Simple and rapid methods for detecting Salmonella enteritidis in raw eggs

The Centers for Disease Control and Prevention estimates there were 300,000 cases of Salmonella enteritidis (SE) in 1997. Egg products were associated with many of the cases. To address this problem, many producers implemented flock surveillance of the SE situation at their facilities. A rapid and simple method for detecting SE from poultry samples is critical for the effective implementation of such testing strategies. A lateral flow device for the detection of SE utilized in this study was manufactured by Neogen, Lansing, MI. The test panel is a presumptive qualitative test system that detects only members of Group D1 Salmonella species. A series of studies were conducted to optimize the test procedure for raw eggs with different sample preparations. A novel antigen extraction method was developed for use with the test panel kit. The detection limit of the test panel kit was increased approximately tenfold when the extraction method was used. Detection of SE was 100% in raw egg pools inoculated with 10 SE cells per ml of egg and incubated at a 1:10 ratio in buffered peptone water (BPW) or tetrathionate brilliant green broth (TBG) for 24 h at 37 degrees C. The developed lateral flow test kit could provide a simple, rapid, and inexpensive method for egg producers and processors to test specifically for Salmonella group D1 serovars, such as SE, in egg samples.     

Simultaneous detection of Listeria monocytogenes and Salmonella by multiplex PCR in cooked ham

A multiplex PCR (m-PCR) assay with an internal amplification control (IAC) was developed for the simultaneous detection of Salmonella spp. and Listeria monocytogenes through invA and prfA genes, respectively. To ensure the detection of the pathogens in cooked ham, samples were enriched in both buffered peptone-water and Half Fraser broth. Subsequently, equal volumes of enrichment broths were mixed and DNA purification was performed prior to m-PCR reaction, saving considerable time and effort. The m-PCR also proved to be very useful as a simple and ready-to-go method for simultaneous confirmation of presumptive L. monocytogenes and Salmonella spp. colonies directly from agar plates without any DNA extraction steps.     

Evaluation of a two-step protocol for rapid detection of Salmonella in ice-cream and Cheddar cheese

A two-step protocol for repair, multiplication and rapid detection of Salmonella species in ice-cream and Cheddar cheese was evaluated. The first step involves selective preenrichment using lactose broth supplemented with sodium pyruvate and yeast extract for the repair of injured cells and brilliant green for repression of the competing flora (LBPYEBG). This enrichment is incubated for 7 h at 40°C. The second step involves direct plating of 7 h selective preenrichment onto XLD agar and simultaneous inoculations into Salmonella 1–2 Test^®for both isolation and presumptive identification of Salmonella the next day. The two-step protocol requires only 26±2 h for isolation and presumptive identification of Salmonella. This protocol was successfully used in detecting as few as 2 colony forming units (cfu) of non-stressed S. enteritidis per ml in 250 ml of enrichment. Initial inocula of 3 cfu ml⁻¹of freeze-thaw-injured S. enteritidis inoculated into various ice-creams grew to significantly (p≤0·05) higher numbers after 6 and 7 h in the LBPYEBG enrichment than in the conventional lactose broth. This was also found to be true with a naturally contaminated ice-cream (MPN of 0·09 g⁻¹of S. enteritidis).S. typhimurium HF artificially incorporated into Cheddar cheese grew more rapidly in the LBPYEBG enrichment than in the conventional lactose broth. This indicates the usefulness of this protocol for rapid detection of Salmonella spp. from ice-cream and Cheddar cheese.     

Several MPN models for serial dilutions with suppressed growth at low dilutions

Most probable number (MPN) estimates of microbial concentrations from serial dilutions employ a usual model of probabilities of the various possible outcomes. Outcomes challenge the usual model when they occur at markedly different rates from what the usual model predicts. Too many outcomes that suggest suppression of growth at low dilutions have occurred in some foods. These challenge the usual model and suggest new models. This paper provides four new models: growth suppression by a toxicant released from the food product by sample preparation; interference by a non-target species of microbe; disaggregation of clumped microbes at a single dilution step; disaggregation spread evenly over several dilution steps. The assumptions and discussion of how to find the maximum likelihood estimate for the parameters are given.     

Comparison of different most-probable-number methods for enumeration of Listeria in poultry

To estimate levels of Listeria spp. in poultry and to select the most appropriate enumeration method for routine analysis, 40 naturally contaminated retail chicken carcasses were tested in Ponferrada (León, N.W. Spain) using the direct plate count technique and various most-probable-number (MPN) designs (UVM I [University of Vermont modified Listeria enrichment broth], Fraser enrichment broth, or both were used in 3-, 5-, and 10-tube MPN techniques). MPN estimation was obtained from the number of tubes with Listeria confirmed (after streaking on PALCAM and modified Oxford agars: "true" MPN) and from the number of dark Fraser broth tubes ("predictive" MPN). Samples were analyzed in duplicate. Low levels of Listeria were found (< 110 CFU/g). The direct plate count technique was totally ineffective for enumerating Listeria in poultry. The single-step (UVM I) and the two-step (UVM I-Fraser) MPN methods gave comparable estimations and a low number of significantly discrepant predictions. Using a single-step method with Fraser broth, lower true MPNs were obtained. The number of tubes used (3, 5, or 10) did not have a substantial influence on the results. Similar estimations, highly correlated (r = 0.538 to 0.968; P < 0.001), were found with (true MPN) and without (predictive MPN) plating confirmation when using the two-step MPN method. The statistical evaluation of the differential character of Fraser broth as part of the two-step MPN method showed high sensitivity (87.5 to 92.5%), specificity (95.2 to 98.6%), efficiency (94.2 to 97.6%), and predictive values (73.6 to 89.9% for a positive test and 98.0 to 98.9% for a negative test). Taking into account these results, we suggest the convenience of using a 3- or 5-tube two-step (UVM I-Fraser) MPN method with estimations obtained from the number of tubes with darkening, without confirmation, in order to achieve great savings in time and money.     

OXYRASETM ENZYME AND MOTILITY ENRICHMENT FUNG-YU TUBE FOR RAPID DETECTION OF LISTERIA MONOCYTOGENES AND LISTERIA SPECIES1

A rapid and simple method using a U-shaped glass apparatus (Fung-Yu tube) for early determination of the presence of Listeria monocytogenes and Listeria species in mixed cultures and inoculated meat samples has been developed. This system utilizes unique biochemical and physical properties of Listeria for selective enrichment. Fraser broth was used as a selective enrichment broth especially for observation of esculin hydrolysis (blackening of broth), and semisolid Modified Oxford agar was used for selective detection of motility of Listeria. When Fung-Yu tubes containing 0.1 unit/mL of Oxyrase^TM (membrane fractions of Escherichia coli) were inoculated with L. monocytogenes, an enhanced early growth of L. monocytogenes occurred. A presumptive positive result for low numbers of L. monocytogenes (1–100 CFU/g) in the presence of large numbers of competitive microflora in pre-enriched (24 h) ground beef samples using the Fung-Yu tube method with the aid of Oxyrase^TMwas obtainable within 10 h. Using this system, isolation of Listeria in the presence of mixed bacterial flora (44 species), such as Bacillus, Escherichia, Klebsiella, Proteus, Salmonella, Shigella, Staphylococcus, and Streptococcus, and in inoculated ground beef was successful in 24–48 h. The Fung-Yu tube procedure is a highly sensitive, selective, and easy-to-use method to separate and isolate L. monocytogenes and other Listeria spp. from other contaminating microorganisms in meats.     

红茶菌中优势微生物发酵儿茶素的变化研究

以10%蔗糖、0.7%绿茶为主要原料制成的糖茶水为培养基质,传统的红茶菌菌膜分离得到的优势微生物裂殖酵母菌、产膜醋酸菌、乳酸菌A、乳酸菌P为试验菌种,在28℃恒温静止进行单一和混合培养12d,每4d取样分析。结果表明:不同菌种单一和混合培养不同时间,其培养液中的各儿茶素及总量存在明显差异,产膜醋酸菌混合培养有促进EGCG、ECG降解的作用。酵母菌、醋酸菌和乳酸菌A、P混合培养,其培养液中酯型儿茶素含量远远高于酵母菌、醋酸菌混合培养液,乳酸菌A、P有抑制酯型儿茶素降解的作用。     

A model of the effect of temperature on the growth of pathogenic and nonpathogenic Vibrio parahaemolyticus isolated from oysters in Korea

Vibrio parahaemolyticus is recognized as the leading cause of human gastroenteritis associated with the consumption of seafood. The objective of this study was to model the growth kinetics of pathogenic and nonpathogenic V. parahaemolyticus in broth and oyster slurry. Primary growth models of V. parahaemolyticus in broth and oyster slurry fit well to a modified Gomperz equation (broth R(2)=0.99; oyster slurry R(2)=0.96). The lag time (LT), specific growth rate (SGR), and maximum population density (MPD) of each primary model were compared. The growth of nonpathogenic V. parahaemolyticus was found to be more rapid than that of pathogenic V. parahaemolyticus, regardless of the model medium. In addition, significant (P<0.05) differences in the growth kinetics between pathogenic and nonpathogenic V. parahaemolyticus in broth were observed at 10 degrees C. When compared to growth in broth, the growth of V. parahaemolyticus was delayed in oyster slurry, and growth was not observed at 10 or 15 degrees C. The Davey and square root models were identified as appropriate secondary models for predicting the LT and SGR, respectively. For the broth model, the average B(f) and A(f) values for LT were found to be 0.97 and 1.3, respectively, whereas the average B(f) and A(f) values for SGR were 1.05 and 1.11, respectively. The model generated in this study predicted an LT that was shorter and an SGR that was similar to those that were actually observed, which indicates that these models provide a reliable and safe prediction of V. parahaemolyticus growth.     

Alternative indicator bacteria analyses for evaluating the sanitary condition of beef carcasses

Sponge samples were obtained from 47 (study 1) and 32 (study 2) beef carcasses in a small plant over 6 months. In study 2, slaughter equipment surfaces were also sampled. In study 1, the Petrifilm method was used to count presumptive Escherichia coli and spread plating on kanamycin esculin azide (KEA) agar with and without 40% added bile was used to count presumptive Enterococcus spp. Qualitative testing for presumptive E. coli and Enterococcus spp. in study 1 was done using lauryl sulfate tryptone broth (LST) + 4-methylumbelliferyl-beta-D-glucuronide (MUG) and KEA + 40% bile broth, respectively. In study 2, LST + MUG was used as a most probable number (MPN) method along with the Petrifilm method. In the two studies, 8 (17.0%) and 11 (34.4%) carcasses were contaminated with presumptive E. coli; all but one contaminated carcass contained <1 CFU/cm2. Presumptive Enterococcus spp. were recovered from 15 carcasses (31.9%) in study 1, but the KEA + 40% bile agar method lacked specificity (only 31.3% of isolates confirmed as Enterococcus spp.) The LST + MUG and Petrifilm methods were significantly (P < 0.05) related in terms of detecting presumptive E. coli, but the presence of presumptive Enterococcus spp. was not significantly related to the presence of presumptive E. coli. However, on slaughter plant equipment in Study 2 there was a statistically significant (P < 0.05) relationship between the presence of presumptive E. coli and presumptive Enterococcus spp. In study 2, there was no significant (P < 0.05) difference in numbers of presumptive E. coli (obtained using Petrifilm) on carcasses chilled 1 day (n = 16) and 7 days (n = 16), although more of the 7-day carcasses were contaminated (five and seven carcasses, respectively). For samples testing positive for presumptive E. coli, the 95% confidence intervals obtained using the LST + MUG MPN method included the Petrifilm value for all but one sample.     

Efficacy of chromocult coliform agar for coliform and Escherichia coil detection in foods

Chromocult coliform agar (CCA) was compared with Petrifilm Escherichia coli count plate (PEC) for identifying coliforms and E. coli in a variety of meat products. Products examined included 45 raw beef samples, 12 sausage emulsion samples, 11 samples of meat-based ready-to-eat appetizers, and 8 pork trimming samples. Coliforms from CCA and PEC were confirmed by gassing in brilliant green lactose broth plus a positive reaction on purple broth agar plus lactose after incubation at 35 degrees C for 48 h. Lauryl sulfate tryptose plus methylumbelliferyl-beta-glucuronide and tryptophan broth were used to confirm E. coli from CCA and PEC with 48-h incubations at 35 and 42.5 degrees C, respectively. API 20E test strips were inoculated for final confirmation. The overall respective confirmation percentages (CFU/g) for the PEC and the CCA methods were 93.1 and 93.7% for coliforms and 99.8 and 98.1% for E. coli, although the CCA method yielded significantly (P < 0.001) higher mean CFU/g values for both coliforms and E. coli. Regression analyses of these data indicated a strong positive linear relationship existed between the two methods over a wide CFU/g range for both coliforms and E. coli. The respective correlation coefficients obtained for coliforms and E. coli of 0.89 and 0.86 indicate that the CCA method provides a reliable optional method for these determinations in meat products.     

HEAT STABILITY OF STAPHYLOCOAGULASE MEASURED BY A CAPILLARY TUBE METHOD

Staphylocoagulase is highly heat resistant. Inactivation profiles of crude staphylocoagulase at 80, 100 and 121°C showed that total inactivation occurred after heating for 5 h,2 h and 30 min, respectively. Heat treated coagulase has the ability to reactivate when placed at 25°C for 24 hrs similar to previous observations on staphylococcal enterotoxins B and C. Staphylocoagulase could be recovered from beef broth and chicken broth before or after heating at 80°C for 5 min. Coagulase activities were measured by a quantitative capillary tube method.     

Determination of the occurrence of Arcobacter butzleri in beef and dairy cattle from Texas by various isolation methods

Arcobacter butzleri is a pathogenic bacterium that has been found in dairy cattle, pigs, poultry, and humans. As of this writing, there are no data on the incidence of A. butzleri in beef cattle. Given the differences in rearing practices used for feedlot cattle and those used for dairy cattle, differences in the incidences of this organism in various types of cattle may also exist. Numerous culture methods have been used to isolate A. butzleri, but there are few data on the comparative efficacies of these methods. The objectives of this study were to determine the incidence of A. butzleri in cattle from Texas and to compare the effectiveness levels of the Johnson-Murano (JM) method (consisting of enrichment in JM broth followed by plating on JM agar) and the Collins method (consisting of enrichment in EMJH-P80 broth followed by plating on Cephalothin, Vancomycin, and Amphotericin B [CVA] agar) in the isolation of this organism. Fifty cattle each from two feedlots, a dairy, and a stocker yard were sampled. Fecal swabs were obtained from cattle, and each sample was cultured by the JM method, the Collins method, and combinations of the two methods with the broth of one method being used with the agar of the other. Polymerase chain reaction was used to identify the isolates for confirmation of A. butzleri. Samples from 18 of 200 cattle tested positive for A. butzleri. This organism was detected by the JM method in 4.5% of the samples and by the Collins method in 2.5% of the samples. An incidence of 4.0% was found when JM broth was used with CVA agar, while no samples tested positive for A. butzleri when EMJH-P80 broth was used with JM agar.     

分光光度法测定发酵液中L-精氨酸含量

建立了定量测定发酵液中L-精氨酸含量的方法。以百里酚的次溴酸钠溶液为显色剂,用正丁醇萃取显色产物,以分光光度计比色测定有机相的吸光度值。测定发酵液中L-精氨酸的最佳条件为:取待测物稀释液5.0mL,依次加入0.03%的百里酚溶液2.0mL,0.7%的次溴酸钠溶液0.5mL,摇匀,30s内加入正丁醇5.0mL,除去水相;向有机相中加入0.4mL无水乙醇,室温放置1min,用分光光度计测定OD480。方法的检出限为2.5μg/mL,摩尔吸光系数ε为1.2×104L/(mol.cm),发酵液样品相对标准偏差为0.89%～1.10%,回收率为97.3%～102.0%。此方法简便、快速、准确可靠,适合用于发酵液中L-精氨酸含量的定量检测。     

Estimation of the Thurstonian model for the 2-AC protocol

The 2-AC protocol is a 2-AFC protocol with a “no-difference” option and is technically identical to the paired preference test with a “no-preference” option. The Thurstonian model for the 2-AC protocol is parameterized by δ and a decision parameter τ, the estimates of which can be obtained by fairly simple well-known methods. In this paper we describe how standard errors of the parameters can be obtained and how exact power computations can be performed. We also show how the Thurstonian model for the 2-AC protocol is closely related to a statistical model known as a cumulative probit model. This relationship makes it possible to extract estimates and standard errors of δ and τ from general statistical software, and furthermore, it makes it possible to combine standard regression modelling with the Thurstonian model for the 2-AC protocol. A model for replicated 2-AC data is proposed using cumulative link mixed models. Examples will be given throughout the paper and the methodology is implemented in the authors’ free R-packages sensR and ordinal.     

分光光度法测定乳酸菌发酵体系中甘露醇的含量

建立了一种比较简便和精确的分光光度分析法,用以测定乳酸菌发酵体系中的甘露醇含量。通过与盐酸共热脱水反应去除发酵体系中果糖对甘露醇分析测定的干扰和影响。精密度试验和回收率试验表明,此法准确可靠     

Recovery of presumptive Alicyclobacillus strains from orange fruit surfacest

Objectives of this research were to investigate the detection frequency of presumptive Alicyclobacillus strains, also known as thermoacidophilic or acidothermophilic bacteria, on oranges entering juice-processing facilities and to compare results from three common isolation agars (acidified potato dextrose agar, Ali agar, and K agar). A total of 1,575 fruits were sampled from three points (ungraded fruits, graded sound fruits, and graded defective fruits) at two juice-processing facilities during two harvest seasons. Buffer used to rinse individual fruits was assayed for the presence of thermoacidophilic bacteria using an enrichment procedure. Isolates were considered presumptive Alicyclobacillus if they were gram-positive, sporogenous, rod-shaped bacteria, with growth at 45 degrees C and no or slight growth at 25 degrees C on a low pH medium (pH 3.7) coupled with lack of growth on a neutral pH medium (pH 7.0) at both temperatures and a lack of growth in Sulfobacillus broth medium (pH 2.0). More than one third of all fruits sampled at the two facilities were contaminated with presumptive Alicyclobacillus strains. Therefore, incoming fruits are a substantial means by which these organisms gain entrance to the processing facility. Significantly (P < or = 0.05) more detection was observed with a mineral-containing medium (Ali agar) than with nonmineral containing media (K agar and acidified potato dextrose agar).     

外界条件对冬虫夏草胞外多糖构象的影响

以人工培养冬虫夏草菌丝发酵液中分离纯化的胞外多糖EPS-1A为原料,利用紫外光谱和圆二色谱研究不同外界条件如处理温度、金属离子(Ca2+)、变性剂(尿素)及化学修饰剂(刚果红)对胞外多糖EPS-1A构象的影响。结果表明：刚果红反应揭示胞外多糖EPS-1A为无规线团链构象;外界条件均不能改变胞外多糖EPS-1A在溶液中的无规线团链构象,只是在不同程度上影响多糖分子的分子内和分子间氢键相互作用,使多糖变得松散、无序。     

Short communication: Heritability of susceptibility to infection by Mycobacterium avium ssp. paratuberculosis in Holstein cattle

Johne's disease in cattle is the result of infection of the small intestine by Mycobacterium avium ssp. paratuberculosis (MAP), leading to an incurable inflammatory bowel disease (Johne's disease or paratuberculosis). The disease is a concern both for its direct cost to dairy producers and for its zoonotic potential. The objective of this study was to estimate the heritability for susceptibility to infection of cattle by MAP using Johne's testing records (ELISA test for presence of antibodies to MAP in milk or blood) from US Holstein cattle from 2009 to 2016. Data sets were edited to include records from herds with 100 or more total records and sires with 50 or more daughters. Data sets were further edited to include (1) only herds with at least 1 positive test, (2) herds with at least 2.5% positive test results, and (3) herds with at least 5% positive test results to examine the effect of data from herds with higher proportions of positive tests, and presumably higher pathogen exposure, on heritability estimates. Two models were used in this study, a linear sire model and a binary threshold-probit sire model. Both were mixed models considering fixed effects of herd and age at test, the latter as a covariate accounting for linear and quadratic effects; random effects included sire and residual. Analyses were conducted using a restricted maximum likelihood method. Heritability estimates (±standard error) from the linear model were 0.041 ± 0.004, 0.050 ± 0.004, and 0.062 ± 0.007 for data from herds with at least 1 positive test, ≥2.5% positive tests, and ≥5% positive tests, respectively. Heritability estimates from the threshold model were 0.157 ± 0.014, 0.174 ± 0.016, and 0.186 ± 0.021 for data from herds with at least 1 positive test, ≥2.5% positive tests, and ≥5% positive tests, respectively. Heritability estimates from the linear model were affected by population incidence for positive tests, in contrast to estimates from the threshold model, likely accounting for the difference in magnitude of heritability estimates between models and suggesting that the threshold model analysis is the better choice. Heritability estimates increased as data were restricted to herds with presumed higher MAP exposure for both linear model and threshold model analyses. These estimates are similar to previous estimates in other dairy cattle populations and suggest the potential for selection to lessen susceptibility to MAP infection.     

Development of a Simple Method for Detecting Presumptive Escherichia coli on Fresh Retail Beef

ABSTRACT: A simple method of detecting Escherichia coli on retail beef was developed. Diluent used in sponge-sampling of meat was added to a tube containing double-strength lauryl sulfate tryptone broth + 5-bromo-4-chloro-3-indolyl β-D-glucuronide (X-GlcA) and a Durham tube. Tubes with blue color and gas after 24 h incubation at 35 °C were presumptive-positive. Chi-squared analysis showed a significant ( P < 0.05) relationship between the method and the Petrifilm™ E. coli /Coliform Count Plate method for E. coli in analysis of 66 retail beef samples and the 2 methods had a similar proportion of confirmed isolates (61 and 63%). The method developed is qualitatively comparable to the Petrifilm™ method and provides small-scale beef processors a simple way to monitor sanitary conditions in fabrication and grinding operations.     

野生酵母菌在膜生物反应器中的适应性培养与发酵动力学研究

进行了野生酵母菌在硅橡胶膜生物反应器长期封闭循环连续发酵环境中的多轮发酵-优势菌株筛选-转移培养的适应性进化实验,研究了菌株的发酵性能变化和发酵动力学.发酵实验共进行了5轮,每轮发酵持续时间500h,且采用前一轮发酵残液中筛选出的优势菌株作为发酵菌种.第4轮、第5轮发酵实验在乙醇产率、乙醇生成比速率、细胞比生长率等发酵性能上较第一轮有显著提高.多轮适应性培养有效增强了酵母细胞对乙醇的耐受力以及缩短了发酵适应期.