Evaluation of methods for DNA extraction from Clostridium tyrobutyricum spores and its detection by qPCR.

Clostridium tyrobutyricum is the major agent that causes the blowing defect in cheese due to the germination of its dormant spores during the ripening stage. As a result, many of the affected cheeses show cavities and cracks, which cause the product loss in most cases. Nowadays, there is not a fast method capable of detecting milk contaminated with C. tyrobutyricum spores. The aim of this study has been to develop a fast and reliable method based on real time PCR (qPCR) to detect C. tyrobutyricum spores in raw milk. One of the main limitations has been to find a good procedure for the spore disruption to extract the DNA due to its high resistance. For this reason, different disruption methods have been tested, including chemical agents, bead beating, enzymatic and microwave treatment. Furthermore, an enzymatic treatment with subtilisin was applied for milk clarification and recovery of spores. The comparison of the assayed methods has been made using sterile milk spiked with C. tyrobutyricum spores, obtained in solid or liquid medium. The results showed that microwave treatment followed by a standard DNA purification step was found to be the best disruption method. The Ct values obtained for spores were higher than those found for vegetative cells by qPCR, for the same quantity of DNA. This difference could be due to the action of the Small Acid Soluble Proteins (SASP) in the DNA packaging of spores. Moreover, spores obtained in agar plate were found more resistant to disruption than those obtained in liquid medium. Subtilisin and microwave treatments were found to be successful for DNA extraction from C. tyrobutyricum spores in milk and subsequent identification by qPCR. However, the differences observed between the amplification of DNA from spores obtained in different media and from vegetative cells have to be taken into account to optimize a method for C. tyrobutyricum detection.

Dairy farm management practices and the risk of contamination of tank milk from Clostridium spp. and Paenibacillus spp. spores in silage, total mixed ration, dairy cow feces, and raw milk.

The occurrence of Paenibacillus and Clostridium spores in silage is of great concern for dairy producers because their spores can contaminate milk and damage processed milk and semi-hard cheeses. Spoiled silage is considered to be the main contamination source of the total mixed ration (TMR), feces of dairy cows, and consequently bulk tank milk via the contamination of cow teats by dirt during milking. The presence of an anaerobic and facultative anaerobic sporeformer population in different matrices (soil, corn silage, other feeds, TMR, feces, and milk) and its transmission pathway has been studied on 49 dairy farms by coupling plate count data with 16S-DNA identification. The different matrices have shown a high variability in the anaerobic and facultative anaerobic spore count, with the highest values being found in the aerobically deteriorated areas of corn silages. Clostridium tyrobutyricum, Paenibacillus macerans, and Paenibacillus thermophilus were detected in all the matrices. The TMR spore count was influenced by the amount of spoiled corn silage in the TMR and by the care taken when cleaning the spoiled silage before feed-out. Most of the farms that prevent the presence of visible moldy silage in the silo and carefully clean to remove molded spots were able to maintain their TMR spore counts below 4.0 log spores/g. When a level of 4.5 log spores/g of TMR was exceeded, the feces presented a greater contamination than 3.0 log spores/g. Moreover, the higher the number of spores in the feces was, the higher the number of spores in the milk. Most of the farms that presented a feces contamination greater than 5.0 log spores/g had a higher milk spore contamination than 1,000 spores/L. Careful animal cleaning and good milking practices have been found to be essential to maintain low levels of contamination in bulk tank milk, but it has emerged that only by coupling these practices with a correct silage management and cleaning during TMR preparation can the contamination of milk by spores be kept at a low level. It has been found that aerobically deteriorated silage has a great capacity to contaminate TMR and consequently to increase the risk of milk spore contamination, even when routine milking practices are adopted correctly.

Effect of hot season on blood parameters, fecal fermentative parameters, and occurrence of Clostridium tyrobutyricum spores in feces of lactating dairy cows.

High temperature influences rumen and gut health, passage rate, and diet digestibility, with effects on fermentative processes. The main aim of the study was to investigate the effect of hot season on hindgut fermentation, the occurrence of Clostridium tyrobutyricum spores in bovine feces, and on their relationship with metabolic conditions in dairy cows producing milk used for Grana Padano cheese. The study was carried out on 7 dairy farms located in the Po Valley (Italy), involving 1,950 Italian Friesian dairy cows. The study was carried out from November 2013 till the end of July 2014. Temperature and relative humidity were recorded daily by weather stations. Constant management conditions were maintained during the experimental period. Feed and diet characteristics, metabolic conditions, and fecal characteristics were recorded in winter (from late November 2013 to the end of January 2014), spring (from April to May 2014), and summer (July 2014) season. In each season, blood samples were collected from 14 multiparous lactating dairy cows per herd to measure biochemical indices related to energy, protein, and mineral metabolism, as well as markers of inflammation and some enzyme activities. Fecal samples were also collected and measurements of moisture, pH and volatile fatty acids (VFA) were performed. The DNA extracted and purified from fecal samples was used to detect Clostridium tyrobutyricum spores in a quantitative real-time PCR assay. The daily mean temperature-humidity index was 40.7 ± 4.6 (range 25 to 55), 61.2 ± 3.7 (range 39 to 77), and 70.8 ± 3.2 (range 54 to 83) in winter, spring, and summer, respectively. Total VFA concentration in feces progressively decreased from winter to summer. The seasonal changes of acetate and propionate followed the same trend of total VFA; conversely, butyrate did not show any difference between seasons, and its molar proportion was greater in summer compared with winter. A greater occurrence of Cl. tyrobutyricum spores in summer compared with the other seasons was observed. The plasma concentrations of glucose, urea, albumin, Ca, Mg, Cl, Zn, and alkaline phosphatase activity were lower in summer compared with winter, whereas the opposite occurred for bilirubin and Na. Our results show that summer season, through direct and indirect effect of heat stress, affected fecal fermentative parameters and hindgut buffering capacity, and was responsible for the increasing occurrence of Cl. tyrobutyricum spores in feces.

Multilocus variable-number of tandem repeat analysis (MLVA) for Clostridium tyrobutyricum strains isolated from cheese production environment.

Clostridium tyrobutyricum is a gram-positive spore-forming anaerobe that is considered as the main causative agent for late blowing in cheese due to butyric acid fermentation. In this study, multilocus variable-number of tandem repeat (VNTR) analysis (MLVA) for C. tyrobutyricum was developed to identify the source of contamination by C. tyrobutyricum spores in the cheese production environment. For each contig constructed from the results of a whole genome draft sequence of C. tyrobutyricum JCM11008(T) based on next-generation sequencing, VNTR loci that were effective for typing were searched using the Tandem Repeat Finder program. Five VNTR loci were amplified by polymerase chain reaction (PCR) to determine their number of repeats by sequencing, and MLVA was conducted. 25 strains of C. tyrobutyricum isolated from the environment, raw milk, and silage were classified into 18 MLVA types (DI=0.963). Of the C. tyrobutyricum strains isolated from raw milk, natural cheese, and blown processed cheese, strains with identical MLVA type were detected, which suggested that these strains might have shifted from natural cheese to blown processed cheese. MLVA could be an effective tool for monitoring contamination of natural cheese with C. tyrobutyricum in the processed cheese production environment because of its high discriminability, thereby allowing the analyst to trace the source of contamination.

Detection of Clostridium tyrobutyricum in milk to prevent late blowing in cheese by automated ribosomal intergenic spacer analysis.

Clostridium tyrobutyricum has been identified as the main causal agent of the late blowing defect in cheese, with major effects on quality and commercial value. In this work, for the first time, we applied automated ribosomal intergenic spacer analysis (ARISA) approach to diagnose the presence of C. tyrobutyricum in raw milk before cheesemaking. A species-specific primer set was designed and used for this original application of the ARISA. Sensitivity of detection, reproducibility of the fluorescent PCR assay, and repeatability of the capillary electrophoretic analysis of amplicons were evaluated using DNA extracted from milk added with known amounts of C. tyrobutyricum genome copies, ranging from 3 × 10(6) to 3. Results indicated that the sensitivity of the technique permits to detect the bacterium in all the samples. The reproducibility, evaluated by analyzing 3 sets of serial dilutions, resulted satisfactory, with little deviation within PCR reactions amplifying the same starting amount of template (standard deviations ≤ 0.1, coefficients of variation ≤ 3%). The peaks' fluorescence displayed an evident correspondence with the number of genome copies contained in each dilution. The capillary electrophoretic analysis, tested by running a single PCR product per dilution point in 10 repeats, resulted efficient and highly repeatable, with excellent coefficients of variation ≤ 2% and standard deviations ≤ 0.1 in all the sample sets. This application of ARISA gives good estimates of the total C. tyrobutyricum DNA content allowing a specific, fine-scale resolution of this pollutant species in a complex system as milk. A further advantage linked to the automatization of the process.

Identification of Clostridium beijerinckii, Cl. butyricum, Cl. sporogenes, Cl. tyrobutyricum isolated from silage, raw milk and hard cheese by a multiplex PCR assay.

Late blowing, caused by the outgrowth of clostridial spores present in raw milk and originating from silage, can create considerable product loss, especially in the production of hard and semi-hard cheeses. The conventional method for the isolation of Clostridium spp. from cheeses with late-blowing symptoms is very complicated and the identification of isolates is problematic. The aim of this work was the development of a multiplex PCR method for the detection of the main dairy-related clostridia such as: Cl. beijerinckii, Cl. butyricum, Cl. sporogenes, Cl. tyrobutyricum. Samples derived from silage, raw milk and hard cheese were analysed by the most probable number (MPN) enumeration. Forty-four bacterial strains isolated from gas positive tubes were used to check the reliability of the multiplex PCR assay. The specificity of the primers was tested by individually analysing each primer pair and the primer pair combined in the multiplex PCR. It was interesting to note that the samples not identified by the multiplex PCR assay were amplified by V2-V3 16S rRNA primer pair and the sequencing revealed the aligned 16S rRNA sequences to be Paenibacillus and Bacillus spp. This new molecular assay provides a simple promising alternative to traditional microbiological methods for a rapid, sensitive detection of clostridia in dairy products.

Specific peptides as alternative to antibody ligands for biomagnetic separation of Clostridium tyrobutyricum spores.

Nowadays, the reference method for the detection of Clostridium tyrobutyricum in milk is the most-probable-number method, a very time-consuming and non-specific method. In this work, the suitability of the use of superparamagnetic beads coated with specific antibodies and peptides for bioseparation and concentration of spores of C. tyrobutyricum has been assessed. Peptide or antibody functionalized nanoparticles were able to specifically bind C. tyrobutyricum spores and concentrate them up to detectable levels. Moreover, several factors, such as particle size (200 nm and 1 µm), particle derivatization (aminated and carboxylated beads), coating method, and type of ligand have been studied in order to establish the most appropriate conditions for spore separation. Results show that concentration of spore is favored by a smaller bead size due to the wider surface of interaction in relation to particle volume. Antibody orientation, related to the binding method, is also critical in spore recovery. However, specific peptides seem to be a better ligand than antibodies, not only due to the higher recovery ratio of spores obtained but also due to the prolonged stability over time, allowing an optimal recovery of spores up to 3 weeks after bead coating. These results demonstrate that specific peptides bound to magnetic nanoparticles can be used instead of traditional antibodies to specifically bind C. tyrobutyricum spores being a potential basis for a rapid method to detect this bacterial target.

Immunological response in egg-sensitive adults challenged with cheese containing or not containing lysozyme.

OBJECTIVE: Lysozyme is an enzyme that hydrolyzes bacterial peptidoglicans. For this reason, it is used in cheese manufacturing in order to prevent a defect of long-ripened hard cheese called "late blowing" due to the outgrowth of spores of Clostridium tyrobutyricum and Clostridium butyricum. Moreover, germination of Listeria monocytogenes spores into vegetative cells is also sensitive to lysozyme. The enzyme can be an allergenic molecule, and for this reason there are concerns about its use in food industry. The immunological and clinical response of consumption of lysozyme-containing cheese has been evaluated in 25 egg-sensitive subjects with or without lysozyme sensitization. METHODS: A total of 25 egg-sensitive subjects were enrolled in this study. All the subjects were already treated for egg-sensitization and presented a positive skin prick test. All the subjects had a body mass index ≤ 25 kg/m(2) and were in the age range of 20-50 years. Each subject was studied twice and received randomly 30 g of Grana Padano (containing lysozyme) or TrentinGrana cheese (lysozyme-free) of two different aging periods: 16 or 24 months. A washout period of 1 week between each cheese intake was adopted. Blood samples were taken in fasting conditions and 1 hour after cheese intake and IgA, total IgE, and lysozyme-, ovomucoid-, and ovalbumin-specific IgE were measured. RESULTS: No adverse reactions were observed in both groups of patients after cheese samples were given. Lysozyme did not determine any variation of specific IgE compared with basal level. In lysozyme-sensitive patients a significant relationship between IgA and lysozyme-specific IgE was observed when lysozyme-containing cheese was given, confirming that lysozyme can pass the gut barrier. CONCLUSIONS: Neither adverse events nor immunological responses were observed after ingestion of cheese containing lysozyme. However, the immunological properties of peptides deriving from cheese protein hydrolysis need to be clarified, as does the effect of lysozyme on bacterial proteolytic activity.

Detection of Clostridium tyrobutyricum spores using polyclonal antibodies and flow cytometry.

AIMS: The present work investigates the feasibility of using flow cytometry (FCM) combined with fluorescent-labelled specific polyclonal antibodies for the detection and presumptive identification of Clostridium tyrobutyricum spores in bovine milk. METHODS AND RESULTS: Two fluorescent molecules (fluorescein isothiocyanate and Alexa Fluor 488) were conjugated to antispores polyclonal antibodies. Side scatter and forward scatter profiles of the Cl. tyrobutyricum spores marked with fluorescent antibodies permitted the detection of spores and differentiated them from other related microbial species. The detection limit of this method was 10(3) spores per 100 ml of milk, and results could be achieved in 2 h. CONCLUSIONS: FCM combined with fluorochrome-conjugated antibodies, especially Alexa Fluor, could be an efficacious means to detect and provide presumptive identification of Cl. tyrobutyricum spores, as well as differentiation from other Clostridium species that can also cause late blowing in cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the basis for the development of a method suitable for analysis of milk destined for cheese manufacture that would permit the detection of Cl. tyrobutyricum spores in a short period. This would enable the industry to use contaminated milk for dairy products other than cheese where Cl. tyrobutyricum does not cause a problem.

Selection of high affine peptide ligands for detection of Clostridium Tyrobutyricum spores.

Clostridium tyrobutyricum is the main agent responsible for "late blowing" in cheese, which causes severe economic losses. Nowadays, the reference method for its detection is the Most-Probable-Number (MPN); however, it is time consuming and non-specific. Thus, in order to check milk contamination with spores of C. tyrobutyricum, a more specific and rapid method would be required. The objective of this work was to obtain a ligand to establish the basis to develop a biomagnetic separation method for detection of C. tyrobutyricum spores. This study describes the selection of thirteen highly affine peptides to C. tyrobutyricum spores from a phage-display peptide library. In order to test the ability of the peptides attached to a solid support to bind the spores, the most frequent peptide was synthesised and used to coat paramagnetic beads.

Low permeability to oxygen of a new barrier film prevents butyric acid bacteria spore formation in farm corn silage.

The outgrowth of Clostridium spore-forming bacteria causes late blowing in cheeses. Recently, the role of air diffusion during storage and feed-out and the role of aerobic deterioration has been shown to indirectly favor butyric acid bacteria (BAB) growth and to determine the presence of high concentrations of BAB spores in farm tank milk. A new oxygen barrier (OB) film was tested and compared with conventional polyethylene (ST). The objective was to verify whether the OB film could prevent BAB spore formation in whole-crop corn silage during storage on 2 commercial farms with different potential silage spoilage risks. Two bunkers (farms 1 and 2) were divided into 2 parts along the length so that half the feed-out face would be covered with ST film and the other half with OB film. Plastic net bags with freshly chopped corn were buried in the upper layer and in the central part (CORE) of the bunkers. The silos were opened in summer and fed out at different removal rates (19 vs. 33 cm/d). Herbage at ensiling, silage at unloading, and silage after air exposure (6 and 15 d) were analyzed for pH, nitrate, BAB spores, yeasts, and molds. The BAB spores in herbages at ensiling were 2.84 log(10) most probable number (MPN)/g, with no differences between treatments or farms. Nitrate was below the detection limit on farm 1 and exceeded 2,300 mg/kg of fresh matter on farm 2. At unloading, the BAB spores in the ST silage on farm 1 were greater than 5 log(10) MPN/g, whereas in the CORE and the OB silages, they were approximately 2 log(10) MPN/g. The ST silage had the greatest pH (5.89), the greatest mold count (5.07 log(10) cfu/g), and the greatest difference between silage temperature and ambient temperature (dT(section-ambient)). On farm 2, the ST silage had the greatest concentration of BAB spores (2.19 log(10) MPN/g), the greatest pH (4.05), and the least nitrate concentration compared with the CORE and the OB silages. Pooled data on BAB spores collected from aerobically deteriorated samples showed a positive relationship with pH, mold count, and dT(section-ambient) and a negative relationship with nitrate concentration. A high concentration of BAB spores (>5 log MPN/g) was associated with visible spoilage, high pH values (>5.00), high mold counts (>5 log cfu/g), high dT(section-ambient), and nitrate below 1,000 mg/kg of fresh matter. We concluded that the use of a film with reduced oxygen permeability prevented the outgrowth of BAB spores during conservation and feed-out, and it could improve the microbiological quality of corn silage by eliminating the fractions of silage with high BAB spore concentrations.

Bactericidal effect of bovicin HC5 and nisin against Clostridium tyrobutyricum isolated from spoiled mango pulp.

AIMS: To test the effect of bovicin HC5--a bacteriocin from Streptococcus bovis HC5--against the strains of Clostridium tyrobutyricum isolated from canned spoiled mango pulp. METHODS AND RESULTS: Bovicin HC5 [40-160 arbitrary unit (AU) ml(-1)] reduced the specific growth rate and increased the lag phase duration of the bacterial isolates inoculated in brain heart infusion media at 30 degrees C. The inhibitory activity of bovicin HC5 (100 AU ml(-1)) in mango pulp was bactericidal and more pronounced at acidic conditions. When C. tyrobutyricum was inoculated into mango pulp with bovicin HC5, gas production was not observed. Cultures that were successively transferred in the presence of sublethal doses of bovicin HC5 did not become resistant. CONCLUSIONS: The addition of bovicin HC5 to mango pulp might be effective in preventing deterioration by spoilage bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Bovicin HC5 and nisin have the potential to increase the shelf life of canned fruit pulps.

Quantitative detection of Clostridium tyrobutyricum in milk by real-time PCR.

We developed a real-time PCR assay for the quantitative detection of Clostridium tyrobutyricum, which has been identified as the major causal agent of late blowing in cheese. The assay was 100% specific, with an analytical sensitivity of 1 genome equivalent in 40% of the reactions. The quantification was linear (R(2) > 0.9995) over a 5-log dynamic range, down to 10 genome equivalents, with a PCR efficiency of >0.946. With optimized detergent treatment and enzymatic pretreatment of the sample before centrifugation and nucleic acid extraction, the assay counted down to 300 C. tyrobutyricum spores, with a relative accuracy of 82.98 to 107.68, and detected as few as 25 spores in 25 ml of artificially contaminated raw or ultrahigh-temperature-treated whole milk.

Effect of different premilking manual teat-cleaning methods on bacterial spores in milk.

Different teat-cleaning methods were evaluated to determine their effect on the presence of spores from anaerobic bacterial spore-formers in the milk. Artificial contamination was used to achieve uniform contamination of teats to reduce the number of cows and samples needed in the experiments and still obtain adequate power to detect differences among tested methods. Teats were contaminated experimentally with a large amount of Clostridium tyrobutyricum spores in a manure-water slurry. Various types of dry and moistened towels and different combinations of methods using soap or 2 types of towels, together with cleaning times of 10 or 20 s, were compared in 2 Latin square-designed experiments with 7 cows, 7 treatments, and 4 replications in each experiment. In comparison with control (no cleaning and no forestripping), cleaning teats with dry paper towels for 10 s reduced concentration of spores in milk by 45 to 50%. A 50 to 74% reduction was achieved using different types of moist towels for 10 s. Methods using 2 towels, soap, or a longer cleaning time reduced bacterial contamination by 85 to 91%. The most effective methods in reducing milk spore content (96% reduction) were use of a moist washable towel with or without soap followed by drying with a dry paper towel, for a total time of 20 s per cow. One of the best cleaning methods was studied in an additional experiment to determine the effect of different teat contamination mixtures. The Latin square-designed experiment with 8 cows, 8 treatments, and 2 replications showed that cleaning was independent of the tested contamination matrix (manure, soil, or sawdust), type of spores (Cl. tyrobutyricum and Bacillus cereus), or degree of contamination (manure or extra manure).