Prevalence of Mycobacterium avium subspecies paratuberculosis in Swiss raw milk cheeses collected at the retail level

A total of 143 raw milk cheese samples (soft cheese, n = 9; semihard cheese, n = 133; hard cheese, n = 1), collected at the retail level throughout Switzerland, were tested for Mycobacterium avium ssp. paratuberculosis (MAP) by immunomagnetic capture plus culture on 7H10-PANTA medium and in supplemented BAC-TEC 12B medium, as well as by an F57-based real-time PCR system. Furthermore, pH and water activity values were determined for each sample. Although no viable MAP cells could be cultured, 4.2% of the raw milk cheese samples tested positive with the F57-based real-time PCR system, providing evidence for the presence of MAP in the raw material. As long as the link between MAP and Crohn's disease in humans remains unclear, measures designed to minimize public exposure should also include a focus on milk products.     

Short communication: Detection of Mycobacterium avium subspecies paratuberculosis by polymerase chain reaction in bovine milk in Brazil

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis, or Johne's disease, a chronic granulomatous enteritis that affects all ruminants worldwide. Since the isolation of MAP from intestinal tissue of human patients bearing Crohn's disease, there has been a debate on the possibility of this agent playing a role in the etiology of Crohn's disease. Milk could be the potential vehicle for transmission to humans. Mycobacterium avium ssp. paratuberculosis has already been detected in milk samples worldwide. In Brazil, detection of MAP is uncommon; however, it has already been detected by bacterial isolation and serological test. The aim of this study was to investigate the presence of MAP, by PCR, in raw milk samples in the region of Viçosa, Minas Gerais State, Brazil. Of 222 milk samples evaluated, 8 (3.6%) quarter milk samples amplified fragments of similar size to that expected of 626 bp. These fragments were cloned and sequenced. The genetic analysis revealed a 99% identity match between the sequences obtained in this study and the insertion sequence IS900 deposited in the GenBank. In the analyzed milk samples, MAP DNA was detected, confirming its presence in dairy cattle in the region of Viçosa. This is the first report of MAP presence in raw milk samples in Brazil.     

Detection of Mycobacterium avium subspecies paratuberculosis in naturally exposed dairy heifers and associated risk factors

An observational prospective study was conducted to identify risk factors associated with fecal shedding of Mycobacterium avium ssp. paratuberculosis (MAP) in naturally exposed dairy heifers. The study population consisted of heifers from 8 dairy herds in Michigan participating in a MAP control demonstration project. Ten heifers from 4 age groups (0 to 3, 4 to 6, 7 to 14, and 15 to 24 mo) were selected from each herd every 4 mo for 28 mo and tested for the presence of MAP by fecal culture (FC). Heifers from dams testing positive for MAP by serum ELISA or FC were preferentially selected, with the remainder of the age cohort filled with randomly selected heifers. Logistic regression using generalized estimating equations to account for clustering of data within herds and repeated measures across heifers was used to evaluate the relationship between MAP FC status of heifers and herd risk factors. In total, 1,842 fecal samples were collected from 1,202 heifers. Thirty-six (2%) fecal samples, representing 27 individual heifers, cultured positive for MAP. Heifers shedding MAP were more likely to occur in herds with adult-cow MAP ELISA prevalence >10% (odds ratio = 4.7; 95% confidence interval: 2.0-11.1) and herds milking >300 cows (odds ratio = 5.7; 95% confidence interval: 2.4-13.4). Mycobacterium avium ssp. paratuberculosis can be cultured from the feces of naturally infected dairy heifers. The future performance of these MAP FC-positive heifers is unknown and needs to be explored.     

Effects of seasonal climatic conditions on the diagnosis of Mycobacterium avium subspecies paratuberculosis in dairy cattle

Validity of Johne's disease programs and control protocols that rely on established cut points [e.g., specified sample-to-positive (S/P) ratios] for ELISA serological tests depends on interpreted results that are not susceptible to variable test accuracy. It was hypothesized that seasonal variability exists in serological response to Mycobacterium avium subsp. paratuberculosis (MAP) infection. Further, a reciprocal response may occur, resulting in greater risk of fecal shedding in subclinically infected animals. A testing regimen was invoked that included multiple testing of individual adult cows during the 4 seasons. Serum was collected on a cyclic, monthly basis from 3 randomly selected cohorts of dairy cows, and fecal samples were collected from the 20% of cows with the greatest ELISA test S/P ratios. Staggered, quarterly sampling was continued for 1 yr, and at the conclusion, serum was analyzed en masse. The ELISA outcome values (i.e., S/P ratio) were treated both as categorical and continuous variables. The potential lagged effects of temperature-related seasonality on S/P ratio, as well as the potential for a change in test result caused by temperature were assessed. Results for fecal culture were analyzed on a categorical scale and compared with the ELISA results to explore the possibility of reciprocal fecal shedding. No significant seasonal effects on either S/P ratios or the proportion of cows seropositive to MAP were observed. Furthermore, no evidence was found linking temperature-related seasonality to a reciprocal increase in the risk of fecal culture positivity for MAP.     

Osteopontin: a novel cytokine involved in the regulation of Mycobacterium avium subspecies paratuberculosis infection in periparturient dairy cattle

Osteopontin (Opn), an important mediator of the cell-mediated immune response, enhances the host immune response against mycobacterial infections. Infections caused by Mycobacterium avium ssp. paratuberculosis (MAP) have a devastating effect on the dairy industry. We sought to characterize Opn at the level of gene and protein expression in periparturient dairy cows naturally infected with MAP. Peripheral blood mononuclear cells (PBMC) were isolated from control, subclinical, and clinical periparturient dairy cows naturally infected with MAP beginning 3 wk precalving to 5 wk postcalving and incubated with medium alone (non-stimulated: NS), concanavalin A (ConA), or a whole-cell sonicate of MAP (MPS). Real-time PCR was performed to evaluate expression of Opn and classical Th1 and Th2 cytokines. Results demonstrated greater Opn expression in nonstimulated PBMC isolated from subclinical cows compared with control and clinical cows. For clinical cows, there was a strong correlation between Opn expression and expression of the Th1 cytokines IFN-γ and IL-1α for nonstimulated PBMC and IFN-γ and IL-12 for PBMC stimulated with MPS. Expression of tumor necrosis factor-α was greater in clinical cows than the other groups. Nonstimulated, ConA, and MPS-stimulated PBMC from subclinical cows secreted more IFN-γ, and MPS-stimulated PBMC from clinical cows secreted more IL-4 compared with the other groups. Immunoblot analysis of PBMC detected 4 Opn proteins at 60, 52, 34, and 27 kDa. This is the first study to evaluate the role of Opn on the immune response of dairy cows naturally infected with MAP, and results suggest Opn may be a key regulator against MAP infection.     

The distribution of Mycobacterium avium ssp. paratuberculosis in the environment surrounding Minnesota dairy farms

The objective of this study was to characterize the distribution of Mycobacterium paratuberculosis (Map) in the environment of infected and uninfected Minnesota dairy farms. Eighty herds known to be infected from Minnesota's Johne's Disease Control Program (JDCP) and 28 herds known to be uninfected from Minnesota Voluntary Johne's Disease Herd Status Program (VJDHSP) were sampled. Fecal samples from up to 100 cows in each herd were cultured in pools of 5 cows. Two environmental samples were obtained from each farm from various locations. All samples were tested using bacterial culture for Map. Eighty percent of the JDCP herds had at least one positive pool. Environmental samples were cultured positive in 78% of the JDCP herds. Two (7%) of the VJDHSP herds had one positive pool, and one herd had one positive environmental sample. Environmental samples were cultured positive in cow alleyways (77% of the herds), manure storage (68%), calving area (21%), sick cow pen (18%), water runoff (6%), and postweaned calves areas (3%). There was an association between maximum level of colonies per tube from cow alleyways and manure storage and fecal pool prevalence. Herds with both areas cultured negative were estimated to have 0.3 to 4% fecal pool prevalence. Herds with both areas having a heavy load of bacteria were estimated to have 53 to 73% fecal pool prevalence. The study results indicate that targeted sampling of cow alleyways and manure storage areas appears to be an alternative strategy for herd screening and Johne's infection status assessment and for estimating herd fecal prevalence.     

Application of IS900 PCR for detection of Mycobacterium avium subsp. paratuberculosis directly from raw milk

A polymerase chain reaction (PCR)-based assay was developed for detection of insertion sequence 900 (IS900) of Mycobacterium avium subsp. paratuberculosis in raw milk. This IS900 PCR assay included DNA extraction and PCR assay using commercially available kits. The DNA extraction and PCR assay were optimized to detect the IS900 sequence directly from raw milk. The IS900 PCR assay was evaluated by inoculating raw bulk milk and Middlebrook's 7H9 broth with 0 to 10(8) cfu/ml of each of four American Type Culture Collection strains of M. paratuberculosis. Under experimental conditions, both milk culture on Herrold's egg yolk medium slants, and IS900 PCR could detect 10 to 100 cfu/ml of M. paratuberculosis. Detection of M. paratuberculosis by IS900 PCR was consistent (24/24 PCR assays) when about 100 cfu/ml were present, whereas detection was variable (12/24 PCR assays) at concentrations as low as 10 cfu/ml. Based on the findings of the experimental study, IS900 PCR was further evaluated with pooled quarter milk samples from 211 cows from five herds with known history of Johne's disease. Out of 211 animals examined, nine (4%) and 69 (33%) were positive for M. paratuberculosis by milk culture and IS900 PCR from milk, respectively. A total of 20 bulk tank milk sample aliquots (one sample, four aliquots from each herd) were also examined, of which 10 (50%) were positive for M. paratuberculosis by IS900 PCR. By contrast, only one out of 20 (5%) bulk tank milk sample aliquots was positive by culture. The IS900 PCR amplified product of 229-bp obtained on testing of quarter milk and bulk tank milk samples was confirmed to be the IS900 of M. paratuberculosis by DNA sequence analysis. The results of this study suggest that M. paratuberculosis can be detected directly from quarter milk and bulk tank milk by IS900 PCR.     

Environmental sampling for detection of Mycobacterium avium ssp. paratuberculosis on large California dairies

Environmental samples collected from each of 3 locations on 23 large California dairies were cultured to evaluate the utility of this approach for identifying herds infected with Mycobacterium avium ssp. paratuberculosis. Results were compared with concurrent ELISA testing of ≥ 60 animals in each herd, and with previously performed individual and pooled fecal cultures of 60 animals. The estimated proportions of infected herds did not differ significantly among the testing methods (environmental sampling, 74%; previous fecal culture, 70%; and concurrent ELISA testing, 65%). Measures of agreement between environmental sampling and the results of previous fecal cultures were 70% (observed agreement), 85% (positive agreement), 62% (negative agreement), and 0.47 (kappa), whereas agreement between environmental sampling and concurrent ELISA testing was 65, 75, and 43%, and 0.19, for the same measures, respectively. The proportion of positive environmental samples on each farm was significantly correlated with the proportion of seropositive animals (r = 0.53), suggesting that environmental sampling may also provide a qualitative estimate of within-herd prevalence. Of the sampling locations that were evaluated, samples of lagoon water (15/23; 65%) were significantly more likely to yield a positive result than were composite manure samples (8/22; 36%) collected from the sick/fresh cow pen or from the alleyway (9/23; 39%) where cows exited from the milking parlor. Environmental sampling was an effective and inexpensive method of identifying herds infected with Mycobacterium avium ssp. paratuberculosis.     

Scenario analysis of changes in consumption of dairy products caused by a hypothetical causal link between Mycobacterium avium subspecies paratuberculosis and Crohn's disease

Johne's disease is an infectious disease of cattle caused by Mycobacterium avium subspecies paratuberculosis (MAP). Crohn's disease (CD) is a human disease of unknown etiology that is characterized by chronic bowel inflammation. No causal link has been scientifically established between MAP and CD, but it is important to understand possible impacts on society should such a causal link be established. The goal of this paper is to analyze the implications and the possible economic impacts that finding such a link would have on milk demand in the dairy industry and to provide a framework for further discussion among stakeholders. Three scenarios were developed based on the effectiveness of possible risk-mitigation strategies. In the first scenario, it was assumed that an effective strategy exists; therefore, a negligible demand decrease in the consumption of dairy products was expected. In the second scenario, it was assumed that new risk mitigation would need to be implemented to minimize the health hazard for humans. In this case, a small milk demand decrease was expected, but larger demand decreases were also possible. The third scenario assumed that no fully effective risk mitigation was available, and this resulted in a considerable demand decrease and a potential reduction in milk supply as a result of regulatory measures. A milk demand reduction of 1 or 5% resulted in a reduction in consumer surplus of $600 million and $2.9 billion, and a reduction in dairy farm income of $270 million and $1.3 billion, respectively. A decrease in milk supply would cause a slight increase in total losses, but would cause the greatest losses to test-positive dairy farms. Given the current scientific knowledge about MAP and CD, we conclude that if a link were established, it is most likely that the first or second scenario would occur. Thus, consumer response and economic consequences to the discovery of such a link are expected to be limited, but could be large if the consumer's perception of risk is large or if risk-mitigation strategies were ineffective.     

Effect of pasteurization on survival of Mycobacterium paratuberculosis in milk

Mycobacterium paratuberculosis (Mptb) is the causative agent of Johne's disease of ruminant animals including cattle, goats, and sheep. It has been suggested that this organism is associated with Crohn's disease in humans, and milk is a potential source of human exposure to this organism. A total of 18, including 7 regular batch and 11 high temperature short time (HTST) pasteurization experiments, were conducted in this study. Raw milk or ultra-high temperature pasteurized milk samples were spiked at levels of 10(3), 10(5), and 10(7) cfu of Mptb/ml. Escherichia coli and Mycobacterium bovis BCG strains at 10(7) cfu/ml were used as controls. Pasteurization experiments were conducted using time and temperature standards specified in the Canadian National Dairy Code: regular batch pasteurization method: 63 degrees C for 30 min, and HTST method: 72 degrees C for 15 s. The death curve of this organism was assessed at 63 degrees C. No survivors were detected after 15 min. Each spiked sample was cultured in Middlebrook 7H9 culture broth and Middlebrook 7H11 agar slants. Samples selected from 15 experiments were also subjected to BACTEC culture procedure. Survival of Mptb was confirmed by IS900-based PCR of colonies recovered on slants. No survivors were detected from any of the slants or broths corresponding to the seven regular batch pasteurization trials. Mptb survivors were detected in two of the 11 HTST experiments. One was by both slant and broth culture for the sample spiked to 10(7) cfu/ml of Mptb, while the other was detected by BACTEC for the sample spiked to 10(5) cfu/ml. These results indicate that Mptb may survive HTST pasteurization when present at > or = 10(5) cfu/ml in milk. A total of 710 retail milk samples collected from retail store and dairy plants in southwest Ontario were tested by nested IS900 PCR for the presence of Mptb. Fifteen percent of these samples (n = 110) were positive. However, no survivors were isolated from the broth and agar cultures of 44 PCR positive and 200 PCR negative retail milk samples. The lack of recovery of live Mptb from the retail milk samples tested may be due to either the absence of live Mptb in the retail milk samples tested or the presence of low number of viable Mptb which were undetected by the culture method used in this study.     

An inter-laboratory ring trial for the detection and isolation of Mycobacterium avium subsp. paratuberculosis from raw milk artificially contaminated with naturally infected faeces

There is a need for standardised, robust, reproducible molecular and culture methods to achieve clarification of the inactivation of Mycobacterium avium subsp. paratuberculosis (Map), the causative microbial agent of Johne's disease, in (faecally) contaminated milk and other food products such as meat. This study assessed the performance of a commercially available Map DNA extraction kit for milk Adiapure and accompanying PCR detection kit Adiavet alongside 'in-house' molecular and culture methods in an inter-laboratory ring trial using raw milk spiked with Map-infected faeces. The combined Adiapure-Adiavet Map DNA extraction and detection kit consistently detected 30 copies of IS900 (equivalent to approximately 2 cells) ml(-1) raw milk, when used in four different laboratories. Improvements in sensitivity and ease of use for 'in-house' Map detection were observed when the Adiapure extraction kit was combined with 'in-house' detection assays. Detection by real-time PCR methods, using the commercial extraction and detection systems, resulted in an overall detection rate of 100%, 90%, 85% and 25% for respective Map concentrations of 300, 30, 3 and 0.3 copies of IS900ml(-1) raw milk. Map, at 300 copies of IS900 (equivalent to approximately 20 Map cells) ml(-1) raw milk, was recovered from all samples cultured in mycobacteria growth indicator tube (MGIT) medium, from 10 of 12 samples on Herrold's egg yolk medium (HEYM) and not recovered from any samples using BACTEC medium. In conclusion, the Adiapure DNA extraction kit allows for sensitive and easy detection of Map in raw milk. The extraction method can form a candidate part of essential methodology and real-time PCR can further increase the sensitivity of the detection method. Moreover, MGIT medium is promising for culture-dependent detection of Map from raw milk.     

Reliability of environmental sampling to quantify Mycobacterium avium subspecies paratuberculosis on California free-stall dairies

The reliability of environmental sampling to quantify Mycobacterium avium ssp. paratuberculosis (MAP) based on collector and time was evaluated. Fecal slurry samples were collected using a standardized protocol simultaneously by 2 collectors of different experience levels. Samples were collected from 30 cow pens on 4 dairies every other day on 3 occasions while cow movements between pens were minimal. The 4 study herds had moderate MAP seroprevalence and were housed in free-stall dairies in central California. Results of testing the environmental samples for MAP using PCR and culture were strongly correlated. The reliability of environmental sampling simultaneously by different collectors as estimated by the intraclass correlation coefficient was excellent (81%) for PCR and good (67%) for culture and may justify comparison of quantitative results of samples collected by different investigators. The reliability of environmental sampling over a 5-d period was good (67 and 64% for PCR and culture results, respectively), which justifies the utility of environmental sampling to identify pens with a high MAP bioburden between routine cow pen changes on a dairy. Environmental sampling of free-stall pens using the standardized sampling protocol yielded comparable PCR and culture results across collectors with different experience levels and at different times within a 5-d period.     

High-throughput genotyping of filamentous fungus Aspergillus oryzae based on colony direct polymerase chain reaction

High-throughput genotyping of Aspergillus oryzae was achieved using an FTA card for the extraction of a genomic DNA template for polymerase chain reaction from a fungal colony growing on an agar plate. This method was then applied to detect other fungal species from agar slants and food materials. This method offers a convenient tool for the genotyping of filamentous fungi without using an organic solvent or specialized equipment.     

Case-control study: Productivity and longevity of dairy cows that tested positive for infection with Mycobacterium avium ssp. paratuberculosis as heifers compared to age-matched controls

A case-control study was performed to determine if dairy heifers testing positive for Mycobacterium avium ssp. paratuberculosis (MAP) before 2 yr of age by either fecal culture or serum ELISA had decreased productivity and longevity as cows compared with age-matched herdmates. Cases were individually matched with 4 controls. Survival analysis was conducted to determine differences in longevity between cases and controls. Conditional logistic regression was used to assess differences in mean 3.5% fat-corrected 305-d mature-equivalent milk, milk fat, and milk protein production, linear somatic cell count, and MAP test and clinical status as mature cows. No significant difference was found between cases and controls for any parameter assessed. Herd production performance and longevity did not appear to be impaired; therefore, testing immature dairy heifers for MAP is not economically justifiable, using currently available culture methods and commercial serum ELISA tests.     

Short communication: Recovery of viable Mycobacterium avium subspecies paratuberculosis from retail pasteurized whole milk in Brazil

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic granulomatous enteritis that affects all ruminants worldwide. Some researchers have indicated a possible role of MAP in Crohn's disease. Despite extensive research and large and important advances in the past few decades, the etiology of Crohn's disease remains indefinite. The most probable transmission route of MAP from animals to humans is milk and dairy products. Mycobacterium avium ssp. paratuberculosis has already been detected in milk samples worldwide, and some studies have reported that MAP is resistant to pasteurization. In Brazil, MAP has been reported in raw milk samples; however, Brazilian retail pasteurized milk has not yet been tested for viable MAP. The aim of this study was to investigate MAP in pasteurized milk in the region of Viçosa (Minas Gerais, Brazil). Thirty-seven samples were collected and processed for culture of MAP. One colony similar to MAP was observed and confirmed by IS900-nested PCR and sequencing. Analysis revealed 97 to 99% identity with the MAP K-10 strain. This study is the first report of the presence of MAP in retail pasteurized whole milk in Brazil.     

利用PCR方法检测水稻及其加工产品中转基因水稻

市场上的转基因产品以及深加工产品越来越多,其安全性引起全世界的极大争论,很多国家的检验机构相继开展了转基因产品的检测工作。以转基因水稻为材料,以聚合酶链式反应(PCR)方法为基础,选择适用于转基因食品安全性检验的核酸检测技术,针对转入苏云金杆菌(Bacillus thuringiensis,简写为Bt)基因水稻的Cry1Ac片段进行PCR检测,建立适合转基因水稻的检测方法。该方法简便快速、检测结果与标准及其他文献资料相符。     

Short communication: Carnobacterium maltaromaticum: the only Carnobacterium species in French ripened soft cheeses as revealed by polymerase chain reaction detection

The PCR technique using Cb1-Cb2R and species-specific primers was applied to various French soft flowered or washed rind cheeses to identify Carnobacterium species. Thirty cheeses made from cow's, ewe's, or goat's milk (raw or pasteurized), of which 20 were Appellation d’Origine Contrôlée, were analyzed in the autumn and spring. The results revealed that, irrespective of the season, the Carnobacterium genus was initially detected in 5 cheeses and was detected in 5 others after an enrichment period. Polymerase chain reaction results using species-specific primers of Carnobacterium showed that these 10 cheeses contained only the species Carnobacterium maltaromaticum. Six different patterns of fermentation were found, and 3 of the 10 cheeses contained C. maltaromaticum isolates with anti-Listeria activity.     

Short communication: Investigation of Coxiella burnetii occurrence in dairy sheep flocks by bulk-tank milk analysis and antibody level determination

To estimate the prevalence of Coxiella burnetii in the dairy sheep population from the Basque Country (northern Spain), a study was carried out combining molecular and serological techniques. First, bulk-tank milk samples from 154 flocks belonging to the Latxa Breed Farmers Association were analyzed by PCR, with 22% of flocks testing positive for C. burnetii. Then, a selection of 34 flocks (7 PCR positive and 17 negative) was investigated for the presence of serum antibodies by ELISA test on 1,011 ewes (approximately 30 ewes per flock). A total of 8.9% of the animals were seropositive, 67.6% of the flocks had at least one seropositive animal, but only in 14.7% of them was seroprevalence greater than 25%. Older ewes showed a significantly greater prevalence (17.5%) compared with yearlings (7.5%) or replacement lambs (1.5%). A marginally significant association was found between seroprevalence and PCR detection of C. burnetii in bulk-tank milk. The widespread distribution of C. burnetii in the region advocates for the implementation of Q fever control strategies and highlights the potential risk of sheep as a reservoir and infection source for other domestic and wildlife species and the human population.     

Incidence of Escherichia coli O157:H7 and E. coli virulence factors in US bulk tank milk as determined by polymerase chain reaction

Samples of bulk tank milk from dairies across the United States, taken as part of the National Animal Health Monitoring Dairy 2002 survey, were analyzed for the presence of several genes encoding virulence factors associated with enterohemorrhagic forms of Escherichia coli (EHEC) using real-time and conventional PCR assays. Samples from 859 farms in 21 states were collected and enriched in EC medium at 42.5°C to amplify any E. coli present, and DNA was isolated from the resulting biomass. The eaeA gene encoding intimin, a virulence factor associated with enteropathogenic forms of E. coli and EHEC, was detected in 199 (23%) of the samples. Thirty-six samples (4.2%) were positive for eaeA, the gamma allele of the translocated intimin receptor (γ-tir), found in EHEC strains of O157:H7, and one or both shiga-like toxin genes (stx1 and stx2), a combination that may be indicative of the presence of O157:H7 EHEC. Testing these 36 samples with a commercially available real-time PCR kit for detection of O157:H7 indicated that 5 samples could be contaminated with O157:H7. A multiplex PCR to detect the presence of fliC, rfbE, and hlyA genes found in O157:H7 reduced to 2 (0.2% of all samples) the number of samples likely to be contaminated with this organism. A strain of O157:H7 (eaeA⁺, γ-tir⁺, stx2⁺, rfbE⁺, fliC⁺, hlyA⁺) was subsequently isolated from one sample. Thirty-four eaeA-positive samples did not contain detectable γ-tir but did contain one or both of the stx genes suggesting the presence of EHEC strains other than O157:H7. These results indicate a low incidence of O157:H7 in bulk tank milk but suggest that a risk from other enteropathogenic and EHEC forms of E. coli may exist and that PCR targeting virulence factors associated with highly pathogenic forms of E. coli may be an effective means of detecting potential dangers in raw milk.     

A novel real-time polymerase chain reaction-based method for the detection and quantification of lactose-fermenting Enterobacteriaceae in the dairy and other food industries

The presence of lactose-fermenting Enterobacteriaceae and coliforms is routinely assessed to determine the hygienic quality of water and foods, particularly dairy products. This paper reports the use of lacZ-specific primers in an SYBR green I-based real-time PCR method for the easy and rapid detection of coliforms in dairy products. A large number of bacterial species were assayed to establish the specificity of the method. The sensitivity of the method was assessed using artificially contaminated cheeses. The limit of detection was 1 coliform cell in cheese samples enriched for 8 h in a culture medium. The entire procedure, including sample processing, enrichment, DNA extraction, and real-time PCR amplification, can be completed within 10 to 12 h, making it a single-day assay.