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Outbreak of Salmonella serotype Anatum infection associated with unpasteurized orange juice.

ABSTRACT: In March 1999, a patient was infected with Salmonella serotype Anatum after having consumed unpasteurized orange juice from a manufacturer in Florida. We conducted a cohort study among customers of the manufacturer, performed pulsed-field gel electrophoresis (PFGE) on isolates, and inspected the manufacturing plant. Surveillance data identified three additional patients infected with Salmonella Anatum showing indistinguishable or closely related PFGE patterns. Three of the four patients had consumed orange juice from the same manufacturer. In the cohort study, 6 of 68 persons (9%) who consumed orange juice and/or orange ice cream from the manufacturer were ill, compared with 1 of 47 (2%) who did not. A positive antigen test for Salmonella species and coliform growth in juice samples taken from the production line suggested contamination during the manufacturing process. Commercially produced orange juice should be pasteurized or otherwise processed to achieve equivalent reduction of pathogens.

UNPASTEURIZED fruit juices and cider have been associated with outbreaks of gastrointestinal illness caused by infections with Salmonella species, Escherichia coli O157:H7, and cryptosporidia. (1-11) Listeria monocytogenes has also been identified from unpasteurized retail apple juice. (12) In 1996, after an outbreak in Florida in which Salmonella serotype Hartford infection was associated with commercially produced unpasteurized orange juice, (8) the Florida Department of Citrus enacted new rules to regulate the production of freshsqueezed, unpasteurized orange juice. (13) However, small producers are regulated by the Florida Department of Agriculture and Consumer Services (FDACS), (14) not the Department of Citrus. We report an outbreak of Salmonella enteritidis serotype Anatum infections associated with unpasteurized orange juice manufactured by a small producer in Sarasota County, Florida.

In the United States, Salmonella Anatum represented only 0.4% of the Salmonella isolates from human sources reported to the National Salmonella Surveillance System at the Centers for Disease Control and Prevention (CDC) in 1998. (15) The annual mean number of reported isolates of Salmonella Anatum in the United States from 1988 to 1998 was 209. (15) In Florida, 3 (0.5%) of 578 isolates reported to the Salmonella Surveillance System during 1998 were Salmonella Anatum.

On March 23, 1999, a 1-year-old boy from Connecticut who had recently returned from a 1-week vacation in Sarasota County had a gastrointestinal illness found to be caused by Salmonella Anatum. The Connecticut Department of Public Health informed the Florida Department of Health about the case. The Florida Public Health Laboratory Information System had identified three additional cases of Salmonella Anatum infections in Florida from February 13 through March 16, 1999, two of them in residents of Sarasota County. Initial interviews indicated that the patient from Connecticut and the two patients in Sarasota County had consumed unpasteurized orange juice made by a roadside juice manufacturer and fruit stand (manufacturer A) in Sarasota County. We conducted an investigation to identify the source of infection.


Descriptive Epidemiology

We reviewed data from the Florida Department of Health surveillance system for a possible increase of Salmonella infections in the state and in the region. Data from the CDC National Salmonella Surveillance System were also reviewed to identify a possible increase of Salmonella Anatum reports in states other than Florida. Also, all county health departments in South Central Florida were asked if additional cases of salmonella infections had been reported. Patients with Salmonella Anatum infections identified through this search were interviewed regarding past food exposures and travel history.

Bacterial Cultures and Subtyping

Stool specimens from the three initial case-patients were cultured using tetrathionate broth with brilliant green dye. (16) Pulsed-field gel electrophoresis was done on isolates from patients with onset dates in January through March 1999 using a standardized protocol. (17)

Cohort Study

A cohort study was conducted among all customers with a Sarasota County billing address who had ordered shipments from manufacturer A from February 26 through March 5, 1999, and who had been at the manufacturer's roadside store at least once from January 1 through April 5, 1999. The previously described patients with culture-confirmed Salmonella Anatum were not part of the cohort study, because none of them were on the manufacturer's customer list. Inter views were done over the telephone. Using a standardized, two-page questionnaire, customers were asked about consumption of any products from manufacturer A and any episodes of illness occurring from February 1 through April 5. For the cohort study, illness was defined as any episode of diarrhea (ie, three or more loose stools in 24 hours) or vomiting. Exposure was defined as consumption of orange juice or orange ice cream made from unpasteurized orange juice by manufacturer A from January 1 to April 5, 1999. Data were analyzed with Epi Info, Version 6.02.

Site Inspection and Environmental Sampling

The Florida Department of Health and FDACS inspected the manufacturing plant on April 2, 1999. The inspection included a review of processing procedures and sanitary conditions of the plant. Thirteen juice samples were taken from various parts of the production line on April 2 and April 13, 1999, and cultured for bacterial growth. (18) Also, an chromatographic test was performed, using the Reveal for Salmonella test system. (19)


Descriptive Epidemiology

Review of data from the CDC National Salmonella Surveillance System indicated no increase of Salmonella Anatum reports from states other than Florida. From January through March 1999, four cases of infection due to Salmonella Anatum were identified: three patients were Florida residents, and one was a Connecticut resident who had visited Florida during that period. County health departments in the region did not report any increase in salmonellosis of any serotypes from January to April 1999. Two of the three infected patients in Florida and the patient from Connecticut had consumed unpasteurized orange juice that was made by manufacturer A. The patient who had not consumed any product from manufacturer A lived in a different Florida county more than 190 miles from Sarasota County and had not travelled to Sarasota County (Table 1).

Bacterial Cultures and Subtypes

Stool samples from all four patients were culture positive for Salmonella Anatum. Pulsed-field electrophoresis revealed that one Florida case-patient had a strain indistinguishable from that of the Connecticut case-patient, whereas the other two case-patients had strains with one and three band differences respective to the strain of the Connecticut patient. The patient who had the strain with the three-band difference had not consumed orange juice from manufacturer A, whereas the other two patients had (Table 1).

Cohort Study

A total of 251 customers of manufacturer A with Sarasota County billing addresses had placed orders for shipment of oranges either to themselves or to third parties. Of these, 134 customers (53%) were available for interview; 117 (47%) either could not be reached or refused to be interviewed. Of the 134 customers available for interview, 19 had not been at a store owned by manufacturer A during the period of interest. Among the 115 customers left for the study, 7 reported an illness episode meeting the case definition of diarrhea or vomiting; two of them saw a physician for treatment, and none submitted a stool sample.

Results of the cohort study analysis are presented in Table 2. Customers who consumed orange juice and/or orange ice cream made by manufacturer A were four times more likely to report illness than those who did not consume any of those products (6 of 68 versus 1 of 46, relative risk: 4.2). The association, however, was not statistically significant (95% confidence interval = 0.5-33.3)

Site Inspection and Environmental Sampling

Manufacturer A was located adjacent to a roadside store in which the fruits and fruit products were sold. Manufacturer A produced unpasteurized orange juice squeezed from approximately 6,000 to 7,000 (234,000 to 284,000 kilos) of oranges per year. Orange juice was sold at the adjacent roadside store and two other stores in Sarasota County belonging to the manufacturer. The manufacturer also produced orange ice cream from the orange juice, which was sold at the three stores. Oranges were shipped in boxes to customers throughout the United States. However orange juice and orange ice cream were available only at the Sarasota stores and were not shipped outside of the county.

The oranges were brought into the processing plant on open trucks and unloaded onto a conveyor system. The oranges went through a cleaning process, in which they were washed, scrubbed by rollers and brushes, waxed, dried, and sorted. The oranges sorted for juicing were then conveyed through a chlorine rinse (200 ppm) and rinsed again with water before juicing. The oranges were then squeezed by a Bertuzzi juicer machine, and the juice was held in stainless-steel holding tanks refrigerated at 3[degrees]C before bottling. The juice was then filled through a filling tap into single-use plastic bottles of 1-pint to 1-gallon capacity. The filled containers were then stored under refrigerated conditions. The filled bottles were not labeled with a filling date, nor was any other labeling used to identify the approximate production date.

Several deficiencies in the processing plant were observed. The processing room was not sealed against the outside, and lizards had reportedly been seen in the room. The conveyor system leading into the juice room had old food and mold residues. Plastic lids used to cover the juice containers were soiled. We also detected mold growth on the product areas of the juicer, in the cooler, and in the room in which the ice cream was made.

Analysis of the 13 juice samples that were taken during the inspection on April 2 and April 13, 1999, showed coliform growth in two samples. One positive sample was taken directly from the tap used to fill the bottles. This sample was also positive on the chromatographic Reveal test for Salmonella species, but Salmonella could not be cultured from that sample. The other positive sample was taken from an unopened bottle of orange juice that contained 0.916 mpn (most probable number of organisms per gram) coliform bacteria.


The common exposure of the three case-patients, the molecular subtyping of their isolates, the poor sanitary conditions in the plant, and the results of the chromatographic test of the orange juice strongly suggest that consumption of orange juice products from manufacturer A caused infection with Salmonella serotype Anatum. Although our results did not achieve statistical significance, the cohort study suggested that customers of manufacturer A who were exposed to orange juice or ice cream made with orange juice were more likely to become ill than customers who did not consume these products.

Various deficiencies at the plant may have contributed to contamination of the product. The reported presence of lizards in the manufacturing area indicates that animals may have contaminated the processing surfaces or the oranges directly. In Florida, free-ranging lizards have been reported to carry a variety of Salmonella species. (20) The finding of old food, mold, and soil on surfaces on which the juice product had direct contact indicates that sanitary conditions in the plant were deficient.

In the past, outbreaks of foodborne pathogens have been linked to various types of unpasteurized fruit juices produced in the United States. (1,5,7-11) In some of the outbreaks, improper foodhandling techniques were described in the production plant. (7,8) In one outbreak, the production plant was described as a "state of the art" facility without any apparent flaws in sanitation practices. (1) In a recently described outbreak, unpasteurized orange juice produced or packaged by an Arizona manufacturer was associated with 207 cases of Salmonella serotype Muenchen infection reported in 15 US states and two Canadian provinces. (9) In Australia, during 1999, an outbreak of Salmonella typhimuriurn affecting 502 persons was associated with consumption of commercial, unpasteurized orange juice (Robert Hall, written communication, December 1999).

In 1998, the Food and Drug Administration (FDA) proposed new regulations that require hazard analysis and critical control point principles be applied to the manufacture of juice products. (21) According to these FDA regulations, packaged juice that has not been processed to achieve a 5-log reduction of pathogens must have labels warning the consumer about the potential risk of consuming the juice.

The Florida Department of Citrus does not require such warnings for juice produced and consumed within Florida, but new regulations implemented in 1996 require each production lot or each day's production to undergo microbiologic monitoring. However, processors who squeeze less than 30,000 boxes (1,215,000 kilos) of oranges annually (eg, manufacturer A) are exempt from these requirements and fall under regulations by the FDACS. Since July 1999, the citrus industry in Florida is required to use warning labels for fruit juices if the manufacturing process does not include a 5-log reduction of pathogens. The FDACS usually inspects the manufacturers that fall under its regulation at least twice a year. The processing plant of manufacturer A, however, had not been inspected in the 2 years before the outbreak.

This study has several limitations. Defining an appropriate and also practical study population proved difficult because the juice was sold at a roadside store. Stool cultures were not available to diagnose illness episodes in the case-patients included in the cohort study. Only a few cases were identified through the cohort study, likely because interviewees might have had difficulty remembering a minor illness that may have occurred 3 months previously Another reason for the low number of cases may be that only a small amount of product was contaminated or that the product was contaminated with a small bacterial load.

Although the Reveal test was positive for Salmonella species, we were not able to culture Salmonella from it, which would have made the comparison of PFGE patterns possible. The chromatographic antigen test cannot prove that viable pathogens were in the juice. However, the test reportedly does not cross-react with other closely related enterobacteriaceae, therefore indicating that juice may have been contaminated with Salmonella species before bottling (package insert for Neogen's Reveal). The fact that no Salmonella species could be cultured from juice samples does not contradict this assumption, because the sample was taken 4 weeks after the last culture-confirmed case-patient had consumed orange juice from the manufacturer. Unfortunately, no product samples were available from the period in which the patients became ill.


This investigation emphasizes that orange juice, if not pasteurized or otherwise processed to reduce pathogens, is a potential source of foodborne pathogens. Regulating agencies need to schedule regular inspections of manufacturing plants to ensure compliance with existing rules for hygienic production. Juice products should be labeled with the date of production. Epidemiologic and laboratory capacity for public health surveillance needs to be maintained at a high level so that similar foodborne outbreaks can be detected and controlled. Most importantly, commercially produced orange juice should be pasteurized or otherwise processed to achieve an equivalent reduction in pathogens before sale.


(1.) Cody SH, Glynn MK, Farrar JA, et al: An outbreak of Escherichia coli O157:H7 infection from unpasteurized commercial apple juice. Ann Intern Med 1999; 130:202-209

(2.) Tamblyn S, deGrosbois J, Taylor D, et al: An outbreak of Escherichia coli O157:H7 infection associated with unpasteurized non-commercial, custom-pressed apple cider--Ontario, 1998. Can Commun Dis Rep 1999;25:113-117

(3.) Thurston H, Stuart J, McDonnell B, et al: Fresh orange juice implicated in an outbreak of Shigella flexneri among visitors to a South African game reserve. J Infect 1998;36:350

(4.) Ooi PL, Goh KT, Neo KS, et al: A shipyard outbreak of salmonellosis traced to contaminated fruits and vegetables. Ann Acad Med Singapore 1997;26:539-543

(5.) Millard PS, Gensheimer KF, Addiss DG, et al: An outbreak of cryptosporidiosis from fresh-pressed apple cider. JAMA 1994;272:1592-1596

(6.) Zhao T, Doyle MP, Besser RE: Fate of enterohemorrhagic Escherichia coli O157:H7 in apple cider with and without preservatives. Appi Environ Microbiol 1993;59:2526-2530

(7.) Besser RE, Lett SM, Weber JT, et al: An outbreak of diarrhea and hemolytic uremic syndrome from Escherichia coli O157:H7 in fresh-pressed apple cider. JAMA 1993;269:2217-2220

(8.) Cook KA, Dobbs TE, Hlady WG, et al: Outbreak of Salmonella serotype Hartford infections associated with unpasteurized orange juice. JAMA 1998;280:1504-1509

(9.) Centers for Disease Control and Prevention: Outbreak of Salmonella serotype Muenchen infections associated with unpasteurized orange juice--United States and Canada, June 1999. MMWR Morb Mortal Wkly Rep 1999;48:582-585

(10.) Centers for Disease Control and Prevention: Outbreaks of Escherichia coli O157:H7 infection and cryptosporidiosis associated with drinking unpasteurized apple cider-- Connecticut and New York, October 1996. MMWR Morb Mortal Wkly Rep 1997;46:4-8

(11.) Centers for Disease Control and Prevention: Outbreak of Escherichia coli O157:H7 infections associated with drinking unpasteurized commercial apple juice--British Columbia, California, Colorado, and Washington, October 1996. MMWR Morb Mortal Wkly Rep 1996;45:975

(12.) Sado PN, Jinneman KC, Husby GJ, et al: Identification of Listeria monocylogenes from unpasteurized apple juice using rapid test kits. J Food Protection 1998;61:1199-1202

(13.) Florida Department of Citrus Rule, chapter 20-64.020, Amendment A (12), Standards for Processed Citrus Products, 1997

(14.) Florida Department of Agriculture and Consumer Services, Chapter 5K-4, Food, 1997

(15.) National Salmonella Surveillance. Atlanta, Centers for Disease Control and Prevention, 1998

(16.) Farmer JJ, Kelly MT: Enterobacteriaceae. Manual of Clinicl Microbiology. Bellows A (ed). Washington, DC, American Society for Microbiology, 5th Ed, 1991, pp 360-383

(17.) Gautom RK: Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli O157:H7 and other gram negative organism in 1 day. J Clin Microbiol 1997;35:2977-2980

(18.) Andrews WH, June GA, Sherrod P: Salmonella. FDA Bacteriological Analytical Manual. Gaithersburg, Md, AOAC International, 8th Ed, 1995, pp 5.01-5.20

(19.) Bird CB, Miller RL, Miller BM: Reveal for Salmonella test system. J AOAC Int 1999;82:625-633

(20.) Hoff GL, White FH: Salmonella in reptiles: isolation from free-ranging lizards (reptilia, Lacerttilia) in Florida. J Herpetol 1977;11:123-129

(21.) Federal Register, July 8, 1998, Vol 63, No.13

Case-Patients With Salmonella Serotye Anatum Infection Reported to the
Florida Department of Health, January through March 1999

 Case- Age (yrs), Residence or Stay Consumption of Orange
Patient Sex in Sarasota County Juice From Manufacturer A

 1 1, M No No

 2 60, F Yes Yes

 3 64, F Yes Yes
 4 4, M Yes Yes

 Case- Onset of DNA DNA Fingerprint
Patient Symptoms Fingerprint Interpretation

 1 Feb 13, 1999 Ib 3 band difference
 compared with I
 2 Feb 26, 1999 Ia 1 band difference
 compared with I
 3 Mar 3, 1999 I Indistinguishable
 4 Mar 6, 1999 I Indistinguishable

Association Between Case-Patients With Gastroenteritis and Exposure to
Products From a Roadside Orange Juice Store in Sarasota County, Florida,
January through March 1999

 No. of Persons Ill/Not Ill
Exposure Among Exposed

Orange juice produced at store A 4/47
Orange ice cream from store A 4/39
Orange juice produced at store A 6/62
 and/or orange ice cream
 from store A

 No. of Persons Ill/Not Ill Relative
Exposure Among Unxposed Risk

Orange juice produced at store A 3/61 1.7
Orange ice cream from store A 3/69 2.2
Orange juice produced at store A 1/46 4.2
 and/or orange ice cream
 from store A

 95% Confidence
Exposure Interval

Orange juice produced at store A 0.4-7.1
Orange ice cream from store A 0.5-9.5
Orange juice produced at store A 0.5-33.3
 and/or orange ice cream
 from store A


* Orange juice, if not pasteurized or otherwise processed to reduce pathogens, is a potential source of foodborne pathogens.

* Juice products should be labeled with the date of production.

* Epidemiologic and laboratory capacity for public health surveillance needs to be maintained at a high level.

* Commercially produced orange juice should be pasteurized or processed to achieve an equivalent reduction in pathogens before sale.
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Author:Hammond, Roberta
Publication:Southern Medical Journal
Article Type:Statistical Data Included
Geographic Code:1USA
Date:Dec 1, 2001
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