Microscopic and molecular studies of the diversity of free-living protozoa in meat-cutting plants.
ABSTRACT: The diversity of free-living protozoa in five meat-cutting plants was determined. Light microscopy after enrichment culturing was combined with sequencing of PCR-amplified, denaturing gradient gel electrophoresis (DGGE)-separated 18S rRNA gene fragments, which was used as a fast screening method. The general results of the survey showed that a protozoan community of amoebae, ciliates, and flagellates was present in all of the plants. Protozoa were detected mainly in floor drains, in standing water on the floor, on soiled bars of cutting tables, on plastic pallets, and in out-of-use hot water knife sanitizers, but they were also detected on surfaces which come into direct contact with meat, such as conveyer belts, working surfaces of cutting tables, and needles of a meat tenderizer. After 7 days of incubation at refrigerator temperature, protozoa were detected in about one-half of the enrichment cultures. Based on microscopic observations, 61 morphospecies were found, and Bodo saltans, Bodo spp., Epistylis spp., Glaucoma scintillans, Petalomonas spp., Prodiscophrya collini, and Vannella sp. were the most frequently encountered identified organisms. Sequencing of DGGE bands resulted in identification of a total of 49 phylotypes, including representatives of the Amoebozoa, Chromalveolata, Excavata, Opisthokonta, and Rhizaria. Sequences of small heterotrophic flagellates were affiliated mainly with the Alveolata (Apicomplexa), Stramenopiles (Chrysophyceae), and Rhizaria (Cercozoa). This survey showed that there is high protozoan species richness in meat-cutting plants and that the species included species related to known hosts of food-borne pathogens.
Project description:The influence of grazing by a mixed assemblage of soil protozoa (seven flagellates and one amoeba) on bacterial community structure was studied in soil microcosms amended with a particulate resource (sterile wheat roots) or a soluble resource (a solution of various organic compounds). Sterilized soil was reinoculated with mixed soil bacteria (obtained by filtering and dilution) or with bacteria and protozoa. Denaturing gradient gel electrophoresis (DGGE) of PCR amplifications of 16S rRNA gene fragments, as well as community level physiological profiling (Biolog plates), suggested that the mixed protozoan community had significant effects on the bacterial community structure. Excising and sequencing of bands from the DGGE gels indicated that high-G+C gram-positive bacteria closely related to Arthrobacter spp. were favored by grazing, whereas the excised bands that decreased in intensity were related to gram-negative bacteria. The percentages of intensity found in bands related to high G+C gram positives increased from 4.5 and 12.6% in the ungrazed microcosms amended with roots and nutrient solution, respectively, to 19.3 and 32.9% in the grazed microcosms. Protozoa reduced the average bacterial cell size in microcosms amended with nutrient solution but not in the treatment amended with roots. Hence, size-selective feeding may explain some but not all of the changes in bacterial community structure. Five different protozoan isolates (Acanthamoeba sp., two species of Cercomonas, Thaumatomonas sp., and Spumella sp.) had different effects on the bacterial communities. This suggests that the composition of protozoan communities is important for the effect of protozoan grazing on bacterial communities.
Project description:The effects of protozoa (heterotrophic flagellates and ciliates) on the morphology and community composition of bacterial biofilms were tested under natural background conditions by applying size fractionation in a river bypass system. Confocal laser scanning microscopy (CLSM) was used to monitor the morphological structure of the biofilm, and fingerprinting methods (single-stranded conformation polymorphism [SSCP] and denaturing gradient gel electrophoresis [DGGE]) were utilized to assess changes in bacterial community composition. Season and internal population dynamics had a greater influence on the bacterial biofilm than the presence of protozoa. Within this general framework, bacterial area coverage and microcolony abundance were nevertheless enhanced by the presence of ciliates (but not by the presence of flagellates). We also found that the richness of bacterial operational taxonomic units was much higher in planktonic founder communities than in the ones establishing the biofilm. Within the first 2 h of colonization of an empty substrate by bacteria, the presence of flagellates additionally altered their biofilm community composition. As the biofilms matured, the number of bacterial operational taxonomic units increased when flagellates were present in high abundances. The additional presence of ciliates tended to at first reduce (days 2 to 7) and later increase (days 14 to 29) bacterial operational taxonomic unit richness. Altogether, the response of the bacterial community to protozoan grazing pressure was small compared to that reported in planktonic studies, but our findings contradict the assumption of a general grazing resistance of bacterial biofilms toward protozoa.
Project description: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.
Project description:Irrigated rice field soil is subjected to frequent changes in oxygen status due to the water regime by agricultural management. In this study, the community response of microeukaryotes in rice field soil to the oxygen status was explored in a microcosm experiment under defined conditions. Water-saturated soil was incubated under a two-level factorial design of oxygen and organic enrichment with plant residue. The eukaryotic microbial community composition, which was either present or potentially active in the soils, was analyzed using denaturing gradient gel electrophoresis (DGGE) targeting the 18S rRNA gene or reverse-transcribed 18S rRNA. Oxygen availability was a primary factor shaping the microeukaryotic community in both DNA- and RNA-based analyses, revealing a shift within a week of incubation. Plant residue also affected the microeukaryotic community, which was more notable in the active community showing rRNA expression with time. Sequences of amplicons in DGGE bands indicated that protozoa (ciliates, flagellates, and amoebae) were the most prominent microeukaryotes in water-saturated rice field soil both in DNA- and RNA-based analyses. The use of a modified primer for soil protozoa suggested the functional importance of Heterolobosea amoeba in rice field soil, particularly in anoxic soil with organic enrichment.
Project description:The microbial spoilage of beef was monitored during storage at 5 degrees C under three different conditions of modified-atmosphere packaging (MAP): (i) air (MAP1), (ii) 60% O2 and 40% CO2 (MAP2), and (iii) 20% O2 and 40% CO2 (MAP3). Pseudomonas, Enterobacteriaceae, Brochothrix thermosphacta, and lactic acid bacteria were monitored by viable counts and PCR-denaturing gradient gel electrophoresis (DGGE) analysis during 14 days of storage. Moreover, headspace gas composition, weight loss, and beef color change were also determined at each sampling time. Overall, MAP2 was shown to have the best protective effect, keeping the microbial loads and color change to acceptable levels in the first 7 days of refrigerated storage. The microbial colonies from the plate counts of each microbial group were identified by PCR-DGGE of the variable V6-V8 region of the 16S rRNA gene. Thirteen different genera and at least 17 different species were identified after sequencing of DGGE fragments that showed a wide diversity of spoilage-related bacteria taking turns during beef storage in the function of the packaging conditions. The countable species for each spoilage-related microbial group were different according to packaging conditions and times of storage. In fact, the DGGE profiles displayed significant changes during time and depending on the initial atmosphere used. The spoilage occurred between 7 and 14 days of storage, and the microbial species found in the spoiled meat varied according to the packaging conditions. Rahnella aquatilis, Rahnella spp., Pseudomonas spp., and Carnobacterium divergens were identified as acting during beef storage in air (MAP1). Pseudomonas spp. and Lactobacillus sakei were found in beef stored under MAP conditions with high oxygen content (MAP2), while Rahnella spp. and L. sakei were the main species found during storage using MAP3. The identification of the spoilage-related microbiota by molecular methods can help in the effective establishment of storage conditions for fresh meat.
Project description:Ticks acquire a wide range of microorganisms as a natural part of their lifecycle. Bacteria, viruses, and protozoa can be transmitted to ticks during feeding and free-living phases. DGGE profiling is a molecular method to describe the microbial population associated with ticks and demonstrate some of the complexity and variety of tick-borne microorganisms. The present study profiled a total of 120 I. ricinus ticks, which were divided into three equally sized groups. We found that B. burgdorferi s.l.-infected ticks presented a pattern consisting of bacterial Pseudomonas spp. (67.5%), Bacillus spp. (50%), and Sphingomonas spp. (77.5%), while A. phagocytophilum-infected ticks were associated with Pseudomonas spp. (82.5%) and Sphingomonas spp. (57.5%). All profiles had one or more Pseudomonas species present, and the intramitochondrial endosymbiont Candidatus Midichloria mitochondrii was present in more than 25% of the samples. Statistical analysis demonstrated that the microbial communities were not significantly different between the groups and that the groups could not be characterised by a specific microbial population.
Project description:Bacterial endosymbiosis has been instrumental in eukaryotic evolution, and includes both mutualistic, dependent and parasitic associations. Here we characterize an intracellular bacterium inhabiting the flagellated protist Bodo saltans (Kinetoplastida). We present a complete bacterial genome comprising a 1.39 Mb circular chromosome with 40.6% GC content. Fluorescent in situ hybridisation confirms that the endosymbiont is located adjacent to the nuclear membrane, and a detailed model of its intracellular niche is generated using serial block-face scanning electron microscopy. Phylogenomic analysis shows that the endosymbiont belongs to the Holosporales, most closely related to other α-proteobacterial endosymbionts of ciliates and amoebae. Comparative genomics indicates that it has a limited metabolism and is nutritionally host-dependent. However, the endosymbiont genome does encode diverse symbiont-specific secretory proteins, including a type VI secretion system and three separate toxin-antitoxin systems. We show that these systems are actively transcribed and hypothesize they represent a mechanism by which B. saltans becomes addicted to its endosymbiont. Consistent with this idea, attempts to cure Bodo of endosymbionts led to rapid and uniform cell death. This study adds kinetoplastid flagellates to ciliates and amoebae as hosts of Paracaedibacter-like bacteria, suggesting that these antagonistic endosymbioses became established very early in Eukaryotic evolution.
Project description:BACKGROUND: To simplify the methodology for the isolation of Campylobacter spp. from retail broiler meat, we evaluated 108 samples (breasts and thighs) using an unpaired sample design. The enrichment broths were incubated under aerobic conditions (subsamples A) and for comparison under microaerobic conditions (subsamples M) as recommended by current reference protocols. Sensors were used to measure the dissolved oxygen (DO) in the broth and the percentage of oxygen (O2) in the head space of the bags used for enrichment. Campylobacter isolates were identified with multiplex PCR assays and typed using pulsed-field gel electrophoresis (PFGE). Ribosomal intergenic spacer analyses (RISA) and denaturing gradient gel electrophoresis (DGGE) were used to study the bacterial communities of subsamples M and A after 48 h enrichment. RESULTS: The number of Campylobacter positive subsamples were similar for A and M when all samples were combined (P = 0.81) and when samples were analyzed by product (breast: P = 0.75; thigh: P = 1.00). Oxygen sensors showed that DO values in the broth were around 6 ppm and O2 values in the head space were 14-16% throughout incubation. PFGE demonstrated high genomic similarity of isolates in the majority of the samples in which isolates were obtained from subsamples A and M. RISA and DGGE results showed a large variability in the bacterial populations that could be attributed to sample-to-sample variations and not enrichment conditions (aerobic or microaerobic). These data also suggested that current sampling protocols are not optimized to determine the true number of Campylobacter positive samples in retail boiler meat. CONCLUSIONS: Decreased DO in enrichment broths is naturally achieved. This simplified, cost-effective enrichment protocol with aerobic incubation could be incorporated into reference methods for the isolation of Campylobacter spp. from retail broiler meat.
Project description:Kinetoplastids are protozoa containing a range of ubiquitous free_living species-pathogens of invertebrates, vertebrates and even some plants. Some of them are causative agents of canine vector-borne diseases. Their diagnosis is often missing in a gold standard. Here, we proposed a molecular approach for the diagnosis and study of Kinetoplastida. The TaqMan qPCR assays target the following genes: 24Sa LSU of Kinetoplastida, 28S LSU of Leishmania/ Trypanosoma spp., 5.8S rRNA of Trypanosoma spp., 18S SSU of Leishmania spp., kinetoplast minicircle DNA (kDNA) of L. donovani complex and kDNA of L. infantum, were designed, validated for their sensitivity (Se) and specificity (Sp) in silico and in vitro using a panel of known DNAs. They were then used to screen 369 blood samples (358 dogs, 2 equids, 9 monkeys). In addition, new 28S LSU primer sets are presented to use for Kinetoplastida's identification by PCR/sequencing. All qPCRs showed consistently high analytical sensitivities and reproducibility. They detect approximately 0.01 parasite/ mL blood for the kDNA based- qPCRs and at least a single cell-equivalent of rDNA for the other systems. Based on the sequencing results, after screening, Se and Sp were: 0. 919 and 0.971, 0.853 and 0.979, 1.00 and 0.987, 0.826 and 0.995 for all of Kinetoplastida, Leishmania/ Trypanosoma, Trypanosoma, Leishmania spp. specific qPCRs, respectively. kDNA based qPCRs were more sensitive and specific (Se: 1.00; Sp: 0.997). PCR/sequencing allowed the detection of Kinetoplastids in animal blood samples such as L. infantum, L. guyanensis, T. congolense, T. evansi and Bodo spp. The molecular approach proposed here is useful for epidemiological studies, fundamental research such as screening for new Kinetoplastida species, diagnosis and therapeutic follow-up. In addition, researchers are free to choose the molecular tools adapted to their aims.
Project description:Detection of urinary parasites is relatively rare and incidental finding in routine urine examination. Common urinary parasitic infections as described in literature include Trichomonas, Schistosoma hematobium and Microfilaria. Trichomonas vaginalis is known to cause vaginitis and urethritis, and may be found in urine sediments. In this study, the spectrum of urinary parasitic infections that had been reported in the last one and a half year was evaluated, and point prevalence in this zone was estimated. Microbiologist opinion had been taken in the difficult cases. Out of the total centrifuged urine sediments examined, urinary parasitic infection was found in 33 cases. The calculated point prevalence is 0.39%. Most common parasitic infection reported was flagellates (27 cases: 25 T. vaginalis, 2 commensal flagellate closest to Chylomastix), followed by three cases showing eggs of Enterobius vermicularis, one case showing larvae of Strongyloides stercoralis and two cases of ciliate protozoa. One of the ciliate protozoa was Balantidium coli and the other one was Balantidium like ciliate morphologically closest to Chilodonella spp. Pyuria was found in 22 out of the 33 cases and hematuria in 17 out of 33 cases. A fairly wide morphological spectrum of parasites may be diagnosed through microscopic examination of centrifuged urine sediment. They may cause pyuria and haematuria, and morphological awareness helps in prompt and effective management in most cases.