Rapid tests for salmonella

Обновлено: 28.03.2024

As well as being the cause of enteric (typhoid) fever, an important infectious disease, Salmonella is perhaps best known as a cause of bacterial food poisoning. Although typhoid fever has been largely eradicated in the developed world, Salmonella food poisoning has long been, and continues to be, an important global public health problem. In much of Europe and North America, Campylobacter is now the most frequent cause of foodborne human infections, but Salmonella remains a very important and widespread pathogen. It is a major cause of concern for the food industry, where its control is vital for products ranging from cooked meats to chocolate and from fresh produce to peanut butter.

Given the long history of foodborne salmonellosis, it is not surprising that the need for microbiological testing of food ingredients and food products is very significant. A substantial number of methods, both traditional and rapid, have been developed over the years for the detection and identification of Salmonella.

Salmonella enterica

Bacteria of the genus Salmonella are Gram-negative, facultatively anaerobic, non-spore forming, usually motile rods belonging to the family Enterobacteriaceae and primarily associated with animals. The genus currently contains just two species, Salmonella enterica (including six subspecies) and Salmonella bongori. Most of the Salmonella isolates from cases of human infection belong to Salmonella enterica subspecies enterica. The genus is also further subdivided into approximately 2,500 serovars (or serotypes), characterised on the basis of their somatic (O) and flagellar (H) antigens.

Until recently, individual serovars were referred to as if they were species, for example Salmonella typhimurium. However the current convention is to refer to this serovar as Salmonella enterica subsp. enterica serovar Typhimurium. Fortunately, it is customary to shorten this to Salmonella Typhimurium. The commonest serovars associated with human disease are S. Typhimurium and S. Enteritidis, but many others have been shown to cause disease, notably S. Infantis, S. Virchow and S. Newport.

Individual serovars can be further characterised (typed) by a number of methods, including phage typing and antibiotic resistance profiles. The most severe form of Salmonella infection is typhoid fever caused by serovars adapted to a human host, such as S. Typhi and S. Paratyphi. But infection by non-typhoid salmonellae is much more common and usually causes gastroenteritis, with symptoms including diarrhoea, abdominal pain, nausea and vomiting lasting from 1-7 days. Healthy adults rarely suffer other symptoms and the mortality rate is

The incidence of salmonellosis has been falling steadily in Europe since the mid 1990s. In 2011, approximately 95,500 cases of human salmonellosis were recorded. This almost certainly represents considerable under-reporting, and the real number of cases could be a factor of 10 to 100 times greater. A similar pattern has been seen in the USA, but the incidence has remained steady at roughly 15 cases per 100,000 people since 2001. One reason for the decline in cases in the late 1990s was better control of S. Enteritidis in egg production. Outbreaks of foodborne salmonellosis are still common and have been associated with a very wide variety of foods, including dairy products, eggs, fruit juice, fresh produce, herbs and spices, chocolate confectionery, cereals, cooked and cured meats and ice cream. For example, a outbreak in the USA in 2009 was caused by S. Typhimurium in peanut butter and peanut paste and affected nearly 700 people nationwide. Salmonella is a very common component of the gut microflora of animals, including humans, other mammals, birds, reptiles and amphibians, and is thus found in their faeces. Faecal pollution is the main route by which food and water supplies become contaminated and largely accounts for the ubiquity of Salmonella in the food supply chain. Food animals, especially poultry and pigs, can also become infected and act as reservoirs of Salmonella.

Salmonellae are not heat resistant and do not grow at low temperatures, but they are surprisingly tough and are not killed by freezing. They may also survive well in acid foods and resist dehydration. This means that, while not able to multiply in many processed foods, if contamination is present, it can be difficult to eradicate. For example, fatty foods can protect the cells from quite severe heat treatments making pasteurisation ineffective.

Detection and Isolation

Storage and Preparation of Samples - Stool samples are the most frequently tested clinical materials for Salmonella. Animal faeces and water sources may also be tested. Large numbers of food ingredients and food products are routinely tested by the food industry, since the presence of Salmonella in any ready-to-eat food is not acceptable. A wide variety of foods may be tested, but meat products, eggs and dairy products are a particular concern. Other foods and ingredients where regular tests are required include, chocolate confectionary, herbs and spices, fresh salads, fruits, seeds and nuts, flour and shellfish. Sampling from animal carcases at slaughter may also be carried out.

Salmonella is not able to grow at low temperatures and samples should be refrigerated if they cannot be sent for analysis immediately. The cells survive well in frozen foods and other materials, but samples should be kept frozen prior to testing. Typically, 25g food samples are cultured in detection testing, but dried foods require a resuscitation stage for sub-lethally damaged cells in non-selective pre-enrichment media, such as buffered peptone water, before further culture. Furthermore, some dried foods, notably herbs and spices and dried onions, contain compounds that could inhibit Salmonella growth in enrichment cultures. These compounds should be neutralised, either by the addition of a suitable neutralising agent, or by additional dilution.

There is a current ISO horizontal method, ISO 6579: 2002, for the detection Salmonella spp. in food and animal feed. The method was amended in 2007 to include testing of animal faeces and environmental samples from primary production. Similar standard methods have been published elsewhere by other bodies, notably in the USFDA Bacteriological Analytical Manual (BAM). The first stage in traditional detection methods for most food samples is usually a pre-enrichment culture in a non-selective liquid medium such as buffered peptone water, incubated at 37 o C for 18 hours. Modified pre-enrichment methods may be necessary for samples containing inhibitory compounds. The pre-enrichment culture is then typically subcultured into two different selective enrichment media, such as Rappaport Vasiliadis Soy broth (RVS) and Muller-Kauffmann Tetrathionate-Novobiocin (MKTTn) broth, and incubated for a further 24 hours at 41.5 o C (RVS) or 37 o C (MKTTn).

The selective enrichment culture is usually inoculated on to at least two selective agar media and incubated at 37 o C for 24 hours. The ISO method specifies the XLD agar and one optional selective medium. A variety of alternatives are available, including Bismuth Sulphite agar, Brilliant Green agar and Hektoen Enteric agar. A number of selective chromogenic agar media specifically designed for the differentiation of Salmonella colonies are commercially available. Typical Salmonella colonies on selective agar are subcultured onto non-selective media prior to confirmatory testing.

Rapid Methods - It can take at least three to five days to obtain a result using traditional methods of detection for Salmonella spp. For this reason a substantial number of alternative rapid screening methods have been developed to produce results more quickly for food and environmental samples. Many of these are available commercially and have been successfully validated by the AOAC and/or AFNOR. The AOAC database of performance tested methods contains more than 40 products for the rapid detection of Salmonella.

Salmonella rapid test and screening kits utilise several different technologies, including novel culture techniques, immunomagnetic separation, EIA- and ELISA-based assays incorporating fluorescent or colorimetric detection, simple lateral flow assays incorporating immunochromatographic technology, and molecular techniques such as DNA hybridisation and PCR-based assays, many of which now include real-time detection. Some methods can be automated to screen large numbers of samples. Almost all rapid test protocols include a selective enrichment stage, and then apply concentration and/or rapid detection techniques to replace culture on selective agars and further confirmatory tests. Most can claim to produce a result in approximately 48 hours or less, depending on the enrichment protocol.

Confirmation and Identification

Traditional Methods - There are well established confirmation and identification procedures for Salmonella spp. Preliminary identification based on colony appearance on chromogenic and other selective agar media is traditionally confirmed using classical biochemical tests for Salmonella and serological testing. Key biochemical tests are fermentation of glucose, negative urease reaction, lysine decarboxylase, negative indole test, H2S production, and fermentation of dulcitol. Serological confirmation tests typically use polyvalent antisera for flagellar (H) and somatic (O) antigens. Isolates with a typical biochemical profile, which agglutinate with both H and O antisera are identified as Salmonella spp. Where results are inconclusive, it may be necessary to perform additional biochemical tests for Salmonella.

Positive isolates are often sent for further serotyping to identify the serovar using specific antisera. However, this is normally a task for specialist reference laboratories and is rarely undertaken in routine food or clinical laboratories. Reference labs are also able to type isolates further using techniques such a phage typing, antibiotic susceptibility and pulsed-field gel electrophoresis (PFGE).

Rapid methods - Many rapid confirmation and identification methods have been developed for Salmonella and a large number have been developed into commercial products. In addition many of the rapid detection kits mentioned above can also be used for confirmatory testing. Biochemical confirmation can be accomplished using commercial identification systems.

Rapid immunological identification and confirmation tests based on latex agglutination, enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) have been developed for Salmonella, and simple-to-use lateral flow test strips using immunochromatographic technology have also been developed into commercial products by a number of manufacturers.

Molecular methods are also available, notably DNA hybridisation and PCR assays for the identification of Salmonella enterica. However, these are generally designed for use as part of a method for rapid detection and screening rather than for confirmation. A range of DNA-based typing techniques have also been developed for use by specialist laboratories involved in the investigation of food-borne disease outbreaks. Even whole genome sequencing has become a practical tool for the characterisation of Salmonella strains implicated in outbreaks.

Salmonella growing on SS agar

Article by Dr. Suzanne Jordan, Molecular Methods Manager, Campden BRI

The foodborne pathogen Salmonella has been reported to cause approximately 100,000 human cases of salmonellosis annually in the EU. Salmonella spp. have been isolated from many sources including water, soil, food contact surfaces, raw meats, poultry, fresh produce, nuts and seeds, and infant formula. The foods associated with salmonellosis include meat and poultry, eggs, chocolate, smoked salmon, infant formula, peanut butter and fresh produce.

To demonstrate effective control of this pathogen, food businesses analyse products for the presence of Salmonella. The current European recognised detection method for Salmonella spp. is ISO 6759:1 2017. ISO 6759:1 2017 is a culture based method with four consecutive steps:

  • Non-selective enrichment to resuscitate injured or stressed Salmonella
  • Selective enrichment to inhibit background flora
  • Subculture of the selective enrichment onto selective differential agars to allow growth of Salmonella and distinguish them from other background flora
  • Confirmation of presumptive positive Salmonella colonies

Confirmation includes biochemical tests and the detection of Salmonella specific structures using antibodies (known as serology). The ISO method gives a presumptive positive result in three days and confirmed results in five days.

Advances in methodology have resulted in a raft of rapid techniques for Salmonella detection. The benefits of rapid methods include the potential for automation, reducing the time to result, and ease of interpretation of results. Rapid methods can be split into three categories:

  • Chromogenic media
  • Antibody-based techniques
  • Molecular-based detection assays.
  • Chromogenic media detect specific biochemical characteristics for Salmonella spp, which are typically enzyme breakdown of unique chemicals (substrates). In chromogenic agar, the substrate of interest is attached to a dye molecule that remains ‘dark’ when the complex is intact. As Salmonella grows, it produces the specific enzyme which degrades the link between the substrate and dye and forms coloured colonies. Chromogenic media improves the ability of laboratory staff to highlight presumptive colonies, which limits the number of confirmation tests needed.
  • Immunological methods detect unique Salmonella molecules using two antibodies; a surface-bound primary antibody to capture the target molecule and a reporter antibody to detect the antibody target complex. Immunological techniques can replace isolation agars, lowering the time to presumptive positive result to one to two days. The immunological assays available for Salmonella detection are lateral flow devices (LFD), enzyme linked immunosorbant assay (ELISA), and Enzyme-linked fluorescent assay (ELFA). LFDs follow the format of pregnancy test kits, giving results within 20 minutes of the enriched sample being loaded into the device. ELISA and ELFA are more complex and incorporate washes between capture and reporter steps to remove non-target molecules that cause false positive results.The adoption of automated ELISA and ELFA systems enables high sample throughput with presumptive results within 19 to 25 hours. ELFA sensitivity has been increased in recent years by using bacteriophage as capture molecules in place of antibodies.Antibodies can also be coated onto magnetic beads in a technique known as immunomagnetic separation (IMS). IMS concentrates Salmonella from food samples for either subculture for colony isolation or as part of the sample preparation for molecular-based detection assays. The IMS removes inhibitors from food matrices and other background flora that can impede assay performance.
  • Molecular-based detection of Salmonella spp. delivers one of the fastest times to result, with run times after enrichment and DNA extraction of 45 min to 3h. The predominant molecular approach is polymerase chain reaction (PCR) that detects DNA regions specific to Salmonella. Commercial PCR detection kits are usually in a user-friendly format for sample set up and result interpretation. More recent advances in molecular assays have introduced isothermal amplification methods which operate at a single temperature rather than cycling through a set temperature profile. Isothermal amplification systems tend to be cheaper, take up less bench space, and have a time to detection of 45 to 90 minutes from the enrichment culture.

A range of ‘off the shelf’ Salmonella detection assays are available for food analysis. When choosing a new method, several factors need to be considered, including ensuring that they are fit for purpose. Method performance is a key point in this decision-making process. Several Salmonella detection kits possess third party method assessments by organisations including AOAC, MicroVal, and NordVal. Campden BRI is an expert laboratory for these validation organisations and carries out validations to recognised ISO16140 standards.

All commercially produced Salmonella detection kits still require confirmation of presumptive positive results. Advances in Salmonella confirmation techniques have improved turn around time and result interpretation. Miniaturised biochemical galleries were the first development for Salmonella confirmation. These galleries rely on either fermentation of a set of substrates, or the detection of characteristic enzyme activities. Commercial automated biochemical identification systems have further reduced the set-up time and the subjectivity when interpreting results because colour changes are read by computers rather than by eye.

In recent years, another instrument based identification system Matrix Assisted Desorption Ionisation time of Flight (MALDI-ToF) has been introduced. (MALDI Biotyper Receives OMA for Identification of Food Pathogens.) The MALDI-ToF instrument directs a laser onto the prepared colony sample which has been dried onto a metal plate. This generates a profile of fragments that are detected within the system. Each isolate specific pattern created is compared to a library to identify the bacteria. MALDI ToF provides a very rapid time to result of approx 15 minutes and the potential for high sample throughput. Campden BRI is now offering a rapid Salmonella confirmation service to genus level using the MALDI-ToF.

The evolution of DNA sequencing has significantly reduced analysis costs, making whole genome sequencing (WGS) possible for Salmonella spp. WGS provides the full genetic code for a pure culture which can be analysed in depth to provide sub -type strain analysis to answer different questions, such as:

  • Are two organisms the same or different?
  • Does an organism contain a particular gene (such as antibiotic resistance genes)?

Salmonella detection methodology is continually evolving, with many advances becoming available in the last decade. The improvement in time to results for both detection and confirmation enable industry to act in a timely response to safety and contamination incidents.

About the author: Dr. Suzanne Jordan joined Campden BRI as Molecular Methods Manager in 2005 following nine years research experience in molecular biology of food microorganisms. Suzanne is involved in several research projects developing and evaluating new methodology as well as lecturing on a range of in house and custom microbiology courses. In addition to research work, Suzanne provides consultancy in molecular based detection, identification and tracking methods to the food and allied industries.

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Poultry Food Safety Testing - Salmonella and Campylobacter

Combined detection of Salmonella and Campylobacter in poultry

Poultry has become one of the largest animal-based protein sources globally, with industrial-scale broiler houses and processors dealing with a huge tonnage of material on a daily basis. Ensuring a microbiologically safe, reliable end product involves vigilance at every stage of the poultry supply chain from farm to fork.

The top causative organisms of food poisoning - Salmonella and Campylobacter are commonly found in poultry meat and outbreaks can often be traced back to these type of products. Whilst the introduction of HACCP and HARPC quality systems can be effective, it is difficult to eliminate Salmonella and Campylobacter from processing areas as they are continually being reintroduced via leakage of intestinal contents/faeces during processing, contaminated processing equipment, water, and the hands of processing workers.

Whilst washes using chlorine have been since been replaced with other antimicrobials such as peracetic acid, cetylpyridinium chloride, and acidified sodium chlorite, but biofilms of Salmonella and Campylobacter can be an issue therefore even with hygiene monitoring systems in place, in-process and final product testing for Salmonella, Campylobacter, E.coli and Enterobacteriaceae is a vital component of delivering safe food and maintaining brand reputation. In 2016, USDA Food Safety and Inspection Service (FSIS) implemented the use of neutralizing Buffered Peptone Water (nBPW) containing lecithin or equivalent and sodium thiosulfate, whilst the use of nBPW is not a requirement for in-house pathogen testing, producers may choose to use it for carcass rinses and parts sampling to be consistent with the FSIS testing regime.

Campylobacter in Poultry

The most common Campylobacter species associated with gastroenteritis in humans are C. jejuni and C. coli, but there are also other emerging species such as C. consicus, C. upsaliensis, C. ureolyticus, C. hyointestinalis and C.sputorum. Camplyobacters are gram-negative, motile, curved rods that are microaerophilic and present in the intestinal tract of birds where it can be passed between chicks within a flock through the faecal-oral route. Some studies have shown C. jejuni to be present in three-quarters of supermarket chickens. Food poisoning from Campylobacter requires a small infectious dose, as few as 500 cells with its pathogenicity depending on the host's age/health and strain of the pathogen.

Traditional Campylobacter testing involves broth enrichment followed by detection on selective agar growing in microaerophilic atmospheric conditions at 42ºC which as they are thermophiles, favours the growth of Campylobacter spp. These modified atmospheres require use of either jars, pouches, incubators or workstations. Visit our 'Anaerobic and Microaerophilic - Systems for Creating Modified Atmospheres' test method for suppliers.

Salmonella in Poultry

The Salmonella genus has only two species S. enterica and S. bongori but there are many, many Salmonella serovars. The most common serovars associated with food poisoning outbreaks are two S. enterica serovars: - S. ser. Enteritidis and S. ser. Typhimurium. In order to track the source of a positive result or an outbreak, the serovar has to be identified, traditionally this is done using a slide based anti-sera agglutination test, whilst this will determine the serovar, more up to date molecular based approaches will better identify exact strain type.

Prevalence of Salmonella in chicken at retail is at approximately 5% in Europe with Salmonella enterica been the most prevalent pathogen. Its infectious dose is as little as 15-20 cells, and its pathogenesis depends on age and health of host as well as the strain of the pathogen.

Indicator Organisms - What Are They and Why Use Them?

Because detection of specific food pathogens can be time consuming and expensive, it can be useful to do an easier, more generic test for indicator organisms or Enterobacteriaceae which are a large family of bacteria containing coliforms and E.coli, commonly found in faecal material - basically the same family as many kinds of pathogens such as Salmonella and E.coli. These tests can be easily done via dry-film (ready to rehydrate) plates or ready-to-use chromogenic media, the chromogen in the media will give an approximate indication of numbers of Enteros present which can be used as a trending tool, showing that the right conditions for actual pathogens may exist, so alerting the need for proactive remedial measures such as cleaning.

Choosing a Rapid Microbiology Method for Testing of Poultry

The early detection and identification of the presence of contaminant microorganisms through leveraging rapid pathogen testing methods help avoid pathogen-carrying products from reaching the consumer and also prevents quality or productivity bottlenecks in product processing. The aim of any new approach to pathogen testing in broiler chickens and turkeys should be to:

  • improve the accuracy of the results - poultry meats are a complex matrix with high background competitive flora
  • reduce the need for confirmation of presumptive positives
  • improve workflow and be capable of handling high throughput workloads
  • reduce technical staff input that may introduce subjective variability
  • reduce time to result so that remedial action can be taken sooner - sensitive molecular methods mean a shorter enrichment time can be used, with total time to result of less than 20 hours

When considering a new technology for your poultry testing lab, find out what steps are required before the sample is added to the automated molecular platform, does the protocol fit in with your work practices - will the faster results be delivered same-day/same-shift (no point in paying a higher cost per test if the results are not actionable until the following day), is the supplier working towards a multiplex option whereby one assay will detect more than one organism? Do different assays require different run protocols which can lead to workflow blockages?

Molecular based kits use either PCR or LAMP technology - What's the difference between PCR and LAMP (Loop Mediated Isothermal Amplification)? watch the rapidmicrobiology video here

Ji Youn Lim at AMDIlabs

The PBM Salmonella (PBMS) test kit based on an mmunochromatographic method was evaluated for the screening of Salmonella spp. in pure cultures, and 80, 15, and 10 artificially and naturally contaminated, and negative controlled food samples, respectively. The PBMS test involves presumptive qualitative procedures, detecting the presence of Salmonella spp. in foods within 26 h total testing period and allowing the user to release negative products 70 h earlier than the conventional methods. The PBMS test using Buffered Peptone Water and Rappaport-Vassiliadis broth was evaluated for 10 different food types for various Salmonella spp. It showed detection limits of 1 to 25 colony forming units (CFU)/25 g. No cross-reaction was observed, particularly to other gramnegative bacteria. These results indicate the PBMS test is a rapid and inexpensive procedure for the screening of Salmonella spp. present at low concentrations (1 to 25 CFU/25 g) in foods.

. Analysis of artificially contaminated samples by PBMS test

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Department of Microbiology , College of V eterinary Medicine and School of A gricultural Biotechnology

ABSTRACT : The PBM BioSign TM Salmonella (PBMS) test kit ba sed on an immunochromatographic method was evaluated for the

screening of Salmonella spp. in pure cultures, and 80, 15, and 10 artificial ly an d naturally contaminated, a nd negative controlled food

samples, respectively . The PBMS test involves pres umptive qualitativ e procedures, detecting the presence of Salmonella spp. in foods

within 26 h total testing period and allowi ng the user to release negative products 70 h earlier than the conv entional methods. The PBMS

test using Buffered Peptone W ater and Rappaport-V assiliadis broth was evaluated for 10 different food types for various Salmonella spp.

It showed detection limits of 1 to 25 colo ny forming units (CFU)/25 g. No cross-reac tion was observed, particularly to other gr am-

negative bacteria. These results indicate the PBMS test is a rap id and inexpensive procedure for the screening of Salmonella spp. present

at low concentrations (1 to 25 CFU/25 g) in foods. (Asian-Aust. J. Anim. Sci. 2004. V ol 17, No. 12 : 1746-1750)

Figure 1. Procedures of conventional method a nd the PBMS test. The major differences between the two methods are the pre-

enrichment incubation period and th e amount and level of RV broth. 1 Lactose broth (LB, Difco), 2 Hektoen enteric (HE, Difco) agar,

This book is written by leading researchers of Salmonella from Europe, North America and Australia. It provides an up-to-date review of work on all aspects of Salmonella in farm and companion animals, including characteristics and biology, virulence, effects on the host, antibacterial resistance, epidemiology, disease prevention and laboratory methods. This book is an essential reference for researches in animal science, veterinary medicine and microbiology and those working in the public health sector.

The objective of this study was to evaluate the effects of various organic acids on microbial characteristics and Salmonella typhimurium in pork loins. Fresh pork loins were sprayed with various organic acids such as lactic acid, citric acid and acetic acid at various concentrations (0.5, 1, 1.5 and 2%). After spraying, the samples were packaged by HDPE film under air and stored at for 14 days, and analyzed. Microbial deterioration of pork loins during the aerobic cold storage was delayed by organic acid spray. The bactericidal effect of acids increased with the increasing concentration. However, the inhibitory activity of organic acids during the storage varied with the kinds and concentrations of the acids. As for total plate counts, acetic acid was found to have the highest bactericidal activity, whereas citric acid was found to be the most inhibitory for coliform and S. typhimurium.

In vivo antagonistic effect of Lactobacillus helveticus CU 631 and Lactobacillus spp. against typical enteritis causing pathogen Salmonella enteritidis KU 101 have been determined, which showed an increase in survival rate and the decline in viable cell numbers of pathogen in liver and spleen at sacrifice. A signifcant difference in the antagonistic effect against KU 101 were observed, which was species and/or strain dependent of Lactobacillus (p<0.01), the survival rate of the mice in the Salmonella infection by feeding L. helveticus CU 631 has been shown to be 157%, whereas those of L. rhamnosus GG ATCC 53103, L. acidophilus ATCC 4356, L. johnsonii C-4 were 137%, 132%, 119% respectively on the basis of lactobacilli non-associated control KU101 fed mice to be 100%. Viable cells of S. enteritidis KU101 in the liver and in the spleen at sacrifice were decreased in Lactobacillus spp. fed group with no significant difference. The higher level of total secretory IgA concentration in the intestinal fluid of lactobacilli fed mice than control mice have been observed. In vitro antagonistic activity of Lactobacillus spp. against KU101 have been determined, a prominent antagonistic activity of CU 631 against KU 101 were demonstrated.

A collaborative study was performed in 13 laboratories to validate an enzyme immunoassay (EIA) procedure for rapid detection of Salmonella in foods. The EIA was compared with the standard culture procedure for detection of Salmonella in 6 food types: ground black pepper, soy flour, dried whole eggs, milk chocolate, nonfat dry milk, and raw deboned turkey. Uninoculated and inoculated samples were included in each food group analyzed. There was no significant difference in the proportion of samples positive by the EIA and culture procedures at the 5% level for any of the 6 foods. The enzyme immunoassay screening method has been adopted official first action as a rapid screening method for detection of Salmonella.

A new method for the detection of Salmonella in foods and feeds, 'The EiaFoss Salmonella Method' has been performance tested in comparison with a traditional technique, the Rappaport-Vassiliadis cultural procedure, as reference method. The new method is based on a two step enrichment procedure (19 and 3 h) using two different pre-enrichment broths (Salmonella enrichment broth, SEB I and SEB II) for raw and processed foods, followed by an automated enzyme immunoassay (EIA) carried out in the 'EiaFoss analyser'. A total of 161 food and nine feed samples were investigated. Only naturally contaminated samples were used. Viable Salmonella were recovered in 30 samples using either method. When using the EiaFoss Salmonella Method 13% more samples (27:24) were proved salmonella-positive compared with the reference method. The explanation for the better performance of the EiaFoss Salmonella Method is discussed and attributed in part to better enrichment procedures when using SEB I or SEB II.

Five commercially available screening methods, the Oxoid MSRV, Merck SALMOSYST-RAMBACH AGAR combination, Organon Teknika SALMONELLA-TEKTM, Dynal DYNABEADS ANTI-SALMONELLA and Foss Electric EIAFOSS, were compared to the conventional culture procedure for the detection of Salmonella in naturally contaminated feed samples. A total of 217 feed samples from animal as well as from vegetable origin were examined. Twenty one samples were found to be positive for Salmonella by all methods combined. The conventional culture method detected 17 (81,0%), MSRV 19 (90,5%), SALMOSYST-RAMBACH 8 (38,1%), SALMONELLA-TEK 19 (90,5%), DYNABEADS ANTI-SALMONELLA 7 (33,3%) and EIAFOSS 21 (100%) of the 21 total Salmonella contaminated samples.

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Lessons Learned and Experiences Gained in Developing the Waterflooding Concept of a Fractured Baseme.

Ngoc Nguyen

Wisup Bae

Huong Phung

Naturally fractured reservoirs (NFRs) represent more than 20% of the world's oil and gas reserves. However, their characterization is complex and presents unique challenges in comparison with conventional reservoirs. It is immensely difficult to achieve the best results in the secondary-recovery process for NFRs. This paper presents a successful development of waterflooding to overcome the . [Show full abstract] complex geological characterization of the White Tiger field, the largest fractured basement reservoir to date on the continental shelf of Vietnam. This reservoir has a complicated geological structure, with high heterogeneity, high temperature, and high closure stress. The total oil initially in place (OIIP) of this field reached nearly 4 billion bbl from 2000 m of oil-bearing thickness, and the field has been produced by more than 100 wells, 10 of which have flowed at the rate of approximately 1,000 B/D. The geological study and fractured model have been carefully investigated in both micro- and macroscale to improve waterflooding performance. The authors have analyzed the advantages and disadvantages of injection systems in this basement reservoir during 20 years of production history, and an artificial water buffer solution has been proposed to improve the waterflooding process. The authors have described the establishment and association of local artificial water buffer in the basement reservoir. An effective method to optimize the injected-water volume has also been discussed. Promising results from the White Tiger field have shown that the average reservoir pressure and total oil recovery have increased significantly in comparison with previous injection schemes. This paper presents useful guidelines to solve some typical problems of waterflooding in fractured basement reservoirs: What can be applied in waterflooding for a fractured basement reservoir? What is the optimal injection rate and injected volume for the fractured basement reservoir? How do we evaluate the probability of high water cut in production wells during the waterflooding process? How do we predict the rise of an artificial water/oil contact (AWOC)?

Determination of diphacinone in pollard baits by paired ion high-performance liquid chromatography

Diphacinone (I) is extracted from the pulverized bait with methanol, and the extract is mixed with pairing-ion buffer (7.8 g l-1 of NaH2PO4 and 5 ml l-1 of 1M-tetrabutylammonium hydroxide adjusted to pH 7.5). The mixture is analysed on a column (25 cm × 4.6 mm) of Partisil-10 ODS, with methanol - pairing-ion buffer (2:3) as mobile phase and detection at 286 nm. I concn. is calculated from the . [Show full abstract] relative peak areas for the sample and the standard I soln. Recoveries are >90% for I added to pollard baits (coeff. of variation <3.5%). The detection limit is 2 mg kg-1

Joaquin Ruiz at Universidad Católica San Antonio de Murcia

Two rapid test for early detection of Salmonella enterica, the cytochrome oxidase test and a fluorescence test, were evaluated in 1,200 colonies which had been isolated from human feces and cultured on MacConkey and Salmonella-Shigella media. Using the fluorescence test there were no false negative results (sensitivity 100%) and of 205 positive cases 62 did not correspond biochemically to salmonella (specificity 94.1%); 44 of these 62 were positive in the cytochrome oxidase test, raising the specificity to 98.2%. In conclusion, the combination of the two tests could be very useful due to the ease of performance, low cost and excellent results obtained.

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. Durante la prueba, los antígenos presentes en la muestra de heces reaccionan con los anticuerpos anti-Salmonella previamente fijados en el test. La inmunofluorescencia en heces tiene una sensibilidad del superior al 95 % y una especificidad superior al 90 %, de acuerdo con múltiples estudios realizados de 1960 a 2000 [18][19][20] [21] . Por lo anterior, es una prueba útil al orientar a iniciar una terapia antimicrobiana de manera inmediata 22 . .

El diagnóstico de la fiebre tifoidea siempre es complejo. No hay sintomatología patognomónica. Históricamente la reacción de Widal (contenida en los antígenos febriles) era útil para su diagnóstico y manejo. Actualmente dicha práctica se perpetúa solo en países en vías de desarrollo, por ser una prueba rápida y barata, sin embargo, poco útil. El diagnóstico de fiebre tifoidea puede efectuarse solo mediante dos pruebas: aislando a la Salmonella typhi o detectando su ADN, mediante cultivo o reacción en cadena de polimerasa, respectivamente. La inmunofluorescencia en heces es una prueba útil, pues orienta a iniciar una terapia antimicrobiana de manera inmediata.

. The MUCAP test applied in this way is also a rapid method for the presumptive diagnosis of Salmonella spp. when it is used in conjunction with the oxidase test (6) . Although other rapid methods have been described (2,4,5), they are not as fast and economical as this one. .

agarandthe752isolated onHektoen agarandidentified aspositive for Salmonella spp.withconventional biochemical tests werefoundpositive withtheMUCAP test(the sensitivity was 100%andthenegative predictive value was 100%forbothmedia). Moreover, onlysixisolates identified byconventional biochemical tests asProteus vulgaris wereMUCAP testpositive (the specificity was99.8%and thepositive predictive value was 99.2%forbothmedia). Onthebasis ofthese results, we proposetheuseof theMUCAP test asamethodforthescreening ofH2S-positive colonies andonlysubculturing on Kliger agar ofthose colonies which areMUCAP testpositive. TheMUCAP testisa rapid methodforthepresumptive detection ofSalmonella spp.andreduces theworkandmaterial involved intesting. Gastrointestinal infections arestill a worldwide public health problem, being amongtheprimary causes ofmedical consultation (3,8).Amongthebacteria responsible forthese syndromes, Salmonella spp.areoneofthemostcommon causes ofbacterial diarrheal disease (7). Theprocessing ofa stool culture inthesearch forSalmonella spp.andother enteric pathogens bymeansoftheusual method isslowand cumbersome. Accordingly, inthis study wetried toreduce boththetimeandtheeffort neededforthediagnosis of Salmonella spp.byusing afluorescent reagent (MUCAP test; Biolife Italiana S.r.l., Milan, Italy) onH2S-positive colonies isolated onsolid media. TheMUCAPtest reagent detects theC8esterase enzyme present inSalmonella spp.andother bacteria. Theenzyme actsonasubstrate formed byanester of8atomsofcarbon conjugated with4-methylumbelliferone, liberating this last compound, whichisstrongly fluorescent at365nm.The reagent issterile andhasnoinfluence ontheviability ofthe colonies towhichitisadded. Thecostofeach5 ,ulis approximately $0.09. Theroutine technique which wefollowed fortheisolation ofSalmonella spp.istheclassical methodofsubculturing H2S-positive orlactose-negative colonies isolated onmedia forenteric pathogens onKliger agar. Depending onthe reactions produced, we continued withbiochemical tests using API20Egalleries (Biomerieux, Montelieu Vercieu, France) andantisera (Difco, Detroit, Mich.) forserogroup identification. Ourstudy concentrated onlyonthose H2S- producing colonies whichgrewonsalmonella-shigella (SS)

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