VECTOR/PATHOGEN/HOST INTERACTION,TRANSMISSION External Surface Disinfection of the Lesser Mealworm (Coleoptera: Tenebrionidae)

TAWNI L. CRIPPEN AND CYNTHIA SHEFFIELD

Southern Plains Area Research Center, USDAÐARS, 2881 F and B Road, College Station, TX 77845

J. Med. Entomol. 43(5): 916Ð923 (2006) ABSTRACT Understanding the dynamics of movement of bacteria within the environment and between is crucial to unraveling the epidemiology of bacterial diseases and to developing biosecurity measures to prevent dissemination. Many , some beneÞcial and some detri- mental, inhabit poultry houses. The lesser mealworm, (Panzer) (Coleoptera: Tenebrionidae), is a pest commonly found in poultry litter that can harbor pathogens involved in both human and health issues. Current farm management practices perpetuate persistent infesta- tions contributing to the dispersal of and pathogens. To study the dissemination of bacteria by this , we require the ability to differentiate internal from external sources of bacteria carried by the beetle. In this study, we tested previously described methods to externally disinfect beetles and found disinfectant efÞcacies between 40 and 98%. The irregular surface of the posed a challenge to cleansing procedures because the surface offered many recesses able to sequester bacteria. Complete bacterial disinfection was achieved with a serial treatment of ethanol and hydrogen peroxide or hydrogen peroxide/peracetic acid.

KEY WORDS lesser mealworm, external disinfection, Alphitobius diaperinus, bacteria, poultry litter beetle

Lesser mealworms, Alphitobius diaperinus (Panzer) Studies exploring the transfer of bacteria by litter (Coleoptera: Tenebrionidae), at all life stages, inhabit beetles have been reported, but there is limited as- manure and feed in commercial poultry operations sessment of how a transferred bacterium is harbored and are one of the most abundant insect species re- by a beetle. This task requires the ability to differen- covered from broiler chicken and turkey litter samples tiate bacteria carried externally or internally. Al- (Pfeiffer and Axtell 1980, Stafford et al. 1988, Axtell though several previous studies described protocols to and Arends 1990, Rueda and Axtell 1997). Lesser meal- disinfect the surface of beetles, no data were pre- worms are omnivorous scavengers that feed on ma- sented validating the efÞcacy of the techniques (De nure, spilled chicken feed, cracked eggs, chicken car- las Casas et al. 1968, 1972; Harein and De las Casas casses, house ßy maggots, and detritus (Pfeiffer and 1968; Harein et al. 1970, 1972; McAllister et al. 1994, Axtell 1980, Axtell and Arends 1990, Rueda and Axtell 1995, 1996; Hald et al. 1998; Gray et al. 1999). 1997). In turn, these beetles are often fodder for chick- Beetle morphology can reduce the effectiveness of ens, wild birds, and opportunistic rodents. In addition, surface sterilization by preventing adequate access to the beetles are inadvertently dispersed to neighboring bacteria. The insect integument and presumably the residences by the spreading of beetle-containing ma- fecal material adhering to the exoskeleton provide nure on nearby Þelds (Armitage 1986). They are a refuge to bacterial organisms. Flexible joints, wings, prime candidate for participating in the transmission and spiracles occurring between hard plates offer an- of bacteria among fauna inhabiting the poultry house chorage to microbes, whereas structures such as the environment and have previously been implicated in elytra and the cuticle serve as protective covers the transmission of several disease agents, including (Chapman 1982a). In addition, ectodermal invagina- bacterial pathogens (De las Casas et al. 1968, 1973, tions, such as sutures or sulci, may act to shield bac- 1976; Despins et al. 1994; McAllister et al. 1994, 1995, terial organisms from access and displacement during 1996; Watson et al. 2000). disinfectant procedures (Chapman 1982b). This study assesses the efÞcacy of previously described and newly developed methods to disinfect the external surface of beetles. We have devised a speciÞc method Mention of trade names, companies, or commercial products in this resulting in complete surface bacterial disinfection for publication is solely for the purpose of providing speciÞc information and does not imply recommendation or endorsement of the products use in future studies to explore the environmental by the U.S. Department of Agriculture. transfer of bacteria by this beetle. September 2006 CRIPPEN AND SHEFFIELD:BEETLE SURFACE DISINFECTION 917

Materials and Methods EtOH. The beetle was then rinsed in sterile dH2O for 30 s followed by sonication for 2 min; the rinse was Beetles. The Southern Plains Agricultural Research repeated twice. (F) Each beetle was immersed in 2% Center (SPARC) starter colony of A. diaperinus was a NaOCl/10% Tween 80, allowed to soak an additional generous gift from a colony originally isolated from a 8 min, and then immersed in 95% EtOH and the EtOH poultry farm located in Wake County, North Carolina, evaporated for 5 min. (G) Each beetle was immersed and maintained by Dr. D. W. Watson (North Carolina in 95% EtOH, followed by immersion in 2% NaOCl: State University, Raleigh, NC). The SPARC colony 10% Tween 80, and allowed to soak an additional 8 was initiated and has remained in production since min. The beetle was then immersed in 70% EtOH and 2004. Beetles were reared in 1,000-ml wheat bran the EtOH evaporated for 5 min. (Morrison Milling Co., Denton, TX) in plastic con- H2O2-Based Protocols. (H) Each beetle was im- tainers (15 by 15 by 30 cm) with screen tops and held mersed in 20% H O , followed by immersion in sterile at 30ЊC under a photoperiod of 8:16 (L:D) h. Addi- 2 2 2 dH2O. (I) Each beetle was immersed in 20% H2O2, tionally within each cage, a 6-cm sponge moistened followed by immersion in 95% EtOH and the EtOH with deionized water (dH2O) and a 0.5-cm-thick slice evaporated for 5 min. (J) Each beetle was immersed of a medium-sized apple were replenished twice per in 95% EtOH and the EtOH evaporated for 5 min, week, and 30 ml of Þshmeal (Omega Protein, Inc., followed by immersion in 20% H2O2. (K) Each beetle Hammond, LA) was added to the wheat bran once per was immersed in 95% EtOH and the EtOH evaporated week. for 5 min, followed by immersion in 7.35% H2O2/0.23% Disinfecting Agents. Hydrogen peroxide (H2O2) peracetic acid. (Sigma-Aldrich, St. Louis, MO), 95% ethanol (EtOH) Experimental Design. Three replications of each (EMD Chemicals, Gibbstown, NJ), Tween 80 (Am- experimental wash protocol were conducted using 30 resco, Solon, OH), and sodium hypochlorite (NaOCl) beetles per protocol per replication. To collect a sam- (Sigma-Aldrich) were diluted to working concentra- ple of the resident bacteria present on the outer sur- tions with sterile dH2O. The 7.35% H2O2/0.23% per- face of the beetles before external disinfection (PRE- acetic acid was a commercially available formulation, wash), each beetle was immersed in 1 ml of tryptic soy SporGon (Decon Labs, Inc., Bryn Mawr, PA). broth (TSB; Difco, Sparks, MD) at room temperature. These agents were used individually or in combi- The beetle was removed and immediately subjected to nation to formulate and evaluate 11 disinfection pro- one of the experimental disinfection protocols (see tocols designed for comparison to previously de- above). An aliquot of 0.1 ml of PRE-wash TSB was scribed techniques or to test new combinations for serially diluted, spread on blood agar plates (Becton increased efÞcacy. The protocols were grouped into Dickinson, Sparks, MD), and incubated overnight at EtOH (three), NaOCl (four), and H2O2 (four)-based 37ЊC. Bacterial load, expressed as colony-forming units protocols as described below. All protocols were eval- (CFU), was determined after an 18Ð24-h incubation. uated on individual beetles held and disinfected in After each disinfection protocol was completed, sur- 1.5-ml tubes covered with a sterile barrier Þlm viving residual bacteria were sampled (POST-wash) (ParaÞlm; Sigma-Aldrich). by again immersing the beetle in 1 ml of TSB at room For each protocol, the tube in which the beetle was temperature. The beetle was removed, and an aliquot immersed was covered by the barrier Þlm, inverted of 0.1 ml of POST-wash TSB was serially diluted, three times, and sonicated for 2 min at 40 kHz (model spread on blood agar plates, and incubated overnight 8851-34, Cole Parmer, Vernon Hills, IL) before the at 37ЊC. CFU were determined after an 18Ð24-h in- next step. The beetle was transferred into a new sterile cubation. To ensure detection of bacterial contami- tube for each successive immersion or rinse. Evapo- nation below the plating threshold of 10 CFU, the ration was performed by transferring the beetle into a remaining 0.9 ml of PRE-wash and POST-wash TSB sterile tube, which was placed into a sterile, biosafety was enriched by incubation overnight at 37ЊC hood to allow the EtOH to evaporate. Sonication was (ENRICH). After incubation, an aliquot of 0.1 ml of used to assist in dislodging of the bacteria and to TSB was spread on a blood agar plate and incubated ensure uniformity of the agitation procedure for com- overnight at 37ЊC. The presence or absence of bacteria parison of protocols. was recorded after 18Ð24-h incubation at 37ЊC. CFU EtOH-Based Protocols. (A) Each beetle was im- data were analyzed by logistic regression and enrich- mersed in 95% EtOH and then rinsed in sterile dH2O ment data were analyzed by exact logistic regression for 30 s followed by sonication for 2 min; the rinse was in PROC LOGISTIC (SAS Institute, Cary, NC; Agresti repeated. (B) Each beetle was immersed in 70% EtOH 2002). Representative colonies of bacteria present and the EtOH evaporated for 5 min. (C) Each beetle were collected from PRE- and POST-wash blood was immersed in 95% EtOH, and the EtOH evaporated plates for identiÞcation by growth on selective media for 5 min. or by ribotyping. NaOCl-Based Protocols. (D) Each beetle was im- For PRE-TSB control, 60 beetles were taken from mersed in 2% NaOCl/10% Tween 80 and then rinsed the same cage and split into two groups of 30 beetles. in sterile dH2O for 30 s followed by sonication for 2 From one group, beetles were placed into individual min; the rinse was repeated. (E) Each beetle was tubes containing TSB. These served as PRE-wash sam- immersed in 2% NaOCl/10% Tween 80, allowed to ples for this control study to ensure that the tested soak an additional 8 min, and then immersed in 70% beetles were contaminated with bacteria. Beetles 918 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 43, no. 5

Table 1. Comparison of the efficacy of lesser mealworm surface disinfection protocols

CFU Enrichment Disinfection Protocol PRE-Wash POST-wash Clean beetles Meana CFU Ϯ SE Meana CFU Ϯ SE %a Ϯ SE Ϯ Ϯ b Ϯ EtOH-based protocols A 95% EtOH-H2O 10,997.00 36.66 9.14 0.97 48.52 0.07 B 70% EtOH-Evaporation 3,992.59 Ϯ 14.66 14.23 Ϯ 0.50 60.19b Ϯ 0.07 C 95% EtOH-Evaporation 1,271.91 Ϯ 6.27 1.27 Ϯ 0.15 78.26b Ϯ 0.05 Ϯ Ϯ b Ϯ NaOCl-based protocols D NaOCl-H2O 13,228.00 48.71 222.16 2.63 66.72 0.06 Ϯ Ϯ b Ϯ E NaOCl-70% EtOH-H2O 2,981.10 9.75 0.00 0.02 90.85 0.04 F NaOCl-95% EtOH-Evaporation 1,884.10 Ϯ 7.42 0.01 Ϯ 0.11 93.21b Ϯ 0.04 G 95% EtOH-NaOCl-95% EtOH 1,769.25 Ϯ 6.13 0.00 Ϯ 0.00 95.15b Ϯ 0.03 Ϯ Ϯ b Ϯ H2O2-based protocols HH2O-H2O2 1,698.40 5.76 0.00 0.01 97.78 0.00 Ϯ Ϯ b Ϯ IH2O2-95% EtOH-Evaporation 1,181.57 5.69 18.16 0.46 91.45 0.03 Ϯ Ϯ b Ϯ J 95% EtOH-Evaporation-H2O2 1,197.19 7.40 0.00 0.00 100.00 0.00 Ϯ Ϯ b Ϯ K 95% EtOH-Evaporation-H2O2/peracetic 1,524.35 6.44 0.00 0.00 100.00 0.00

a Average of three replicates, 30 beetles per replicate. b POST-wash is signiÞcantly different from PRE-wash (P Ͻ 0.001). from the second group, without prior immersion in STAPH (Analytical ProÞle Index, bioMerieux, Inc., TSB, were subjected to protocol K as described above. Durham, NC) manual identiÞcation test strips. Bacterial Isolation and Preliminary Identification Culture Methods. Individual bacteria from mixed cul- Results tures were grown in TSB and then subcultured onto blood agar plates for selection of isolated colonies. A comparison of external disinfection protocols Individual isolates were identiÞed by growing them on demonstrated large variation in treatment efÞcacy a bank of selective and differential media and then (Table 1). External disinfection using 95% EtOH fol- comparing the results from the unknown isolate to lowed by a dH2O rinse (protocol A), disinfected less data for known species (Atlas 1997). To identify aer- than one-half of the beetles. However, allowing ex- obic bacteria, 10-␮l aliquots were streaked onto Bril- ternal surface evaporation after treatment with EtOH liant Green agar (BGA; Becton Dickinson), Mac- instead of immediately performing a dH2O rinse, sig- Conkey (Becton Dickinson), m Enterococcus (ME; niÞcantly improved disinfection by 61% (protocol C Becton Dickinson), Rogosa (Becton Dickinson), versus A; P Ͻ 0.0001). Using 2% NaOCl/10% Tween 80

CHROMagar E. coli, and CHROMagar Orientation followed by a dH2O rinse (protocol D) disinfected (CHROMagar, Paris, France) plates, and then incu- slightly more than one-half of the beetles. Adding a bated at 37ЊC for 24 h. Each initial colony selection was 70% EtOH treatment to the protocol signiÞcantly im- streaked onto fresh media to ensure cultural purity. A proved disinfection by 36% (protocol E verses D; P Ͻ bacterial lawn of each pure isolate culture was pre- 0.0003). Removing the dH2O rinse completely and pared on blood agar for deÞnitive identiÞcation using adding a higher strength 95% EtOH treatment, which automated ribotyping. was subsequently allowed to evaporate, also signiÞ- Ribotype Characterization. Bacterial isolates from cantly improved disinfection by 40% (protocol F ver- blood agar plates were incubated at 37ЊC for 24 h to sus D; P Ͻ 0.002). Prior treatment of 95% EtOH with allow a bacterial lawn to form. Samples were collected NaOCl/Tween 80 improved disinfection by 19% (pro- while in log phase growth. Bacteria were suspended in tocol C versus F; P Ͻ 0.03). However, the addition of a neutral pH buffer (Qualicon, Inc., Wilmington, DE), a second 95% EtOH treatment before the NaOCl/ heated at 90ЊC for 10 min, combined with two addi- Tween 80 and 95% EtOH treatments did not signiÞ- tional lytic enzymes (Qualicon, Inc.), and analyzed cantly increase efÞcacy (protocol G verses F). A dH2O according to manufacturerÕs instructions using the re- rinse to wet the beetle followed by 20% H2O2 (pro- striction enzymes EcoRI or PvuII. The RiboPrinter tocol H) disinfected almost all of the beetles. The microbial characterization system (Qualicon, Inc.) substitution of a 95% EtOH, which was subsequently characterizes the 5, 16, and 23S rRNA and ßanking allowed to evaporate, before treatment with 20% H2O2 regions of a bacterial sample by using speciÞed re- or 7.35% H2O2/0.23% peracetic acid, disinfected 100% striction enzymes. The resulting rRNA pattern (Ri- of the beetles (protocols J and K). However statisti- boPrint pattern) was automatically characterized and cally nonsigniÞcant the increase between protocols H identiÞed by comparing the pattern to reference pat- versus J or K (2%), it gave us the outcome we desired, terns in the bacterial database provided by Qualicon, which was consistently reproducible complete disin- Inc. (containing Ͼ6000 isolates) and a custom, food fection of culturable, aerobic bacteria. animal-speciÞc database (containing Ͼ400 isolates) The measuring of resident bacteria before execut- developed by C.S. IdentiÞcation requires that the ri- ing the disinfection protocol required a pretreatment bopattern be an 85% or greater match to an existing of beetles by immersion in TSB. As this would not be static ribopattern. When ribotype identiÞcation match a step normally included in the external disinfection was Ͻ85%, isolate identiÞcation was conÞrmed using, protocol, we demonstrated that this initial immersion API Staph, API 20E, API 20NE, API 20 Strep, ID 32 in TSB did not affect the efÞcacy of the disinfection September 2006 CRIPPEN AND SHEFFIELD:BEETLE SURFACE DISINFECTION 919

Fig. 1. Frequency distribution of the bacterial load carried by individual beetles before disinfection procedures. A log10 transformation of the PRE-wash CFU data from 990 individual beetles was used to create the graph.

protocol by testing protocol K (7.35% H2O2/0.23% match). The bacteria showed varying sensitivities to the peracetic acid) without PRE-wash immersion in TSB. disinfection protocols (Table 2). Acinetobacter spp. and A comparison between beetles exposed to prior TSB S. gallinarium seemed particularly resistant to EtOH- and immersion and those disinfected without prior immer- NaOCl-based washes. M. luteus was also resistant to 70% sion showed no signiÞcant difference (P Ͻ 1.0) in EtOH and NaOCl washes, but it did not survive a 95% wash efÞcacy. This demonstrated that the PRE-wash EtOH wash. Bacillus spp. and Enterobacter cloacae re-

TSB immersion had no affect on the outcome of the sisted sequential washing in dH2O and H2O2, but they disinfectant protocol. were removed by sequential washing in 95% EtOH and

The resident bacterial load of each beetle was mea- H2O2. Although we did not identify the taxa, we noted sured individually before exposure to a disinfection that sequential washing in 95% EtOH and H2O2 also protocol (Fig. 1). A frequency distribution of the log10 disinfected fungi present on the PRE-wash plates (data CFU of the results indicated that bacterial load was not shown). variable on individual beetles. Bacterial load ranged from 0 CFU on 20 beetles to 2.5 ϫ 106 CFU on one Discussion beetle, averaging 7.6 ϫ 103Ϯ 9.0 ϫ 104 CFU, with a median of 1.2 ϫ 103 CFU. Litter beetles, especially the lesser mealworm, have The species of bacteria carried by the beetles was become serious pests within the poultry brooder and relatively consistent. Although beetles were reared in laying industry. Because of their mobility, feeding separate cages, they used feed and bran from identical habits, and prey potential, these beetles are implicated sources and were placed into a common cage as adults as mechanical vectors for diseases (e.g., Mareks dis- before use in experiments; thus, similar contamination ease, avian inßuenza, bacterial diseases, fowl pox, coc- proÞles were found. The beetles were contaminated cidiosis, and New Castle disease). The insect offers with 12 bacterial species, representing nine genera: surfaces to support bacterial, fungal, and viral organ- Acinetobacter spp. (Brisou and Pre´vot; 97% API isms (De las Casas et al. 1968, 1972, 1973; Harien et al. match), Aerococcus spp. (Williams; 100% API match), 1970; McAllister et al. 1994, 1995). These beetles have Bacillus spp. (Meyer and Gottheil; 97% ribotype match), high reproductive rates and are difÞcult to control. Bacillus fusiformis (Cohn; 91% ribotype match), Entero- Ultimately, they are portrayed as a reservoir source bacter cloacae (Jordan; 94% ribotype match), Klebsiella contributing to the persistence and transmission of oxytoca (Flu¨gge; 94% ribotype match), Micrococcus lu- pathogens among individual birds within a poultry teus (Schtoeter; 91% ribotype match), Pseudomonas facility (Harien et al. 1970, 1972; Brown et al. 1992; aeruginosa (Schroeter; 85% ribotype match), Staphylo- Hald et al. 1998). coccus gallinarum (Devrise; 94% ribotype match), Strep- Previous studies have attempted to surface sterilize tococcus kloosii (Schleifer; 92% ribotype match), Strep- beetles to establish the carriage of pathogens inter- tococcus xylosis (Schleifer and Kloos; 91% ribotype nally (De las Casas et al. 1968, 1972; Harein and De las match), and Streptococcus spp. (Rosenbach; 92% API Casas 1968; Harein et al. 1970, 1972; McAllister et al. 2 J 920

Table 2. Comparison of surface disinfection protocols by removal of specific bacterial species UNLOF OURNAL EtOH-based protocols NaOCl-based protocols H2O2-based protocols E F G I J K A B C D H NaOCl NaOCl 95% EtOH H O 95% EtOH 95% EtOH 95% EtOH 70% EtOH 95% EtOH NaOCl H O 2 2 70% EtOH 95% EtOH NaOCl 2 95% EtOH Evaporation Evaporation H2O Evaporation Evaporation H2O H2O2 H2O Evaporation 95% EtOH Evaporation H2O2 H2O2/peracetic M Bacterial spp.a PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST EDICAL Acinetobacter spp. ϩϩϩϩϩϩϩϩϩϩϩÑ ϩ Ñ ϩ Ñ ϩϩϩÑ ϩ Ñ Areococcus spp. ϩϩϩÑ ϩ Ñ ϩ Ñ ϩϩϩÑ

Bacillus spp. ϩ Ñ ϩ Ñ ϩ Ñ ϩ Ñ ϩ Ñ ϩϩ ϩ Ñ ϩ Ñ ϩ Ñ E Bacillus fusiformis ϩ Ñ NTOMOLOGY Enterobacter cloacae ϩ Ñ ϩϩϩÑ ϩ Ñ ϩ Ñ ϩϩ ϩ Ñ ϩ Ñ Klebsiella oxytoca ϩϩϩϩÑ ϩ Ñ ϩ Ñ ϩ Ñ ϩ Ñ ϩ Ñ ϩ Ñ Micrococcus luteus ϩ Ñ ϩϩϩÑ ϩϩϩϩ ϩ ϩ ϩÑ ϩϩϩÑ Pseudomonas aeruginosa ϩ Ñ ϩ Ñ ϩ Ñ ϩ Ñ ϩ Ñ Staphylococcus gallinarum ϩϩϩϩϩϩϩϩϩϩϩϩϩÑ ϩ Ñ ϩϩϩÑ ϩ Ñ Staphylococcus kloosii ϩ Ñ ϩ Ñ ϩϩϩÑ ϩ Ñ ϩ Ñ ϩ Ñ ϩ Ñ Staphylococcus xylosis ϩ Ñ ϩϩϩÑ ϩϩϩϩ ϩÑ ϩ Ñ ϩ Ñ Streptococcus spp. ϩ Ñ ϩ Ñ ϩϩϩÑ ϩϩϩÑ ϩ Ñ ϩϩ ϩ Ñ

a Representative bacteria, selected from PRE- and POST-wash blood agar plates, identiÞed using selective culture methods and ribotype characterization. ϩ, bacteria was present; blank space, bacteria was not present; Ñ, bacteria removed by corresponding disinfection protocol. o.4,n.5 no. 43, Vol. September 2006 CRIPPEN AND SHEFFIELD:BEETLE SURFACE DISINFECTION 921

1994, 1995, 1996; Hald et al. 1998; Gray et al. 1999). formed (De las Casas et al. 1968, 1972; Harein and De However, validation of these sterilization methods las Casas 1968). The authors noted that neither treat- was not presented. In this study, we tested the efÞcacy ment disinfected completely; therefore, the effective- of disinfection protocols to remove culturable, aerobic ness of these techniques was determined by subse- bacteria from the external surface of A. diaperinus. quent individual exposure of each beetle to nutrient The protocols were based on the use of three primary broth and removal of contaminated specimens from chemicals (EtOH, NaOCl, Tween 80, and H2O2), and the study. We determined that washing in 2% NaOCl/ the level of external disinfection was found to range Tween 80 followed by dH2O was only 66.72% effective from 48 to 100%. Sonication was used to assure uni- (protocol D). Similarly, De las Casas et al. (1968) formity of the agitation procedure for comparison of determined that Ϸ50% of the pupae subjected to these protocols. However, it should be noted that sonication disinfection procedures were still contaminated. In has been reported to assist in the destruction of bac- our study, the simple addition of an EtOH rinse step terial cells and thus could enhance the efÞciency of after disinfection by NaOCl/Tween 80 increased ef- the disinfection techniques used (Dehghani 2005). fectiveness to an average of 92.33% (protocol E and F). The amount of bacteria carried by each individual Lesser mealworms have been surface sterilized beetle before disinfection varied greatly. The bacterial against infectious bursal disease virus with 10% H2O2 load is of course subject to many factors, such as followed by rinsing in dH2O (McAllister et al. 1995). moisture, litter and feed present, but it was interesting Ͻ In our study, disinfection with 10% H2O2 was 100% that in the same environment we could culture no effective against bacteria (data not shown). An in- bacterial from the external surface of several beetles. crease to 20% H2O2 (protocol H) was found to be Perhaps the bacteria were sequestered in locations 97.8% effective against bacteria. The addition of a 95% impervious to extrication. De las Casas et al. (1972) EtOH treatment before a 20% H2O2 wash resulted in also observed this phenomena, Þnding that some bee- consistent, complete surface bacterial disinfection. tles carried several thousand colonies, whereas others H2O2 is often used as an antimicrobial or bleaching from the same cage carried few or no bacteria. Given agent. However, it is an unstable compound that the individual variation in bacterial load, the use of a quickly neutralizes itself by reverting to oxygen and large sample size is prudent for transmission studies. water. Stability depends upon many factors, but solu- Hald et al. (1998) investigated bacterial carriage by tions of H O which are kept in dark, inert containers hairy beetle, stercorea L., by bulk dis- 2 2 that are completely free of contamination are rela- infection in 70% EtOH followed by evaporation. In our tively stable. Commercial solutions, however, usually study, only 60% of the A. diaperinus were surface contain minute amounts of impurities, which can disinfected by this method (protocol B). The reservoir cause decomposition; therefore, stabilizers are some- competence of lesser mealworms for Salmonella and times added (Goor 1989, Hess 1995, CHEMINFO for E. coli was investigated by disinfection with dH O 2 2005). Concentrated H O solutions can also react followed by 70% EtOH and a second dH O rinse 2 2 2 exothermically with solvents. Although H O proved (McAllister et al. 1994, 1996). Conclusions indicated 2 2 that adult and larval A. diaperinus maintained a viable to be the best cleansing agent, its instability in pure population of internal (homogenate) and external form and exothermic reactivity at high concentrations bacteria for a period of time sufÞcient to infect suc- made it problematic to use; therefore, we investigated cessive ßocks of broiler chicks placed in the house. a commercially available substitute. The substitute However, no data were presented demonstrating the consisted of a less concentrated formulation of 7.35% effectiveness of the external disinfection procedure H2O2 with the addition of 0.23% peracetic acid, a before homogenation. Our study indicates that this in stabilizer, a surfactant and a corrosion inhibitor. Our an incomplete sterilization procedure. Gray et al. results demonstrate that this formulation was as ef- (1999) investigated the histerid beetle Carcinops pu- fective as 20% H2O2 treatment. milio (Erichson), measuring both surface and internal The thin epicuticle secreted on the outside of the (homogenate) contamination after rinsing for 30 s in cuticle serves to minimize loss of insect body water, contributing to their success in a terrestrial environ- 95% EtOH followed by dH2O. These investigators con- cluded that bacteria were carried both externally and ment (Bursell and Clements 1967). The epicuticle internally. However, no supporting data were pre- consists of several layers, including a superÞcial wax or sented to substantiate the disinfection procedure. In lipid layer of long-chain hydrocarbons and esters of our study, disinfection with 95% EtOH followed by fatty acids and alcohols (Chapman 1982a, Lockey dH2O (protocol A) was only 48.52% effective. It is 1988). This deterrent to desiccation also presumably possible that if the surface disinfection procedures diminishes the effectiveness of surface disinfection by were insufÞcient, then bacteria harbored externally H2O2 or NaOCl treatment due to its hydrophobic would be attributed to internal sources upon culturing nature. We found that prior treatment with EtOH of the insect homogenate. enhanced the efÞcacy H2O2 and NaOCl treatments. In studies investigating recovery of bacteria from Because lipids are soluble in organic solvents, it is granary weevils, Sitophilus granarius (L.), and A. dia- likely that EtOH treatment either diminished the perinus adults, pupae, and larvae, surface disinfection waxy layer or wetted the exterior sufÞciently to allow using 2% NaOCl/Tween 80-based protocols were per- a subsequent agent improved access to the beetle 922 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 43, no. 5 surface and invaginations, and the microorganisms Bursell, E., and A. N. Clements. 1967. The cuticular lipids of residing there. the larva of Tenebrio molitor L. (Coleoptera). J. Insect Surface disinfections that included EtOH or NaOCl Physiol. 13: 1671Ð167. were detrimental to the beetles and often resulted in Chapman, R. F. 1982a. The integument, pp. 501Ð528. In The their death. H O treatment seemed less deleterious , structure and function, 3rd ed. Harvard Univer- 2 2 sity Press, Cambridge, MA. because the beetles usually survived the treatment. Chapman, R. F. 1982b. The tracheal system and respiration For our purposes and for many of the studies dis- in terrestrial insects, pp. 529Ð553. In The insects, structure cussed, survival of the specimens was not required. and function, 3rd ed. Harvard University Press, Cam- However, for future studies where survival is de- bridge, MA. sired, a modiÞcation in the protocols to a protocol CHEMINFO. 2005. Canadian Centre for Occupational less adverse to the survival of the beetles while still Health & Safety. Hamilton, Ontario, Canada. (www. maintaining efÞcacy as a disinfectant will be neces- intox.org). sary. De las Casas, E., B. S. Pomeroy, and P. K. Harein. 1968. In summary, this study assessed the efÞcacy of pre- Infection and quantitative recovery of Salmonella typhi- murium and Escherichia coli from within the lesser meal- viously described and newly developed methods to worm, Alphitobius diaperinus (Panzer). Poult. Sci. 47: disinfect the external surface of A. diaperinus. Al- 1871Ð1875. though many of the bacterial disinfecting procedures De las Casas, E., P. K. Harein, and B. S. Pomeroy. 1972. examined cleaned Ͼ90% of the beetles, we endeav- Bacteria and fungi within the lesser mealworm collected ored to Þnd a protocol that disinfected 100% of the from poultry brooder houses. Environ. Entomol. 1: 27Ð30. insects. When investigating bacterial transfer, com- De las Casas, E., P. K. Harein, D. R. Deshmukh, and B. S. plete exoskeleton disinfection is important to discern Pomeroy. 1973. The relationship between the lesser conclusively an internal source of contamination. Sev- mealworm and avian viruses: I. Reovirus 24. Environ. eral previously reported studies used protocols that Entomol. 2: 1043Ð1047. De las Casas, E., P. K. Harein, D. R. Deshmukh, and B. S. purportedly disinfected the surface of beetles; how- Pomeroy. 1976. Relationship between the lesser meal- ever, we could Þnd no data validating the efÞcacy of worm, fowl pox and Newcastle disease virus in poultry. J. the described techniques. Although it is possible that Econ. Entomol. 69: 775Ð779. the insects harbor bacterial organisms internally, in- Dehghani, M. H. 2005. Effectiveness of ultrasound on the sufÞciently sterilized exoskeletons can contaminate destruction of E. coli. Am. J. Environ. Sci. 1: 187Ð189. subsequent homogenized specimens. Therefore, it Despins, J. L., R. C. Axtell, D. V. Rives, J. S. Guy, and M. D. would be unclear whether the bacteria being mea- Ficken. 1994. Transmission of enteric pathogens of tur- sured were actually being harbored internally. We keys by larva (Alphitobius diaperinus). J. Appl. Poult. Res. 3: 61Ð65. found that a combination of treatment with 95% Goor, G. 1989. Hydrogen peroxide, pp. 443Ð466. In W. Ger- EtOH, allowed to evaporate, followed by a 20% H2O2 hartz and Y. S. Yamamoto [eds.], UllmannÕs encyclopedia or 7.35% H2O2/0.23% peracetic acid wash removed all of industrial chemistry, 5th ed., vol. A 13. VCH Verlags- culturable, aerobic bacteria from the external surface gesellschaft, Weinheim, DeerÞeld Beach, FL. of the adult lesser mealworms. Gray, J. P., C. W. Maddox, P. C. Tobin, D. G. Jennifer, and C. W. Pitts. 1999. Reservoir competence of for Salmonella enteritidis (Eubacteriales: Entero- bacteriaceae). J. Med. Entomol. 36: 888Ð891. Acknowledgments Hald, B., A. Olsen, and M. Madsen. 1998. 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