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.2009 Mar;75(5):1417-26.
doi: 10.1128/AEM.02346-08. Epub 2009 Jan 5.

Diversity and habitat specificity of free-living protozoa in commercial poultry houses

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Diversity and habitat specificity of free-living protozoa in commercial poultry houses

Julie Baré et al. Appl Environ Microbiol.2009 Mar.

Abstract

Despite stringent biosecurity measures, infections by bacterial food pathogens such as Campylobacter are a recurrent problem in industrial poultry houses. As the main transmission route remains unclear, persistence of these infections has been linked to bacterial survival and possibly multiplication within protozoan vectors. To date, however, virtually no information is available on the diversity and occurrence of free-living protozoa in these environments. Using a combination of microscopic analyses of enrichment cultures and molecular methods (denaturing gradient gel electrophoresis [DGGE]) on natural samples, we show that, despite strict hygiene management, free-living protozoa are common and widespread throughout a 6-week rearing period in both water and dry samples from commercial poultry houses. Protozoan communities were highly diverse (over 90 morphotaxa and 22 unique phylotypes from sequenced bands) and included several facultative pathogens and known bacterial vectors. Water samples were consistently more diverse than dry ones and harbored different communities, mainly dominated by flagellates. The morphology-based and molecular methods yielded markedly different results: amoebic and, to a lesser degree, ciliate diversity was seriously underestimated in the DGGE analyses, while some flagellate groups were not found in the microscopic analyses. Some recommendations for improving biosecurity measures in commercial poultry houses are suggested.

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Figures

FIG. 1.
FIG. 1.
Sampling strategy. Arrows indicate the three sampling phases. The bold line corresponds to the period when decontamination and disinfection methods were applied.
FIG. 2.
FIG. 2.
Pie chart showing the relative proportion of hetero- and mixotrophic eukaryotic taxonomic groups as determined by morphology (A) and sequencing of excised DGGE bands (only unique sequences were taken into account) (B).
FIG. 3.
FIG. 3.
Variability in species richness within a farm during the three phases determined by average microhabitat and total habitat protozoan diversity. Diversity was assessed on the basis of morphology per broiler house (shown for the three main protozoan morphogroups: ciliates, flagellates, and amoebae) (A) and genetic profiling (B). *, no samples could be taken; 0, no protozoa were detected. The different broiler houses at any one farm are designated X1 to X4, and X1+X2 stands for samples from sources common to the indicated broiler houses (e.g., water from the well and litter and food from the supply). Error bars indicate the standard deviations.
FIG. 4.
FIG. 4.
DCA ordination plots (axes 1 and 2) summarizing differences and changes in eukaryotic microbial community composition at the three farms, based on morphology (samples in panel A and morphotaxa in panel B) and DGGE (samples in panel C and phylotypes in panel D). In plots A and C, water samples and dry samples are indicated by closed and open circles, respectively, and the sources of the samples are indicated by green, red, and blue for farms A, B, and C, respectively. For taxon abbreviations (plot B) and phylotype numbers (plot D), see Table S1 and Table S2, respectively, in the supplemental material. Ciliates, flagellates, and amoebae are represented in green, red, and black, respectively. In panel D, fungi are indicated in blue, diatoms are in purple, plantae are in turquoise, and Apicomplexa are in pink.
FIG. 5.
FIG. 5.
Dendrogram generated by an UPGMA cluster analysis comparison of DGGE patterns from direct amplification of samples collected from three farms. In the sample code, A, B, and C refer to the farms, and the numbers 1, 2, and 3 immediately following indicate the sampling phases. W and D refer to water or dry habitat, respectively, while X1, X2, X3, and X4 represent the different broiler houses at one farm. Similarity is expressed as a percentage value of the Dice correlation coefficient. The numbers refer to the excised bands (see Table S2 in the supplemental material).
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