<I>Clostridium Perfringens</I>

331

Journal of Food Protection, Vol. 68, No. 2, 2005, Pages 331±335 Copyright ᮊ, International Association for Food Protection

Levels and Enterotoxigenicity of Clostridium perfringens in Pozole, Tamales, and Birria

V. NAVARRO-HIDALGO,1,2 E. CABRERA-DIÂAZ,1 H. ZEPEDA,2 L. MOTA DE LA GARZA,2 A. CASTILLO,3 AND R. TORRES-VITELA1*

1Laboratorio de MicrobiologõÂa Sanitaria, Centro Universitario de Ciencias Exactas e IngenierõÂas, Universidad de Guadalajara, Marcelino GarcõÂa BarragaÂn 1451, Guadalajara 44430, MeÂxico; 2Deptartamento de MicrobiologõÂa, Escuela Nacional de Ciencias BioloÂgicas, Instituto PoliteÂcnico Nacional, Carpio y Plan de Ayala, MeÂxico, D. F.; and 3Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471, USA Downloaded from http://meridian.allenpress.com/jfp/article-pdf/68/2/331/1675907/0362-028x-68_2_331.pdf by guest on 28 September 2021

MS 03-419: Received 29 September 2003/Accepted 24 September 2004

ABSTRACT

A quantitative survey of Clostridium perfringens in typical foods served at local restaurants was conducted for 18 months in Guadalajara, Mexico. A total of 151 samples, including goat's birria (50), pozole (50), and beef tamales (51), were collected from small restaurants in Guadalajara. Samples were tested for C. perfringens by the most probable number (MPN) method and for mesophilic aerobic plate counts (MAPCs) and coliform, yeast, and mold counts by plate count methods. Isolates con®rmed as C. perfringens were further sporulated and tested for cytotoxic or cytotonic effect against Vero cells as an indication of enterotoxin production. C. perfringens was detected in 78 (52%) of all samples at concentrations that ranged from 2.3 to 5.4 log MPN/g. Average MAPCs were 1.3 to 2.7 log CFU/g, depending on the type of dish. Coliform counts ranged from less than 1.0 to 1.5 CFU/g, and yeast and mold counts were less than 1.0 log CFU/g in all cases. A total of 118 isolates of C. perfringens were tested for enterotoxic effect on Vero cells; 82 (70%) showed activity against Vero cells. Of them, 31 isolates induced cell lysis, indicating cytotoxic effect; 41 induced cell elongation, indicating cytotonic effect; and 10 produced both cytotoxic and cytotonic effect. Dilution of the bacterial ®ltrates that were still producing an effect on Vero cells ranged from 1:80 to 1:5,120. These results underscore the importance of determining enterotoxigenicity when testing for C. perfringens in foods.

The reported frequency of cases and outbreaks of food- the high protein and water content and a pH suitable for borne infections and intoxications is increasing in industri- bacterial growth. Other local foods where C. perfringens alized and developing countries. According to the Centers may be a signi®cant hazard if involving temperature abuse for Disease Control and Prevention, most outbreaks are are pozole and tamales. Pozole is a broth that contains hom- caused by biological hazards, especially pathogenic bacte- iny, and, immediately before serving, cooked pork and ria, with Salmonella, Staphylococcus aureus, and Clostrid- shredded cabbage or lettuce are added. The pork is usually ium perfringens being among the most common etiologic held at room temperature to speed the tempering with the agents of foodborne disease (2). In The Netherlands, 2,621 hot broth, which may be a contributing factor for C. per- incidents of foodborne illness caused by different agents fringens spores to germinate and grow. Pozole with meat were reported between 1991 and 1994, involving 7,567 ill and vegetables is usually taken home for further serving, people. Of the outbreaks where the etiologic agent was and temperature abuse may promote the growth of C. per- identi®ed, 11% were caused by C. perfringens (15). Most fringens (3). of the foodborne outbreaks that were caused by C. perfrin- An enterotoxin released from C. perfringens during gens between 1988 and 1992 in the United States were sporulation is responsible for diarrhea (5). The ability of C. related to eating meat products, including beef, chicken, perfringens isolates to produce enterotoxin has been inves- and turkey; at least eight of these outbreaks were linked to tigated using serological or molecular methods or by tissue Mexican food (9). culture assays. African green monkey kidney cells (Vero Two large outbreaks of C. perfringens toxic infection cells) are sensitive to the effect of C. perfringens entero- were recently reported in Mexico, implicating 1,777 cases toxin, and the Vero cell cytotoxin assay has been described (1, 3). In one of them, birria, a typical Mexican dish made as a convenient test for C. perfringens enterotoxin detection with cooked goat or beef and a unique tomato gravy, was (7). The effects induced by C. perfringens enterotoxin on identi®ed as the vehicle of the pathogen. This dish is com- Vero cells include morphological changes, detachment from monly served in gatherings. The meat is cooked overnight glass surfaces, decreased viability and plating ef®ciency, and the gravy boiled for several hours. However, if tem- and altered macromolecular synthesis (8). perature abuse occurs, pathogens can grow rapidly due to Laboratory testing for C. perfringens in foods is usu- * Author for correspondence. Tel: 52(33) 3650-0374; Fax: (0133) ally performed during outbreak studies and not for routine 36500374; E-mail: [email protected]. food testing. Therefore, few studies are available on the 332 NAVARRO-HIDALGO ET AL. J. Food Prot., Vol. 68, No. 2 prevalence or counts of this pathogen in foods. The objec- Becton Dickinson) for further determination of their effect on tive of this work was to determine the frequency and con- Vero cells. centration of C. perfringens in birria, pozole, and tamales Another portion of 15 g of sample was weighed, placed in a sold in Guadalajara City, Mexico, and to determine the abil- sterile stomacher bag, added to 135 ml of sterile 0.1% peptone ity of C. perfringens strains isolated from these foods to water, and then pummeled for 1 min in a stomacher 400 mixer (Tekmar Co.). Decimal dilutions were then plated for mesophilic produce enterotoxin as determined by the observation of aerobic plate count (MAPC) by pour plate in a standard method cytotoxic or cytotonic activity against Vero cells. agar (BBL, Becton Dickinson), incubating at 35ЊC for 48 h; total MATERIALS AND METHODS coliform count by pour plate in violet red bile agar (BBL, Becton Dickinson), incubating at 35ЊC for 24 h; and yeast and mold Sample collection and preparation. One hundred ®fty-one counts by pour plate method in potato dextrose agar (BBL, Becton samples were collected from small restaurants in Guadalajara City, Dickinson) added to ampicillin (200 mg/liter, Sigma Chemical Mexico, including birria (50 samples), pozole (50 samples), and Co., St. Louis, Mo.) and rose bengal (600 mg/liter, Sigma), in- beef tamales (51 samples). These foods are usually served at res- cubating at 20ЊC for 72 to 196 h. The pH of the samples was Downloaded from http://meridian.allenpress.com/jfp/article-pdf/68/2/331/1675907/0362-028x-68_2_331.pdf by guest on 28 September 2021 taurants dedicated only to each particular food commodity; there- determined in a homogenate of 5 g of sample in 10 ml of recently fore, 25 restaurants were sampled twice for each commodity. boiled water at room temperature using a Conductronic 10 pH These restaurants were randomly selected for sampling between meter (Conductronic, Puebla, Mexico). October 1998 and April 2000. The temperature at purchase time was recorded for each sample using a K-type thermocouple con- Vero cell assay for enterotoxin. Each C. perfringens isolate nected to a Fluke 50 thermometer (Fluke Corp., Everett, Wash.). was thawed at room temperature for 1 h and then reactivated by Each sample was placed inside a sterile stomacher bag, sealed growing two consecutive times in tryptic soy broth (BBL, Becton with a rubber band, and transported to the laboratory at room Dickinson) with 0.6% yeast extract, incubating at 37ЊC for 24 h temperature for analysis within 1 h after collection. The likelihood in an anaerobic jar with the gas-pak system. The isolates then for C. perfringens to grow in the samples during 1 h at an esti- were transferred to Duncan and Strong's sporulation medium mated room temperature of 30ЊC was modeled using the U.S. modi®ed (11) and incubated at 37ЊC for 24 h in an anaerobic Department of Agriculture's Pathogen Modeling Program V6.1 atmosphere. A supernatant of these isolates was obtained by cen- and determined to be negligible. In the laboratory, samples were trifuging for 10 min at 1,700 ϫ g in a Centra CL-2 centrifuge weighed as follows: birria, 23 g of meat and 2 ml of gravy; pozole, (Termo IEC, Needham Heights, Mass.) and ®ltering through a 15 g of hominy, 7.5 g of pork, and 2.5 ml of broth; and tamales, 0.22-␮m membrane ®lter (Millipore, Bedford, Mass.). The spore- 25 g of the inside meat. Each 25-g sample was placed in a sterile and cell-free supernatants were stored at 4ЊC for 24 to 48 h. Before stomacher bag and added to 225 ml of sterile 0.1% peptone water testing against Vero cells, the supernatants were centrifuged again and homogenized by manual rubbing and shaking for 90 s. This for 10 min at 4,000 ϫ g in a Microfuge E centrifuge (Beckman homogenization procedure was chosen after preliminary studies Instrument, Inc., Palo Alto, Calif.) and ®ltered through a 0.22-␮m showed that manual sample mixing yielded higher counts than membrane ®lter (Millipore). A strain of enterotoxigenic Esche- pummeling with a stomacher 400 mixer (Tekmar Co., Cincinnati, richia coli (provided by Dr. HeÂctor Zepeda, Escuela Nacional de Ohio). Ciencias BioloÂgicas, Instituto PoliteÂcnico Nacional) was used as Microbiological and pH analysis. Enumeration of C. per- positive control, and noninoculated Eagle's minimum essential fringens was achieved following the method of St. John et al. (16). media (Gibco/BRL, Ontario, Canada) was used as a negative con- One milliliter of the sample mixture was transferred to each of trol. three screw-cap tubes (16 by 150 mm) that contained 10 ml of Vero cells used in this study were African green monkey milk iron medium. Another 1-ml portion was used for further kidney cells (ATCC CCL81). The cells were prepared by the decimal dilutions, which were also inoculated in milk iron me- method described by Gentry and Darlrymple (4). Brie¯y, the cells dium for most probable number (MPN) series, including dilutions were propagated in Eagle's minimum essential media with sup- of 10Ϫ1 through 10Ϫ5. All tubes were incubated with the caps plement. The supplement consisted of 10% heat inactivated fetal tightened in a water bath at 37ЊC for 24 h. From each tube that bovine serum, 2 mM L-glutamine, 4.4% sodium bicarbonate, 100 showed storming milk fermentation, three loopfuls were collected U of penicillin, 10 ␮m of streptomycin, and 10 ␮g of EDTA. The from 0.5 to 1 cm under the surface of the medium and then cells then were dispersed by treating with 0.025% trypsin. Freshly streaked onto plates of Shahidi-Ferguson perfringens agar added trypsinized cells were counted and suspended to desired concen- with egg yolk, overlaid with Shahidi-Ferguson perfringens base tration in Eagle's minimum essential media, and 0.1 ml of sus- with no egg yolk, and then placed in an anaerobic jar (BBL, Bec- pension was placed into 96-well microtiter plates (Costar, Cam- ton Dickinson, Bethesda, Md.). Anaerobic atmosphere was bridge, Mass.). Monolayers were then established by incubating Њ achieved by rehydrating and placing a gas-pak envelope together at 35 C for 18 to 20 h in an automatic CO2 incubator (Lab-Line, with an anaerobic indicator strip (BBL, Becton Dickinson) in the Melrose Park, Ill.) with the atmosphere adjusted to 5% CO2. anaerobic jar before closing and incubating at 37ЊC for 24 to 48 A total of 100 ␮l of each C. perfringens cell-free supernatant h (12). was transferred into each of two wells of a microplate coated with Two to four typical colonies (round, translucent to grayish, Vero cell monolayers obtained as described herein. The micro- Њ shiny, with a diameter of 1 to 2 mm) were subjected to Gram plates were incubated at 37 C for 24 h in a 5% CO2 atmosphere, stain and biochemical tests for identi®cation of species. C. per- then ®xed with cold absolute methanol and stained for 3 min with fringens con®rmation was completed when the isolate was a gram- Loef¯er's methylene blue stain. After staining, each well was mi- positive rod and biochemical test results were typical (i.e., positive croscopically examined to determine the effect of the supernatant test results for lecithinase production, ␤-hemolysis, glucose fer- on the cells. Cell elongation and lysis were indicative of possible mentation, lactose fermentation, nitrate reduction, gelatin hydro- enterotoxin production (8). Cell elongation was indicative of a lysis, and H2S production and negative results for motility). These cytotonic effect, and cell lysis indicated a cytotoxic effect. To isolates then were stored frozen in cooked meat medium (BBL, determine the lowest titer of bacterial ®ltrate still producing an J. Food Prot., Vol. 68, No. 2 C. PERFRINGENS IN MEXICAN FOODS 333

TABLE 1. Populations of Clostridium perfringens found in birria, pozole, and tamale samples collected at small restaurants in Guadalajara, Mexico No. (%) of samples Interval for each food commodity C. perfringens (log MPN/g)a Birria Pozole Tamales

Nondetectable 25 (50)b 25 (50) 21 (41) 2±3 11 (22) 11 (22) 14 (27) 3±4 10 (20) 13 (26) 13 (25) 4±5 0 (0) 0 (0) 0 (0) Ͼ5 4 (8) 1 (2) 3 (6) Total 50 (100) 50 (100) 51 (100) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/68/2/331/1675907/0362-028x-68_2_331.pdf by guest on 28 September 2021 a Counts above the limit of detection (0.5 log MPN/g) ranged from 2.3 and 5.4 log MPN/g. FIGURE 1. Correlation between sample temperature and count b Percent values within rows were not signi®cantly different in all of Clostridium perfringens in samples of birria, tamales, and po- cases (P Ͻ 0.05). zole obtained from small restaurants in Guadalajara, Mexico. effect on Vero cells, different dilutions (ranging from 1:80 to 1: 20,480) of the bacterial supernatant were added to monolayers and and MAPC or coliform, yeast, and mold counts (regression allowed to interact with the Vero cells as described herein. data not shown). Data analysis. Bacterial count data were converted into log The sample temperature at the time of collection value before data analysis. Log counts and sample temperature ranged from 35 to 75ЊC. Temperature of samples that tested values were subjected to regression analysis to determine associ- positive for C. perfringens ranged from 35 to 65ЊC. Re- ation between C. perfringens counts and temperature or counts of gression analysis indicated a signi®cant correlation (P Ͻ background microbiota. Percent detection of C. perfringens was 0.01) between sample temperatures below 60ЊC and the compared among food commodities by the test of difference be- presence of C. perfringens at levels greater than 2.0 log tween binomial proportions. All data analysis was conducted us- MPN/g (Fig. 1). The pH of samples ranged from 5.5 to 6.5, ing StatGraphics Plus statistical software (version 5.1, Manugis- which is suitable for bacterial growth. tics, Inc. Rockville, Md.). For presentation purposes, a chart was Table 3 shows the effect of cell-free supernatants ob- prepared using Excel 2002 (Microsoft Corp., Mexico City, Mex- ico) with linear trend line to indicate the correlation among vari- tained from 118 C. perfringens isolates obtained from bir- ables. ria, pozole, and tamale samples. Cytotonic effect, indicative of enterotoxin production, occurred in 35% of the isolates, RESULTS whereas cytotoxic effect occurred in 26% of the isolates Of the 151 samples analyzed in this study, 78 (52%) and 9% of all isolates showed both cytotonic and cytotoxic yielded detectable counts of C. perfringens, with 50% of effect. More than half of the isolates obtained from tamales birria and pozole samples and 59% of tamale samples test- and more than one third of the isolates obtained from po- ing positive for this pathogen. No signi®cant differences zole were unable to produce any cytotoxic effect, whereas were found in the percent detection of C. perfringens only 5% of the isolates from birria were nontoxigenic. The among food commodities. Counts of C. perfringens ranged proportion of isolates that produced toxic effect against from 2.3 to 5.4 log MPN/g (Table 1). As given in Table 2, Vero cells was not related to the count of C. perfringens the average MAPCs were 2.7, 2.5, and 1.3 log CFU/g for obtained in the samples tested (data not shown). The titer birria, pozole, and tamales, respectively. Coliforms were of bacterial ®ltrate still producing an effect on Vero cells detected at low counts in a few pozole samples and in none ranged from 1:80 to 1:5,120 (Table 4). Only one bacterial of the birria or tamale samples, whereas yeasts and molds ®ltrate continued to show Vero cell activity at titers as high were not detectable in any sample (Table 2). Regression as 1:5,120, and all ®ltrates lost their activity at titers of 1: analysis indicated no correlation between C. perfringens 20,480.

TABLE 2. Mean mesophilic plate count (MPC) and coliform, yeast, and mold counts in birria, pozole, and tamale samples obtained from small restaurants in Guadalajara, Mexico Mean Ϯ SD count (log CFU/g) for each indicator groupa

Food commodity MPC Coliforms Molds Yeasts

Birria (n ϭ 50) 2.7 Ϯ 0.9 Ͻ1.0 Ϯ 0.0 Ͻ1.0 Ϯ 0.0 Ͻ1.0 Ϯ 0.0 Pozole (n ϭ 50) 2.5 Ϯ 1.3 1.5 Ϯ 1.2 Ͻ1.0 Ϯ 0.3 Ͻ1.0 Ϯ 0.0 Tamales (n ϭ 51) 1.3 Ϯ 0.9 Ͻ1.0 Ϯ 0.0 Ͻ1.0 Ϯ 0.0 Ͻ1.0 Ϯ 0.0 a Detection limit was 1.0 log CFU/g. 334 NAVARRO-HIDALGO ET AL. J. Food Prot., Vol. 68, No. 2

TABLE 3. Effect on Vero cells of cell ®ltrates obtained from 118 isolates of Clostridium perfringens isolated from birria, pozole, and tamales at small restaurants in Guadalajara, Mexico No. (%) of isolates producing cytotoxic effect on Vero cells

Cytotonic and Type of food Cytotoxic Cytotonic effect cytotoxic effect No effect Total

Pozole 12 (34) 7 (20) 5 (14) 11 (31) 35 (100) Birria 12 (29) 22 (54) 5 (12) 2 (5) 41 (100) Tamales 7 (17) 12 (29) 0 (0) 23 (55) 42 (100) Total 31 (26) 41 (35) 10 (9) 36 (31) 118 (100)

DISCUSSION and C. perfringens counts above 2 log MPN/g. This might Downloaded from http://meridian.allenpress.com/jfp/article-pdf/68/2/331/1675907/0362-028x-68_2_331.pdf by guest on 28 September 2021 The three types of Mexican food tested include meat indicate that these products are stored at temperatures that as an ingredient and also a thermal treatment during prep- allow bacterial growth, and the absence of coliforms, aration. These factors are important in selecting for spore- yeasts, and molds, as well as the relatively low MAPC, may formers such as C. perfringens. If these foods are stored at be due to the strong heat treatment applied to the food temperatures suitable for bacterial growth, C. perfringens product rather than to the freshness of the food. may grow to levels of concern. In Mexico, birria has been With regard to the effect of bacterial supernatant from involved with outbreaks of C. perfringens disease (3). If C. perfringens isolates on Vero cells, it can be concluded appropriate conditions are given, tamales and pozole may that approximately two third of the isolates seemed to be be likely to transmit the pathogen as well. In this study, C. able to produce enterotoxin. However, the distribution of perfringens was usually found in populations above 2.0 log toxigenic isolates varied between food commodities. The MPN/g and in some cases at levels above 5.0 log MPN/g. ®nding that 95% of the isolates obtained from birria pro- These numbers have been reported to result in outbreaks if duced a cytotoxic effect compatible with the production of the food is consumed without previous reheating to reduce enterotoxin is signi®cant, since this commodity has been the number of vegetative cells (10, 13, 17). In the case of associated with outbreaks of C. perfringens disease (3). Al- samples with MPN below the detectable limit (0.5 log though the cytotoxic effect was observed regardless of the MPN/g), no attempts were made to further detect the pres- counts of C. perfringens in the samples, it is obvious that ence of this organism, because it is clear that only foods the greater the number of cells, perhaps due to temperature with high levels of this pathogen are of concern. The source abuse, the more likely the potential presence of cells that of C. perfringens in these foods is unknown, and most of produce enterotoxin, which increases the risk for the con- the spores may already be present in the raw meat. In a sumers. The large proportion of samples from which en- survey of spices in Mexico, Rodriguez-Romo et al. (14) terotoxigenic isolates were obtained contrasts with the re- isolated C. perfringens from 3 to 5% of samples of garlic, port of Wen and McClane (18), who found that only 1.4% cumin, and black pepper. However, no attempts were made of 900 meat samples produced isolates that possess the cpe to determine the numbers of this organism in the spices, gene, which is responsible for enterotoxin production. De- which would have provided more insight into the role of tection of enterotoxin-producing isolates has been dif®cult, spices as a source of C. perfringens in foodstuffs. Never- as reported elsewhere (6, 14). Since different methods were theless, C. perfringens spores are of no concern in cooked used for detecting indicators of enterotoxin production in foods unless the food is stored at inappropriate temperatures these studies and in our study, our results are dif®cult to and growth occurs. An interesting ®nding was the signi®- compare with others. A follow-up study is under prepara- cant correlation between sample temperatures below 60ЊC tion to determine why the three foods showed large fre-

TABLE 4. Percent ®ltrates obtained from Clostridium perfringens isolates showing cytotoxic effect against Vero cells as affected by titera Percent ®ltrates still showing activity against Vero cells

Pozole Birria Tamales Filtrate titer VF CE VF ϩ CE VF CE VF ϩ CE VF CE VF ϩ CE

1:1 100 100 100 100 100 100 100 100 0 1:80 100 100 100 100 100 100 100 100 0 1:320 92 70 0 100 100 100 80 70 0 1:1,280 92 0 20 83 60 0 0 0 0 1:5,120 0 0 0 83 0 0 0 0 0 1:20,480 0 0 0 0 0 0 0 0 0 a VF, vacuole formation; CE, cytotonic effect. J. Food Prot., Vol. 68, No. 2 C. PERFRINGENS IN MEXICAN FOODS 335 quencies of enterotoxigenic C. perfringens. This may be an 7. Mahony, D. E., E. Gilliatt, S. Dawson, E. Stockdale, and S. H. Lee. environmental issue, and food safety training of food han- 1989. Vero cell assay for rapid detection of Clostridium perfringens enterotoxin. Appl. Environ. Microbiol. 55:2141±2143. dlers must be encouraged to prevent any temperature abuse 8. McClane, B. A., and J. L. McDonel. 1979. The effects of Clostrid- of these foods in the restaurants. From the results of this ium perfringens enterotoxin on morphology, viability, viability and study, it can be concluded that C. perfringens is associated macromolecular synthesis in Vero cells. J. Cell. Physiol. 99:191± with birria, pozole, and tamales and that these three food 200. commodities may be potentially hazardous if not stored at 9. Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. Grif®n, and R. V. Tauxe. 1999. Food-related illness temperatures that prevent germination and growth of C. and death in the United States. Emerg. Infect. Dis. 5:607±625. perfringens spores. 10. Petersen, L. R., R. Mshar, G. H. Cooper, Jr., A. R. Bruce, and J. L. Hadler. 1988. A large Clostridium perfringens foodborne outbreak ACKNOWLEDGMENTS with an unusual attack pattern. Am. J. Epidemiol. 127:605±611. 11. Rhodehamel, E. J., and S. M. Harmon. 2001. Clostridium perfrin- Dr. Navarro-Hidalgo was supported by a scholarship provided by the gens. Available at: http://www.cfsan.fda.gov/ϳebam/m140.html. Ac- National Council for Research and Technology (CONACYT). Financial cessed 20 August 2004. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/68/2/331/1675907/0362-028x-68_2_331.pdf by guest on 28 September 2021 support for this project was provided by the University of Guadalajara 12. Riley, A., D. H. Kang, and D. Y. Fung. 1999. Comparison of ®ve and the National Polytechnic Institute of Mexico. anaerobic incubation methods for enumeration of Clostridium perfringens form foods. J. Food Prot. 62:1041±1044. REFERENCES 13. Roach, R. L., and D. G. Sienko. 1992. Clostridium perfringens outbreak associated with minestrone soup. Am. J. Epidemiol. 136:1288± 1. Anonymous. 1997. BoletõÂn informativo: varios anÄos: 1980±1996. 1291. Department of Epidemiology, Secretary of Health, Mexico City. 14. RodrõÂguez-Romo, L. A., N. L. Heredia, R. G. LabbeÂ, and J. S. 2. Centers for Disease Control and Prevention. 2000. Surveillance for GarcõÂa-Alvarado. 1998. Detection of enterotoxigenic Clostridium foodborne-disease outbreaksÐUnited States, 1993±1997. Morb. perfringens in spices used in Mexico by dot blotting using a DNA Mortal. Wkly. Rep. 49(SS-1):1±72. probe. J. Food. Prot. 61:201±204. 3. De la Alaniz, O. R., J. L. C. VaÂzquez, L. J. Morales, and M. L. 15. Simone, E., M. Goosen, H. ServeÂ, W. Notermans, and M. W. Borg- Barbosa. 1992. Brote de gastroenteritis por Clostridium perfringens dorff. 1997. Investigations of foodborne diseases by food inspection asociado al consumo de birria. In Proceedings of the 23rd National services in The Netherlands, 1991 to 1994. J. Food Prot. 60:442± Congress of Microbiology. Mexican Association for Microbiology, 446. Acapulco Guerrero, Mexico. 16. St. John, W. D., J. R. Matches, and M. M. Wekell. 1982. Use of iron 4. Gentry, M. K., and J. M. Dalrymple. 1980. Quantitative microtiter milk medium for enumeration of Clostridium perfringens. J. Assoc. cytotoxicity assay for Shigella toxin. J. Clin. Microbiol. 12:361±366. Off. Anal. Chem. 65:1129±1234. 5. Hatheway, L. 1990. Toxigenic Clostridia. Clin. Microbiol. Rev. 3: 17. Thomas, M., and N. D. Noah. 1977. Hospital outbreak of Clostrid- 66±98. ium perfringens food-poisoning. Lancet i:1046±1048. 6. Lin, Y. T., and R. Labbe. 2003. Enterotoxigenicity and genetic re- 18. Wen, Q., and B. A. McClane. 2004. Detection of enterotoxigenic latedness of Clostridium perfringens isolates from retail foods in the Clostridium perfringens type A isolates in American retail foods. United States. Appl. Environ. Microbiol. 69:1642±1646. Appl. Environ. Microbiol. 70:2685±2691.