Detection of enterotoxigenic Clostridium perfringens in food and fecal samples with a duplex PCR and the slide latex agglutination test.

A duplex PCR procedure was evaluated for the detection of Clostridium perfringens in food and biological samples and for the identification of enterotoxigenic strains. This method uses two sets of primers which amplify in the same reaction two different DNA fragments simultaneously: the 283-bp C. perfringens phospholipase C gene fragment and the 426-bp enterotoxin gene fragment. Internal primers within the two primer sets confirmed the specificity of the method by DNA-DNA hybridization with the PCR products. No cross-reaction was observed with other Clostridium species or with other bacteria routinely found in food. The detection level was approximately 10(5) C. perfringens cells per g of stool or food sample. When overnight enrichment culture was used, 10 C. perfringens cells per g was detected in 57 artificially contaminated food samples. The duplex PCR is a rapid, sensitive, and reliable method for the detection and identification of enterotoxigenic C. perfringens strains in food samples. A slide latex agglutination test was also evaluated as a rapid, simple technique for the detection of C. perfringens enterotoxin in stool samples.     

APPLICATION OF A COLONY PCR TECHNIQUE WITH FUNG'S DOUBLE TUBE METHOD FOR RAPID DETECTION AND CONFIRMATION OF CLOSTRIDIUM PERFRINGENS

A colony polymerase chain reaction (PCR) technique was applied with an established Fung's double tube (FDT) method for rapid detection and confirmation of Clostridium perfringens. Published sequences of PCR primers for C. perfringens alpha toxin gene were used and PCR conditions were optimized. From the detection of C. perfringens by FDT tube to the confirmation by a colony PCR assay took as short as 16–18 h. The method was applied to 147 isolates of anaerobic sulfite reducing bacteria isolated from foods, sewages and animal clinical specimens. The results were compared with standard methods for the confirmation of C. perfringens. Of those 147 suspected isolates, 97 and 99 were confirmed as C. perfringens by standard methods and the colony PCR technique, respectively. We found the developed method simple, rapid, cost-effective, and most importantly, very reliable for the detection and confirmation of C. perfringens.

PRACTICAL APPLICATIONS

With Fung's double tube method and a colony polymerase chain reaction technique, a completed determination of Clostridium perfringens contamination can be accomplished within 16–18 h. This saves at least 2–3 days when compared with standard methods. Moreover, it minimizes the cost and labor needed since an anaerobic chamber as well as steps of Gram staining and biochemical testing can be avoided. The developed method is a powerful tool and an alternative for the enumeration of C. perfringens . It can be applied to samples from various sources and highly reliable results are expected. Most microbiological laboratories have a thermocycler and reagents as parts of their basic instruments. Therefore, the developed method can be easily applied without massive investment.     

APPLICABILITY OF RAPID METHODS FOR DETECTION OF SALMONELLA SPP. IN POULTRY FEEDS: A REVIEW

The rapid detection of pathogenic microbial species in feed is of paramount importance considering its implications for animal production and food safety. More sensitive and rapid detection of contaminated feedstuffs may lead to more selective and therefore less expensive treatment of feeds, reduced rates of transmission to a poultry host and reduced carcass contamination. In order to interrupt the cycle of Salmonella spp. transmission from feed to poultry to the consumer, more rapid detection methods to monitor these sources are needed that provide conclusive results within the time frame of feed mixing or broiler processing. Within the last decade, new variations of selective media have been investigated to increase selectivity without reducing Salmonella spp. recovery. Immunological assay methods may also decrease assay time from 96 h to within 24–30 h. But all commercially available methods still require 16 to 57 h for preenrichment, enrichment, and in some cases, postenrichment to recover sublethally injured cells before the assay can be performed. Among the molecular methods that are currently available, the polymerase chain reaction (PCR) represents a tremendous potential for the detection of low levels of pathogenic bacteria. Once optimized, rapid methods may be used to quickly, reliably and inexpensively screen a variety of feedstuffs and feed components for the presence of Salmonella spp., with the goal of minimizing both the cost of feed treatment and the horizontal transmission of Salmonella spp. from feed to poultry.     

产气荚膜梭菌实时荧光PCR方法的建立

目的:利用荧光定量PCR技术,建立快速敏感特异的检测产气荚膜梭菌的方法。方法:以产气荚膜梭菌基因为靶序列设计引物和探针,以自产气荚膜梭菌菌株中提取的DNA为模板,优化引物和探针的浓度比,同时验证方法的特异性、敏感性。结果:建立的反应体系在上游引物浓度为0.45μmol/L、下游引物浓度为0.15μmol/L、探针浓度为0.3μmol/L时,具有良好的特异性和敏感性,与创伤弧菌等12种相关细菌均无交叉反应;对纯菌检测的灵敏度低于10 CFU/反应体系。结论:建立的实时荧光PCR方法特异、灵敏、快速,能对战时气性坏疽做出快速准确的报告,实现对这种战时高发疾病的安全、快速和定量检测。     

Diagnosis of Clostridium perfringens intestinal infections in sheep and goats

Clostridium perfringens produces disease in sheep, goats and other animal species, most of which are generically called enterotoxemias. This micro-organism can be a normal inhabitant of the intestine of most animal species including humans, but when the intestinal environment is altered by sudden changes in diet or other factors, C. perfringens proliferates in large numbers and produces several potent toxins that are absorbed into the general circulation or act locally with usually devastating effects on the host. History, clinical signs and gross post-mortem findings are useful tools for establishing a presumptive diagnosis of enterotoxaemia by C. perfringens in sheep and goats, although no definitive diagnosis of these diseases can be made without laboratory confirmation. Because all types of C. perfringens can be normal inhabitants of the intestine of most animals, culture of this micro-organism from intestinal contents of animals has no diagnostic value unless a colony count is performed and large numbers (usually more than 10(4)-10(7)CFU/g) of C. perfringens are found. The most accepted criterion in establishing a definitive diagnosis of enterotoxaemia by C. perfringens is the detection of its toxins in intestinal contents. However, some of the major toxins of C. perfringens (i.e. epsilon toxin) can also be found, albeit in small amounts, in the small intestine of clinically normal sheep, and this poses a diagnostic challenge. In such cases the histopathology of the brain must be used as an alternative diagnostic tool, since the lesions produced by epsilon toxin in the brains of sheep and goats are unique and pathognomonic for C. perfringens type D enterotoxaemia. Ancillary tests, such as measurement of urine glucose or observation of Gram stained smears of intestinal mucosa can be used and, although they have a presumptive diagnostic value when positive, they cannot be used to rule out a diagnosis of enterotoxaemia if they are negative. In conclusion, the diagnosis of C. perfringens infections in animals is complex and it is appropriate to rely on a combination of diagnostic techniques rather than one singe test.     

Distribution of Clostridium perfringens in polluted lake environments

Many of the directives that relate to the prevention of pollution or the improvement of fresh water also relate to lake waters since lake waters ultimately inherit much of the pollution that enters into fresh water. In order to determine the influence of the water depth on Clostridium perfringens, we utilised a new medium, lactose-sulfite (LS) broth, suggested for rapid enumeration and identification of C. perfringens. Duplicate samples were collected at each one of the following sites of the polluted station: surface, 60 cm, 90 cm and bottom (1.18 cm). Membrane filtration equipment was used. All samples were alternatively passed through two membrane filters, the first (20-25 microm pore size) was used for retention of the abundant phytoplankton and the second (porosity 0.45 microm) for C. perfringens. Membranes were placed into the first tube of ten-fold dilutions from 10(1) to 10(4) and incubated aerobically in a waterbath at 46 degrees C for 24 h. The numbers of C. perfringens fluctuated depending on the water depth. Vegetative forms were found only in the bottom sampling; they were never found in surface, 60 cm and 90 cm sampling sites. Sporulated forms were found in all sampling sites with the exception of the surface sampling. Clostridium perfringens as an anaerobic bacterium never occurred in the surface waters in vegetative or spore forms, even if the waters were extremely polluted by domestic or industrial activities. Vegetative forms occurred only in the bottom samples but spore forms which are more resistant to various environmental effects occurred in all depths except for the surface.     

养猪场空气中抗性基因和条件致病菌污染特征

养殖场空气中含有较高浓度的抗生素抗性基因和条件致病菌,对人畜健康具有潜在威胁.采用中流量TSP采样器在某养猪场的生活区、猪舍内和猪舍外3个地点分别采样24 h和48 h,并采集猪舍内的饲料、粪便和饮水添加剂样品.采用普通PCR检测样品中的3类抗生素抗性基因(大环内酯类、β-内酰胺类、四环素类各3个基因)和7种致病菌/条件致病菌基因(弯曲杆菌属、产气荚膜梭菌、肠球菌属、大肠杆菌、小肠结肠炎耶尔森菌、葡萄球菌属和猪链球菌);选取检出率较高的6种基因,采用荧光定量PCR对其浓度进行测定.结果表明: 空气中大环内酯类抗性基因检出了3个,四环素类检出了2个,肠球菌、大肠杆菌、小肠结肠炎耶尔森菌、葡萄球菌等4种条件致病菌在空气样品中和饮水添加剂中都被检测到.绝大部分目的基因的浓度均在10⁴ copies·m^-3以上,并且猪舍附近浓度远高于生活区;猪场内主要的抗生素抗性基因和条件致病菌的可能来源是猪粪便和饮水添加剂.在养猪场内采样24 h即可满足PCR检测要求;在生活区采样48 h的采样效率高于采样24 h,而在猪舍外和猪舍内采样24 h的效率高于采样48 h.     

New media for detection and counting of clostridia in foods

The growth of pure cultures of Clostridium perfringens (ATCC 12922) and Cl. sporogenes (PA 3679) in five non-selective media, fluid thioglycollate medium (FTM), rapid perfringens medium (RPM), Columbia broth Malthus (CBM), reinforced clostridial medium (RCM) and lactose sulphite (LS), was monitored using conductance measurements with a Malthus analyser. Only FTM and CBM gave useful results. The correlation of log₁₀ plate counts on blood agar of the pure strain of Cl. sporogenes with detection times in FTM was highly significant ( r = 0·96, n = 73), and with detection times in CBM less so ( r = −0·909, n = 33). The correlation of log₁₀ counts on tryptose sulphite neomycin medium (TSN) of wild strains of Cl. sporogenes and Cl. perfringens with detection times with FTM in meat was also highly significant ( r = 0·933, n = 54).     

A quantitative electrochemiluminescence assay for Clostridium perfringens alpha toxin

Described is a rapid direct sandwich format electrochemiluminescence assay for identifying and assaying Clostridium perfringens alpha toxin. Biotinylated antibodies to C. perfringens alpha toxin bound to streptavidin paramagnetic beads specifically immunoadsorbed soluble sample alpha toxin which subsequently selectively immunoadsorbed ruthenium (Ru)-labeled detection antibodies. The ruthenium chelate of detection antibodies chemically reacted in the presence of tripropylamine and upon electronic stimulation emitted photons (electrochemiluminescence) that were detected by the photodiode of the detector. Elevated toxin concentrations increased toxin immunoadsorption and the specific immunoadsorption of Ru-labeled antibodies to alpha toxin, which resulted in increased dose-dependent electrochemiluminescent signals. The standardized assay was rapid (single 2.5-h coincubation of all reagents), required no wash steps, and had a sensitivity of about 1 ng/ml of toxin. The assay had excellent accuracy and precision and was validated in buffer, serum, and urine with no apparent matrix effects.     

Rethinking our understanding of the pathogenesis of necrotic enteritis in chickens

For decades, low doses of antibiotics have been used widely in animal production to promote growth. However, there is a trend to reduce this use of antibiotics in feedstuffs, and legislation is now in place in Europe to prohibit their use in this way. As a consequence, economically important diseases, such as necrotic enteritis (NE) of chickens, that are caused by Clostridium perfringens have become more prevalent. Recent research is creating a paradigm shift in our understanding of the pathogenesis of NE and is now providing information that will be necessary to monitor and control the incidence of NE in poultry.     

Investigation of the survival characteristics of Rhodococcus coprophilus and certain fecal indicator bacteria.

Rhodococcus coprophilus and Clostridium perfringens survived in fresh water samples held at 5, 20, and 30 degrees C for over 17 weeks, whereas Escherichia coli and fecal streptococci disappeared after 5 weeks at all three temperatures. R. coprophilus survived for more than 8 months in sterilized sewage and deionized water at all three temperatures, whereas in normal sewage held at 20 degrees C, the survival time was 12 to 26 weeks. In samples held at 30 degrees C, survival times were shorter, probably because of interbacterial competition or protozoal predation. The results indicate that R. coprophilus may be a useful indicator of the presence of remote fecal pollution of farm animal origin, but not of recent pollution, when enumerated alone in polluted waters or wastewaters.     

坏疽病原体快速检测方法的临床应用

目的:利用荧光定量PCR技术,建立一种快速、敏感、特异的检测产气荚膜梭菌的方法,及时用于指导临床治疗。方法:以产气荚膜梭菌16srRNA基因作为靶序列,设计一对特异引物和探针,以伤口分泌物和脓液提取的核酸作为模板,利用已经优化的引物和探针进行PCR反应;同时与细菌培养作比较,验证此方法的快速性、敏感性及特异性。结果:建立的反应体系在上游引物浓度为0.45μmol/L,下游引物浓度为0.15μmol/L,探针浓度为0.3μmol/L时,具有很好的敏感性,与21种其他细菌均无交叉反应,其敏感性为9cfu/反应体系。荧光定量PCR检测结果与细菌培养结果完全一致。结论:所建立的荧光定量PCR方法特异、灵敏、快速,能对产气荚膜梭菌感染做出准确的检测报告,具有对战时高发疾病气性坏疽进行快速和定量检测潜质。     

The protective effect of breast feeding in relation to sudden infant death syndrome (SIDS): II. The effect of human milk and infant formula preparations on binding of Clostridium perfringens to epithelial cells

Breast feeding is known to protect an infant against gastrointestinal pathogens and epidemiological studies indicate that compared to breast fed infants, formula fed infants are at a greater risk of dying from sudden infant death syndrome (SIDS). Many SIDS infants have symptoms of gastrointestinal infections prior to death and one gastrointestinal pathogen associated with SIDS is Clostridium perfringens. Studies have found that a significantly higher number of formula fed SIDS infants have C perfringens and its enterotoxin in their faeces compared to breast fed infants. The aim of the study was to compare the effects of human milk and infant formula on binding of C perfringens to epithelial cells. Two protocols were used to assess the effect of human milk and infant formula to inhibit binding of C perfringens to epithelial cells. Binding was assessed by flow cytometry. For the in vivo protocol which more closely represents interactions on the mucosal surface, breast milk enhanced bacterial binding but infant formula caused inhibition of binding; however for the in vitro method, both human milk and infant formula resulted in consistent enhancement of binding. Flow cytometry studies indicated that enhancement of binding was due to the formation of bacterial aggregates. Lewis(a) and Lewis(b) antigens, found in both breast milk and infant formula, inhibited C. perfringens binding in a dose dependent manner. The Lewis(a) and Lewis(b) antigens in human milk and infant formula can inhibit C. perfringens binding to epithelial cells. While infant formula reduced binding of C. perfringens to epithelial cells in the experiments carried out with the in vivo protocol, the protective effects of breast feeding in relation to colonisation with C. perfringens are more likely to be due to formation of bacterial aggregates. These findings have implications for improving infant formula preparations.     

食品及饲料中马属动物源性成分的PCR检测研究

采用马和驴mtDNA中特异性片段引物 ,利用聚合酶链反应技术 (polymerasechainreaction ,PCR) ,建立了饲料中马属动物源性动物成分的快速检测方法。通过内切酶HaeⅢ、Sau3A和AluⅠ可对扩增结果进行验证并能够区分马成分和驴成分。扩增产物测序结果表明 :驴扩增产物序列与数据库序列完全一致 ,马样品扩增产物与数据库序列的同源性达 99%。该方法的检测低限分别为 0 5 %(w w)和 0 2 5 %(w w) ,可作为食品及饲料中马属动物成分鉴别检测的有效方法。     

Molecular epidemiology of Clostridium perfringens related to food-borne outbreaks of disease in Finland from 1984 to 1999

From 1975 to 1999, Clostridium perfringens caused 238 food-borne disease outbreaks in Finland, which is 20% of all such reported outbreaks during these years. The fact that C. perfringens is commonly found in human and animal stools and that it is also widespread in the environment is a disadvantage when one is searching for the specific cause of a food-borne infection by traditional methods. In order to strengthen the evidence-based diagnostics of food poisonings suspected to be caused by C. perfringens, we retrospectively investigated 47 C. perfringens isolates by PCR for the cpe gene, which encodes enterotoxin; by reversed passive latex agglutination to detect the enterotoxin production; and by pulsed-field gel electrophoresis (PFGE) to compare their genotypes after restriction of DNA by the enzymes SmaI and ApaI. The strains were isolated during 1984 to 1999 from nine food-borne outbreaks of disease originally reported as having been caused by C. perfringens. In seven of the nine outbreaks our results supported the fact that the cause was C. perfringens. Our findings emphasize the importance of a more detailed characterization of C. perfringens isolates than mere identification to the species level in order to verify the cause of an outbreak. Also, to increase the probability of finding the significant cpe-positive C. perfringens strains, it is very important to isolate and investigate more than one colony from the fecal culture of a patient and screen all these isolates for the presence of the cpe gene before further laboratory work is done.     

Direct detection of Clostridium perfringens enterotoxin in patients' stools during an outbreak of food poisoning

An outbreak of diarrhoea in a hotel affected 25 time keepers attending the 1997 Mediterranean Games. Epidemiological investigation implicated a 'pasta al ragù' consumed at the hotel's restaurant and Clostridium perfringens food poisoning was identified by direct detection of C. perfringens enterotoxin in patients' stools. This report confirms that a careful evaluation of epidemiological features, together with the availability of direct and rapid laboratory methods, may lead to a prompt identification of C. perfringens food poisoning.     

Method for Estimating the Presence of Clostridium perfringens in Food

The methods currently used for the enumeration of Clostridium perfringens in food are often inadequate because of the rapid loss of viability of this organism when the sample is frozen or refrigerated. A method for estimating the presence of C. perfringens in food which utilizes the hemolytic and lecithinase activities of alpha toxin was developed. The hemolytic activity was measured in hemolysin indicator plates. Lecithinase activity of the extract was determined by the lecithovitellin test. Of 34 strains of C. perfringens associated with foodborne disease outbreaks, 32 produced sufficient alpha toxin in roast beef with gravy and in chicken broth to permit a reliable estimate of growth in these foods. Alpha toxin was extracted from food with 0.4 m saline buffered (at pH 8.0) with 0.05 mN-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and concentrated by dialysis against 30% polyethylene glycol. A detectable quantity of alpha toxin was produced by approximately 10(6)C. perfringens cells per g of substrate, and the amount increased in proportion to the cell population. Results obtained with food samples responsible for gastroenteritis in humans indicate that a correlation can be made between the amount of alpha toxin present and previous growth of C. perfringens in food regardless of whether the organisms are viable when the examination is performed.     

Survival of Mycobacterium bovis on feedstuffs commonly used as supplemental feed for white-tailed deer (Odocoileus virginianus)

Mycobacterium bovis, the causative agent of bovine tuberculosis, has become established in free-ranging white-tailed deer Odocoileus virginianus in northeastern Michigan. The practice of supplemental feeding of white-tailed deer during the winter is believed to contribute to transmission of M. bovis between deer. The current study was conducted to determine the ability of M. bovis to survive on various feedstuffs commonly used as supplemental feed for deer in northeast Michigan (i.e., apples, corn, carrots, sugar beets, potatoes, and hay) and the effect of maintenance at -20 C, 8 C, and 23 C on survival. Mycobacterium bovis survived on all feedstuffs at all temperatures tested for at least 7 days. At 23 C, M. bovis could still be isolated from samples of apples, corn and potatoes at 112 days. This study suggests that contamination of feedstuffs by M. bovis-infected deer could act as a source of indirect transmission between deer because M. bovis is able to survive in temperatures similar to those recorded during winter months in northeastern Michigan. Current efforts to ban or control supplemental feeding of deer should have a positive effect on decreasing transmission of M. bovis among deer.     

Comparison of cytotoxicity and thin-layer chromatography methods for detection of mycotoxins.

Thirty-three standard mycotoxins were assayed by thin-layer chromatography and by cytotoxicity in HEp-2 and Chang cells. Various levels of detection were found. The cytotoxicity test was significantly more sensitive than thin-layer chromatography for the trichothecenes and should be useful for screening extracts from animal feedstuffs for the presence of unknown mycotoxins.     

Comparison of cytotoxicity and thin-layer chromatography methods for detection of mycotoxins

Thirty-three standard mycotoxins were assayed by thin-layer chromatography and by cytotoxicity in HEp-2 and Chang cells. Various levels of detection were found. The cytotoxicity test was significantly more sensitive than thin-layer chromatography for the trichothecenes and should be useful for screening extracts from animal feedstuffs for the presence of unknown mycotoxins.