinfection resistancea
Protozoan age (days) Location Avg Campylobacter count (CFU/ml) ± SD C. coli NCTC 11366 C. jejuni NCTC 11351 C. jejuni (poultry isolate) T. pyriformis 3 Internal (3.52 × 103) ± 285 (4.28 × 103) ± 730 (5.77 × 103) ± 999 3 External 0 ± 0 132 ± 18 313 ± 22 6 Internal (11.21 × 102) ± 134 (3.25 × 103) ± 164 (4.56 × 103) ± 644 6 External 5 ± 3 16 ± 10 33 ± 10 9 Internal 0 ± 0 0 ± 0 0 ± 0 9 External 0 ± 0 0 ± 0 0 ± 0 A. castellanii 3 Internal 269 ± 33 326 ± 32 375 ± 16 3 External 1 ± 1 3 ± 1 5 ± 1 6 Internal 113 ± 12 154 ± 24 172 ± 20 6 External 0 ± 0 0 ± 0 0 ± 0 9 Internal 0 ± 0 0 ± 0 0 ± 0 9 External 0 ± 0 0 ± 0 0 ± 0 ↵a T. pyriformis (CCAP 1630/14A) and A. castellanii (CCAP 1501/10) were each grown for 3, 6, and 9 days before being cocultured for 3 h at 25°C (1:1 ratio) with Campylobacter spp. in PAS solution. A 1:1,000 dilution of Virudine (1-min contact time) was then used to kill planktonic C. jejuni, followed by neutralization using STS, gravity filtration (0.8-μm pore size), rinsing and resuspension in PAS, sonication (10 s at 40 W), and counts of viable Campylobacter. Results are presented as the average (performed in quadruplicate) numbers of viable Campylobacter recovered ml of PAS.
