958

Journal of Food Protection, Vol. 63, No. 7, 2000, Pages 958±960

Research Note Isolation of Salmonella spp. from the House¯y, Musca domestica L., and the Dump , aenescens (Wiedemann) (Diptera: ), at Caged-Layer Houses

ALAN R. OLSEN* AND THOMAS S. HAMMACK

U.S. Food and Drug Administration, HFS-315, 200 C Street S.W., Washington, D.C. 20204, USA Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/7/958/1671650/0362-028x-63_7_958.pdf by guest on 25 September 2021

MS 99-362: Received 2 December 1999/Accepted 1 February 2000

ABSTRACT

Flies, especially house¯ies, are widely recognized as potential reservoirs and vectors of foodborne Salmonella pathogens. In this study, ¯ies were collected at caged-layer facilities that had produced eggs that were implicated as the food vehicle in two recent outbreaks of Salmonella Enteritidis infections. The ¯ies were separated by species into pools for microbiological testing. A total of 15 species pools of house¯ies, Musca domestica L., and 7 species pools of bronze dump ¯ies, (Wiedemann) (Diptera: Muscidae), were analyzed. Salmonella Enteritidis was isolated from 2 of the 15 pools of house¯ies. Other species of Salmonella were isolated from three pools of ¯ies, including Salmonella Infantis from house¯ies and from dump ¯ies and Salmonella Heidelberg from house¯ies. Salmonella Mbandaka was isolated from a lesser mealworm, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae).

Within the past 10 years, eggs have emerged as a major a potential contributing factor to the spread of Salmonella source of foodborne infections from Salmonella Enteritidis Enteritidis. (14, 15). , especially house¯ies (Musca domestica L.) MATERIALS AND METHODS (Diptera: Muscidae), are proven carriers of foodborne path- ogens that affect humans (9). In the poultry industry, the Collection of ¯ies. Live adult ¯ies were collected at two greatest numbers of house¯ies and other disease-carrying different caged-layer house facilities (facility 1 and facility 2) that ¯ies occur in caged-layer houses (poultry houses with lay- were identi®ed as a potential source of contaminated eggs. The ing hens in cages for commercial egg production), where houses were suspected of housing Salmonella-infected ¯ocks of the ¯ies breed in accumulated manure beneath the cages hens whose eggs were implicated in the outbreaks of salmonel- (6). The ¯ies that breed in layer houses are a potential res- losis. With one exception, the ¯ocks were caged above pits in ervoir of pathogens, including Salmonella Enteritidis (3, 6, which manure from the layer hens was allowed to accumulate. The exception was house A, facility 1, which was equipped with 8). There is no prior research, however, investigating pos- a conveyor system for the immediate removal of manure from the sible links between ¯ies and actual outbreaks of foodborne house. All the houses contained laying hens in cages that were Salmonella Enteritidis infections. This report contains the stacked in tiers up to eight cages high inside the houses. Traps results of microbiological analyses of ¯ies that were cap- were placed at various locations inside and outside each caged- tured at caged-layer house facilities whose poultry eggs layer house. Each trap was exposed for approximately 15 min and were identi®ed as the food vehicle in two recent outbreaks then aseptically placed in a separate sterile plastic container. In of Salmonella Enteritidis infections. addition, separate collections of ¯ies were made at each trap site The two separate outbreaks occurred in 1998, affecting using a hand net. The netted specimens were pinned and identi®ed 46 and 26 people who became ill from Salmonella Enter- to serve as voucher specimens for this research. The voucher spec- itidis after eating foods that contained egg. In both cases, imens were not used in Salmonella analyses. Fly specimens from each glue board trap were aseptically epidemiological investigators identi®ed eggs as the impli- pooled according to species and trap. Specimens were sorted by cated ingredient in the food vehicles for the Salmonella species using morphological characters observed at ϫ10 to ϫ20 Enteritidis infections. The egg ingredients were traced to magni®cation (7, 8). Table 1 summarizes the species, number of speci®c caged-layer facilities. The U.S. Food and Drug Ad- ¯ies, and location of capture for each species pool from the glue ministration (FDA) sent teams to the facilities to collect board traps. environmental samples. During these investigations, ¯ies were collected at each facility to determine if the ¯ies were Isolation of Salmonella from ¯ies. The pools of ¯y speci- mens were separately analyzed for the presence of Salmonella according to the Bacteriological Analytical Manual culture meth- * Author for correspondence. Tel: 202-205-4438; Fax: 202-205-4091; od for raw ¯esh foods, highly contaminated foods, and E-mail: [email protected]. feeds (5). House¯y samples (less than 1 g) were pre-enriched in J. Food Prot., Vol. 63, No. 7 SALMONELLA AND FLIES 959

TABLE 1. Species pools of ¯ies collected at caged-layer houses Pool no. Fly species No. of specimens Collection location

1 M. domestica 4 Facility 1, house A, egg conveyer 2 M. domestica 8 Facility 1, house A, outside rear door 3 H. aenescens 1 Facility 1, house A, outside rear door 4 M. domestica 3 Facility 1, house A, inside rear door 5 M. domestica 4 Facility 1, house B, egg conveyer 6 M. domestica 6 Facility 1, house B, inside manure pit 7 H. aenescens 1 Facility 1, house B, outside manure pit 8 H. aenescens 1 Facility 2, house A, inside house 9 M. domestica 1 Facility 2, house A, outside manure pit 10 H. aenescens 1 Facility 2, house A, outside manure pit

11 M. domestica 5 Facility 2, house A, outside manure pit Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/7/958/1671650/0362-028x-63_7_958.pdf by guest on 25 September 2021 12 H. aenescens 5 Facility 2, house A, outside manure pit 13 M. domestica 19 Facility 2, house A, in manure pit 14 H. aenescens 7 Facility 2, house A, in manure pit 15 M. domestica 1 Facility 2, house B, outside rear door 16 M. domestica 5 Facility 2, house B, outside rear door 17 H. aenescens 1 Facility 2, house B, outside manure pit 18 M. domestica 2 Facility 2, house B, in manure pit 19 M. domestica 1 Facility 2, house C, outside rear door 20 M. domestica 6 Facility 2, house C, outside rear door 21 M. domestica 1 Facility 2, house C, inside house 22 M. domestica 2 Facility 2, house C, in manure pit

10 ml of lactose broth for 24 Ϯ 2hat35Ϯ 2ЊC. After pre- tica, and seven pools consisted of bronze dump ¯ies, Hy- enrichment, 1-ml aliquots were subcultured to 10-ml portions of drotaea (Ophyra) aenescens (Wiedemann) (Diptera: Mus- tetrathionate broth, and 0.1-ml aliquots were subcultured to 10-ml cidae) (Table 1). The 15 pools of M. domestica ranged from portions of Rappaport-Vassiliadis medium. Tetrathionate broth and 1 to 19 adult ¯ies per pool. The seven pools of H. aenes- Ϯ Rappaport-Vassiliadis medium were incubated for 24 2hat43 cens ranged from 1 to 7 adult ¯ies per pool. The ¯ies cap- Ϯ 0.2ЊC and at 42 Ϯ 0.2ЊC, respectively. Incubated tetrathionate tured by netting but not used for Salmonella analysis uni- broth and Rappaport-Vassiliadis medium were streaked to bismuth sul®te, Hektoen enteric, and xylose lysine desoxycholate agar formly consisted of a mixture of M. domestica and H. plates. The plates were incubated for 24 Ϯ 2hat35Ϯ 2ЊC. aenescens. Bismuth sul®te agar plates, without typical Salmonella colonies, Eighteen of the 22 pools of ¯ies that were analyzed were incubated for an additional 24 Ϯ 2hat35Ϯ 2ЊC. Pre- yielded no Salmonella isolates. Four pools tested positive sumptive positive colonies were picked to triple sugar iron and for Salmonella. Salmonella Enteritidis was found in pools lysine iron agar slants. The triple sugar iron and lysine iron slants 2 and 16, from facilities 1 and 2, respectively. Salmonella were incubated for 24 Ϯ 2hat35Ϯ 2ЊC. Presumptive positive Infantis was also found in pool 2 (facility 1) and in pool Salmonella isolates were screened biochemically with API 20E 14 (facility 2). Salmonella Heidelberg was isolated from test kits (bioMeÂrieux, Hazelwood, Mo.). Presumptive positive iso- pool 20 (facility 2). Table 2 summarizes the isolations of lates were con®rmed serologically with polyvalent A-I and Vi Salmonella spp. from the pools of ¯ies. In addition, Sal- somatic (O) antisera, speci®c somatic group (O) antisera, and monella Mbandaka was isolated from a lesser mealworm polyvalent a-z ¯agellar (H) antisera (Difco Laboratories, Sparks, adult, Alphitobius diaperinus (Panzer) (Coleoptera: Tene- Md.). Salmonella isolates were sent to the FDA's Central Labo- brionidae), from the manure pit beneath house B, facility ratory for Microbiological Investigations for de®nitive serotyping. 2. RESULTS AND DISCUSSION Voucher specimens of M. domestica (four male and four female) and H. aenescens (four male and four female) Eighteen traps yielded 22 species pools of ¯ies for that were collected by hand net at the sites are deposited at analysis. Fifteen pools consisted of house¯ies, M. domes- the U.S. National Museum of Natural History in Washing- ton, D.C. Additional voucher specimens are retained in the TABLE 2. Salmonella spp. isolated from pools of ¯ies FDA repository collection in Washington, D.C. Flies are widely recognized as potential reservoirs and No. of ¯ies Salmonella vectors of foodborne Salmonella pathogens (2±4, 12). Pool no. Fly species in pool serotype(s) isolated Compared with other ¯ies, M. domestica is a superior host for Salmonella, especially Salmonella Enteritidis (11, 13). 2 M. domestica 8 Enteritidis and Infantis Salmonella is transmitted by house¯ies through mechanical 14 H. aenescens 7 Infantis transmission, in vomitus, and in feces (11). It is known that 16 M. domestica 5 Enteritidis house¯ies are able to transmit Salmonella Enteritidis and 20 M. domestica 6 Heidelberg Salmonella Typhimurium to human food (10, 13). 960 OLSEN AND HAMMACK J. Food Prot., Vol. 63, No. 7

The role of house¯ies in transmitting Salmonella to 2. Angelotti, R. 1973. The report of the FDA Salmonella task force. uninfected layer hens or to fresh eggs is not clear. However, U.S. Food and Drug Administration, Washington, D.C. the observed diurnal dispersion patterns of house¯ies in 3. Anonymous. 1969. An evaluation of the Salmonella problem. Na- tional Academy of Sciences, Washington, D.C. caged-layer houses is certainly conducive to the transmis- 4. Anonymous. 1980. Urban pest management: a report prepared by sion of pathogens by ¯ies to laying hens and to any exposed the Committee on Urban Pest Management. National Academy feed, water, or eggs. For example, Anderson and Poorbaugh Press, Washington, D.C. (1) report that house¯ies are active in and around cages 5. Anonymous. 1998. Bacteriological Analytical Manual, 8th ed., revi- until mid-morning, at which time they move to the manure sion A. Association of Of®cial Analytical Chemists, Gaithersburg, Md. pit for feeding and ovipositing. In the afternoon, house¯ies 6. Axtell, R. C., and J. J. Arends. 1990. Ecology and management of tend to move outdoors but return in the evening to the layer pests of poultry. Annu. Rev. Entomol. 35:101±126. 7. Gagne, R. J. 1991. Flies (Diptera), p. 269±295. In J. R. Gorham houses, where they rest in the rafters until the next morning (ed.), and mite pests in food, vol. 1. U.S. Department of Ag- (1). This diurnal dispersion pattern ®ts the accepted FDA riculture, Washington, D.C. Agriculture Handbook no. 655. behavioral pro®le for disease-carrying ¯ies (12). 8. Greenberg, B. 1971. Flies and disease, vol. 1: ecology, classi®cation Our results represent the ®rst published report of an and biotic associations. Princeton University Press, Princeton, N.J. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/7/958/1671650/0362-028x-63_7_958.pdf by guest on 25 September 2021 FDA investigation that may link house¯ies to outbreaks of 9. Greenberg, B. 1973. Flies and disease, vol. 2: biology and disease Salmonella Enteritidis involving eggs as the suspected food transmission. Princeton University Press, Princeton, N.J. vehicle. This report combined with what is known about 10. Greenberg, B., and A. A. Bornstein. 1964. Fly dispersion from a rural Mexican slaughterhouse. Am. J. Trop. Med. Hyg. 13:881±886. the capabilities of house¯ies to transmit Salmonella and 11. Greenberg, B., J. A. Kowalski, and M. J. Klowden. 1970. Factors about the observed activity patterns of house¯ies in caged- affecting the transmission of Salmonella by ¯ies: natural resistance layer houses lead us to conclude that house¯ies merit con- to colonization and bacterial interference. Infect. Immun. 2:800±809. sideration as one of the many potential risk factors in the 12. Olsen, A. R. 1998. Regulatory action criteria for ®lth and other ex- prevention of foodborne Salmonella Enteritidis outbreaks traneous materials, III: review of ¯ies and foodborne enteric disease. involving eggs. Reg. Toxicol. Pharmacol. 28:199±211. 13. Ostrolenk, M., and H. Welch. 1942. The common house ¯y (Musca ACKNOWLEDGMENTS domestica) as a source of pollution in food establishments. Food Res. The authors thank Dean E. Wagner for serotyping the isolates and 7:192±200. Wallace H. Andrews, PhD, for technical comments. We also thank Marilyn 14. St. Louis, M. E., D. L. Morse, M. E. Potter, T. M. Demel®, J. J. F. Balmer, VMD, Diane R. McDaniel, and Tyra S. Wisecup for their as- Guzewich, R. V. Tauxe, and P. A. Blake. 1988. The emergence of sistance. grade A eggs as a major source of Salmonella enteritidis infections. JAMA 259:2103±2107. REFERENCES 15. Trepka, M. J., J. R. Archer, S. E. Altekruse, M. E. Proctor, and J. P. 1. Anderson, J. R., and J. H. Poorbaugh. 1964. Observations on the Davis. 1999. An increase in sporadic and outbreak-associated Sal- ethology and ecology of various Diptera associated with northern monella Enteritidis infections in Wisconsin: the role of eggs. J. In- California poultry ranches. J. Med. Entomol. 1:131±147. fect. Dis. 180:1214±1219.