GUIDELINES IN VBZD

Arthropod Containment Guidelines, Version 3.2

American Committee of Medical Entomology; American Society of Tropical Medicine and Hygiene

Abstract

The Arthropod Containment Guidelines are a product of the work of the American Committee of MedicalEntomology, a subcommittee of the American Society of Tropical Medicine and Hygiene. The guidelinesprovide a reference for research laboratories to assess risk and establish protocols for the safe handling ofarthropod vectors of human and animal disease agents. The guidelines were originally published in 2004 andhave been updated here to reflect the spectrum of vector taxa under investigation, and the demands of workingwith vector arthropods in the context of the Select Agent Rule.

Keywords: guidelines, vector-borne disease, laboratory safety, risk assessment, containment, arthropod

Introduction

Laboratories in which living arthropods are reared andmaintained for research purposes have been in existence for

decades with few reports of harm to their workers or to thecommunities in which they are located. Many of these organ-isms are associated with potential risks should they escape, sincemany are vectors of infectious human diseases. When they areexperimentally infected with a human pathogen, the arthropodsrepresent an immediate risk to those who come into contact withthem. Even when they are uninfected, they can represent a risk tothe community if, by escaping, they become the crucial linkcompleting the transmission cycle for a pathogen.

There are two prominent examples of initially small exoticvector introductions that resulted in significant disease in-creases. In the early 1900s, anopheline mosquitoes weredrastically reduced in northeastern South America because oferadication campaigns. The concomitant drop in incidence ofmalaria and other human infectious diseases was reversed afterAnopheles gambiae was discovered in the port city of Natal,Brazil, in 1930 (Soper and Wilson 1943). The African malariavector was accidentally introduced into the area, probably byrapid marine mail service. Although the release was not from alaboratory, the introduction of a highly efficient vector iswidely thought to be responsible for the resurgence of malariain Brazil. Fortunately, an aggressive effort to eradicate An.gambiae by conventional means was successful.

A second example concerns the current distribution of theChagas disease vector, Rhodnius prolixus, throughout rural

Central America. Whereas this insect is considered indigenousto northern South America, it is thought to have been introducedinto Central America through a laboratory escape that occurredin El Salvador in 1915 (Schofield 2000). While there are anumber of other triatomine species throughout Central Americathat transmit the agent of Chagas disease to humans, the estab-lishment of R. prolixus was especially important because of itsclose association with humans and domestic dwellings. R. pro-lixus is considered the most important vector of Chagas diseasein Central America and the species targeted for elimination bythe Central American Chagas Disease Control Initiative.

Past editions of the reference manual ‘‘Biosafety in Micro-biological and Biomedical Laboratories’’ (BMBL) (Centers forDisease Control and Prevention [CDC] 1999) noted that de-spite the long history of accidental laboratory infections ofworkers and their immediate contacts, ‘‘laboratories workingwith infectious agents have not been shown to represent a threatto the community’’ referring to reviews of laboratory-acquiredinfections at CDC and the National Animal Disease Centerwhich documented that there were no instances of secondarytransmission (Richardson 1973, Sullivan et al. 1978). Never-theless, the historical accidental introductions An. gambiae andR. prolixus suggest the possibility for community impacts ofintroduced arthropod vectors, even when uninfected.

The advent of transgenic technology in which vector ar-thropods are now routinely genetically modified requiresarthropod containment recommendations with a particularview to preventing inadvertent escape and establishment.These guidelines are intended to do that. They have been

Presented as a project of the American Committee of Medical Entomology of the American Society of Tropical Medicine and Hygiene.

� American Committee of Medical Entomology and American Society of Tropical Medicine and Hygiene, 2019; Published by MaryAnn Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

VECTOR-BORNE AND ZOONOTIC DISEASESVolume 19, Number 3, 2019Mary Ann Liebert, Inc.DOI: 10.1089/vbz.2018.2431

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drafted by a subcommittee of the American Committee ofMedical Entomology (ACME) and circulated widely amongmedical entomology professionals. The Committee mem-bership and drafting procedures are summarized in Appendix1–3. These guidelines represent the position of the committeeas a whole, not that of all individuals nor of the membership’sindividual institutions.

Transgenic arthropods and those containing microbesmodified by recombinant DNA technology are addressedhere solely in the context of public health significance. Theemphasis is therefore on the phenotypic changes resultingfrom the modification rather than ecological and environ-mental issues, which are addressed elsewhere (WHO/TDRand FNIH 2014, Marshall 2010).

Background

Several documents particularly influenced the effortto create these guidelines and shaped their content

1980. The Subcommittee on Arboviral LaboratorySafety, a subcommittee of the American Committee onArthropod-Borne Viruses (ACAV), a sister organization ofACME, published guidelines in the American Journal ofTropical Medicine and Hygiene (The Subcommittee on Ar-bovirus Laboratory Safety 1980). These were developed onthe basis of a survey of almost 600 laboratories and covered awide range of pathogenic viruses. They addressed issuesrelevant to the safety of laboratory workers and assigned eachvirus to one of four ‘‘levels of containment and practice’’based on the severity of the associated human disease, themethod of transmission, and the amount of experience han-dling the organism. The document addresses containment ofthe arthropod vector for each level, but only when it is in-fected by the viral agent. It did not address nonviral (e.g.,eukaryotic or bacterial) agents or their vectors, transgenicanimals, nor did it take into account biological containmentthat may be provided by the climate or other characteristics ofthe location in which the research is conducted.

1984. BMBL was published in response to a series ofsurveys, beginning, for example, with reports of laboratory-acquired infections (Sulkin et al. 1949, Richardson 1973,Sullivan et al. 1978). Now in its fifth edition, this PublicHealth Service document describes the practices, facilities,and equipment suggested to safely work with potentiallydangerous agents in a laboratory (Centers for Disease Controland Prevention [CDC] 2009).

1995. The American Mosquito Control Associationadopted a position that containment of genetically manipu-lated arthropods be addressed by funding and regulatoryagencies. A letter sent to the Directors of the National Sci-ence Foundation, the CDC, the National Institute of Allergyand Infectious Diseases, the Pan American Health Organi-zation, and the Administrator of the U.S. Department ofAgriculture (USDA) urged that ‘‘guidelines for research bedeveloped to ensure that no exotic agents are accidentallyreleased, and to ascertain the potential for the released or-ganisms to alter vector-borne disease transmission patterns.’’

1996. Hunt and Tabachnick (1996) prepared a forumarticle on containment of very small arthropod vectors. This

provided a means to test the efficacy of containment andaddressed the peculiar containment needs of laboratoriesworking with arthropods too small to be restricted by con-ventional insectary precautions.

Higgs and Beaty (1996) authored a chapter entitled‘‘Rearing and Containment of Mosquito Vectors.’’ Thechapter gives practical advice and illustrations for the design,construction, and operation of insectaries.

1997. The Molecular Biology of Disease Vectors waspublished (Crampton et al. 1997). It contains several chaptersdescribing methods for the experimental infection of a widevariety of insects. Although most of the emphasis of thistextbook is methodological, safety aspects are also addressed.

1998. The USDA, through its Animal and Plant HealthInspection Service (APHIS), developed draft guidelines forthe containment of nonindigenous, phytophagous arthropodsand their parasitoids. These guidelines include standards forconstruction, equipment, and operations when handling ar-thropod pests of plants. While the document focuses onpreventing environmental detriment, the general containmentprinciples for this class of arthropods are relevant to hema-tophagous arthropods.

1999. The Department of Health and Human Services,through the National Institutes of Health (NIH), issued re-vised guidelines for the safe handling of organisms thatcontain recombinant DNA, including arthropods (NIHGuidelines 2016). Paralleling the BMBL, these guidelines alsospecify that an institutional biosafety committee (IBC) reviewall nonexempt research protocols involving recombinant DNAand approve the level and implementation of appropriatecontainment. Section III.D.4 of the Guidelines specificallyaddresses arthropods that contain recombinant DNA and as-sign them to a minimum of biosafety level (BSL)-2.

The American Society of Tropical Medicine and Hygiene(ASTMH), ACME, adopted a resolution to develop theseArthropod Containment Guidelines. The guideline’s form isdirectly based on the structure and wording of BMBL(American Committee of Medical Entomology and Ameri-can Society of Tropical Medicine and Hygiene 2003) (seeAppendix 2).

Intent

This document supplements BMBL by providing safetyprinciples for handling arthropod vectors. Readers shouldrefer to BMBL for standard and special microbiologicalpractices appropriate for the agents with which they work.These have been repeated here when they would apply to thevector alone as well as the agents. Throughout, the authorshave attempted to formulate guidelines that are consistentwith those of BMBL, yet recognize the fact that biologicalcontainment (i.e., location- and season-specific fate of es-caped arthropods in the environment) significantly confoundsrisk assessment and therefore appropriate safety practices.Furthermore, the flying, crawling, burrowing, and reclusivehabits of arthropods, combined with the agents they maycarry, introduce an element of risk-increasing behavior notcovered by BMBL.

ARTHROPOD CONTAINMENT GUIDELINES, VERSION 3.2 153

This document describes the arthropod handling practices,safety equipment, and facilities constituting Arthropod Con-tainment Levels 1–4 (ACL-1 to -4). These are recommendedby the ASTMH/ACME for work with a variety of uninfectedarthropods and those carrying infectious agents, and for workwith transgenic vector arthropods in laboratory settings. Theprinciples of risk assessment, specific practices, and equip-ment will also be useful in nontraditional arthropod researchsettings such as tents, greenhouses, and outdoor cages. Fieldsites at which research with such arthropods is conducted aredefined by the type and duration of activities that occur thereand the risks to the participants and inhabitants, but they arenot the focus of the document. Although plant pathologistsand entomologists may find this document useful, the focus ofthis document is on arthropods that transmit pathogens ofpublic health importance. More details specifying the ar-thropods that are generally excluded from these guidelinescan be found under ‘‘Arthropod Containment Levels.’’

This document is strictly concerned with laboratory researchthat involves arthropods of public health importance and risksassociated with pathogen transmission. Arthropods to be con-sidered include among others: insects (Diptera—mosquitoes,tsetse flies, black flies, sand flies, midges; Hemiptera—kissingbugs; Phthiraptera—lice; Siphonaptera—fleas) and arachnids(Acari—ticks, mites). All life cycle stages, eggs, larvae,nymphs, adults, must be considered under the term arthropod.The small size, highly motile characteristics of some arthro-pods (especially flying and jumping), and relative long life andresistance of some stages make the containment of arthropods aunique problem. The diversity of these organisms and theircomplex life cycles often mean that procedures and practices tosafely contain the animal are species-specific. Conversely, thespecific culture requirements of some species make mainte-nance difficult, but containment relatively straightforwardsince they cannot survive outside of the preferred habitat. Al-though noninsects (ticks and mites) are also considered here,the designated area in which these organisms are maintainedand cultured will hereafter be referred to as an insectary.

From the above, it follows that many arthropods (e.g., fruitflies, cockroaches, and various Lepidoptera and Coleoptera)that have been collected locally, or have been purchased frompet stores or commercial vendors for study or educationalpurposes, are usually exempt from these guidelines. How-ever, experiments planned with nonvector arthropods that aredeliberately infected with a disease agent might be informedby these guidelines.

Arthropods are an important educational tool in manyschools and colleges, and are collected and cultured for manydifferent purposes. These guidelines should not impact in-terest in and research on uninfected arthropods that pose nodanger to the surrounding environment and public health. Inthese circumstances, maintenance and rearing techniques areat the discretion of the student or instructor, and do not fallwithin the containment criteria described in these guidelines.

It should be noted that although these guidelines are in-tended mainly for U.S. research institutions and those pro-grams in other countries receiving NIH funds, there are manyinstitutions outside of the United States or the EuropeanUnion, particularly in resource-limited sites, where admin-istrative constructs such as institutional animal care and usecommittee (IACUC), IBC, biosafety officers, and occupa-tional health clinics do not exist. Small institutions even in

the United States may not have the infrastructure or funds torenovate laboratories, and therefore, any alternatives to mit-igate these realities have to be thoroughly vetted in a riskassessment to ensure that research on vector arthropods issafe and fully contained.

These recommendations are advisory. They provide avoluntary guide or code of practice as well as principles forupgrading operations. They also offer a guide and referencein the construction of new laboratory facilities or the reno-vation of existing facilities. The application of these recom-mendations to any particular laboratory must be based on arisk assessment of the particular vector species, diseaseagents, activities, and geographic location of the laboratory.

Field Sites

Sites at which vector arthropod research is conductednecessarily span a wide range of sophistication and infra-structure from modern structures that are clearly ‘‘laborato-ries’’—the primary focus of this document—to primitivefield sites that do not. To clearly define the characteristics thatdistinguish a laboratory from a field site, we describe char-acteristics of these activities to enable researchers, IBCs, andgranting organizations to determine whether these laboratoryguidelines apply at all. A field site is a temporary facility, thework is performed with indigenous arthropod vectors that arecollected locally, and in the event of escape, there is likelyonly a small risk that released arthropods would modify thegenetic structure of resident arthropod populations or in-crease the risk of human or animal infection with locallytransmitted pathogens. Maintenance of indigenous vectorsfor local research may occur in these facilities. Field labo-ratories usually do not require structural modification of ex-isting buildings for vector containment, rather arthropodescape is minimized by appropriate primary containment(caging) and handling practices.

Field laboratories are often located in places where thespecies under study is involved in pathogen transmission.Because vectors brought from the field into the laboratorymay be naturally infected with a pathogen, appropriate pre-cautions should be taken to minimize researcher exposure toinfectious organisms. On the contrary, the risk within thelaboratory by manipulating collected samples may be lessthan working outside. The risk assessment process will de-termine what precautions, if any, are warranted, for example,vaccines, prophylaxis, and repellents.

Authorities

Importation and transport of exotic arthropods of publichealth importance fall under the purview of the Public HealthService/CDC, Office of Health and Safety (United StatesPublic Health Service [USPHS] 42 Code of Federal Reg-ulations [CFR], Part 71.54). Many arthropods transmit bothhuman and animal diseases and may therefore also be regu-lated by the USDA APHIS. Arthropods modified by recom-binant DNA methods or containing similarly modifiedmicrobes are addressed in the NIH Guidelines and may beregulated by the U.S. Environmental Protection Agency(EPA) and/or the U.S. Food and Drug Administration (FDA).

154 ARTHROPOD CONTAINMENT GUIDELINES, VERSION 3.2

Risk Assessment for Arthropod Vectors

The intent of this section is to provide guidance and toestablish a framework for selecting the appropriate ACL(facilities, equipment, and practices), reducing risks of re-lease and exposure of laboratory workers and the public to avector and associated agents.

‘‘Risk’’ in the context of this document implies the prob-ability that harm, injury, or disease will occur among labor-atorians or the general public because of accidental release ofa competent disease vector and/or associated agents. In thecontext of vector research laboratories, risk assessmentconsiders two kinds of effects: direct effects such as biting,infestations, and myiasis, and indirect effects of morbidityand mortality due to the pathogens transmitted. The latter isby far of higher concern, and direct effects are not consideredhere. Therefore, in this document, ACLs are directly corre-lated with the appropriate BSL of the agents with which theyare naturally or experimentally infected or may transmit inthe event of accidental release (see BMBL Section VI).

While the focus of this document is human health risk,effects on animals because of arthropods known to transmitanimal disease are to be considered. Researchers are en-couraged to consult with the U.S. Fish and Wildlife Serviceand USDA-APHIS regarding risks and regulation beforecompleting a risk assessment.

The laboratory director or principal investigator (PI) hasprimary responsibility for assessing risks to set the appro-priate BSL for the work. This is done in close collaborationwith the IBC, if there is one, to ensure compliance withestablished guidelines and regulations. Development andreview of the risk assessment and the planned safety pre-cautions by consultation with experts in the biology andpublic health significance of the arthropod are essential.

In performing a qualitative risk assessment, all the riskfactors are first identified and explored considering relatedinformation available such as BMBL, the NIH Guidelines, theCanadian Laboratory Biosafety Guidelines (Public HealthAgency of Canada 2004), the WHO Biosafety Guidelines(World Health Organization (WHO) 2004), and the ACAVCatalogue of Arboviruses (Centers for Disease Control andPrevention [CDC] 1985). In many cases, one must rely onother sources of information such as field data, the literatureconcerning aspects of vector competence, and environmentalrequirements through consultation with recognized experts inarthropod and pathogen relationships.

The greatest challenge of risk assessment lies in thosecases where complete information on these factors is un-available. A conservative approach is advisable when insuf-ficient information forces subjective judgment.

Principles of Risk Assessment

Arthropod risk assessment is primarily a qualitative judg-ment that cannot be based on a prescribed algorithm. Severalfactors must be considered in combination: the agents trans-mitted, whether the arthropod is or may be infected, the mo-bility and longevity of the arthropod, its reproductive potential,biological containment, and epidemiological factors influ-encing transmission in the proposed location or region at risk.Arthropod vectors of infectious agents can be assigned to thefollowing discrete categories. Each category has a range ofrisks that need to be assessed.

Arthropods known to be free of specific pathogens

Risk from these materials to laboratorians is similar to thatexperienced by the general public: nuisance due to conse-quences of escape and temporary or permanent establish-ment. Consequently, the public health risk is likely to be lowunless epidemiological conditions exist that could reasonablybe expected to result in an increase in transmission of anendemic disease in that particular region, or establishment ofthe released vector leads to significant risk of future trans-mission potential for an exotic pathogen. In the event thatestablishment is likely, the arthropod must be handled undermore stringent containment conditions.

If an accidental release occurs, followed by even tran-sient establishment of an uninfected arthropod, the proba-bility of increased transmission must be considered in thecontext of the location in which the work will be performedor in regions to which escaped arthropods could likelymigrate. For example, escape of an exotic malaria vector ina malarious region has a significantly higher probability ofincreasing transmission and therefore a higher risk thanescape in a nonmalarious region. The pathogenicity of theagent and availability of treatments and drugs should alsobe considered.

Answers to the following questions will affect the level ofrisk due to accidental escape of uninfected arthropods:

� Is the arthropod species already established in the locale?� If the arthropod is exotic, is it likely that the arthropod

would become temporarily or permanently establishedin the event of accidental escape?� Does the arthropod have a known or characterized

insecticide-resistant genotype or phenotype?� Could the arthropod be realistically controlled or lo-

cally eradicated by traditional methods (e.g., spraying,trapping) in the event of escape?� Are the agents that the arthropod is known to transmit

cycling in the locale, or has the agent been present inthe past?� Are agents that the arthropod could reasonably be ex-

pected to transmit to animals present in the locale?� Would accidental release of the arthropod significantly

increase the risk to humans and animals above thatalready in existence in the event of introduction ofexotic pathogens in the area?� In the case of zoonotic diseases, does the animal res-

ervoir exist in the locale, and, if so, what is the infec-tion status?� Was the exotic arthropod derived from a subpopulation

(strain, geographically distinct form) whose phenotypeis known or suspected to vary in ways that could rea-sonably be expected to significantly increase its vectorcompetence? If so, it should be handled under the morestringent conditions within ACL-2 (described below)even if uninfected.� Are disabled strains available, whose viability after

escape would be limited (e.g., eye-color mutants, cold-sensitive)?

Arthropods known to contain specific pathogens

Arthropods that are known to be, or reasonably suspectedof being, infected with infectious agents always have risks

ARTHROPOD CONTAINMENT GUIDELINES, VERSION 3.2 155

that must be identified, and appropriate precautions must betaken for worker and public health safety. The characteristicsof most known infectious agents have been well-defined andare the starting point for determining risk from these ar-thropods. Information useful to risk assessment can be ob-tained from laboratory investigations, disease surveillance,and epidemiological studies. Infectious agents known tohave caused laboratory-associated infections are included inthe BMBL agent summary statements (Section VII). Othersources include the American Public Health Association’smanual, Control of Communicable Diseases (Benenson1995). Literature reviews on laboratory-acquired infectionsalso may be helpful (Sulkin et al. 1949, Richardson 1973,Sullivan et al. 1978).

The pathogenicity of the infectious or suspected infectiousagent, including disease incidence and severity (i.e., mildmorbidity versus high mortality, acute versus chronic dis-ease), is the most important consideration in assessing therisk due to accidental exposure to an infected arthropodvector. As the initial criterion, it is clear that the more severethe potentially acquired disease, the higher the risk.

Readers will observe that ACL-2 has broad latitude inspecific practices. This reflects, in part, the widely differingdegrees of effects of arthropod-borne agents, many of whichfall within the BSL-2 level. Considerable variation in mor-bidity and mortality exists within the level 2 classification.For example, level 2 arboviruses are exemplified by LaCrosse virus with a 1% or less mortality rate and limited, mildneurological sequelae. Higher containment levels are re-commended for agents that cause disease in humans con-sidered potentially severe, life threatening, or cause residualdamage. Our general approach in formulating these guide-lines has been to include a wide range of ACL-2 features thatreflect this broad range of agent potency. Moreover, thepossible natural and artificial modes of infection (e.g., par-enteral, airborne, ingestion) of the agent are considered. Thisis essential to prevent infections in laboratorians.

The established availability of an effective prophylaxis ortherapeutic intervention is another essential factor to beconsidered. The most common form of prophylaxis is im-munization with an effective vaccine. In some instances,immunization may affect the BSL or ACL; for example,Central European Tick Borne Encephalitis virus has beenreclassified to BSL-3/ABLS3 (from BSL-4/animal biosafetylevel [ABS]-4) when laboratory personnel are vaccinated(BMBL5, p. 236). Although IBCs once could permit cer-tain tasks and procedures with eastern equine encephalitisvirus (EEEV) to be conducted at BSL-2 (from BSL-3) whenpersonnel were vaccinated, recent regulations appear to pro-hibit such a downgrading (www.selectagents.gov/guidance-encephalitis.html). At any rate, the availability of therapeuticsand vaccines only serves as an additional layer of protectionbeyond engineering controls, proper practices, and procedures,and the use of personal protective equipment. Occasionally,immunization or therapeutic intervention (antibiotic or antiviraltherapy) may be particularly important in field conditions. Theoffer of immunizations is part of risk management to protectlaboratory workers and vaccination may be demanded, as acondition of employment, for any laboratory worker workingwith yellow fever virus, or any pathogens for which an effi-cacious vaccine is available. Such a policy may vary frominstitution to institution.

Medical surveillance is encouraged to ensure that the in-stituted safeguards provide the expected health outcomes.Surveillance may include serum banking, monitoring em-ployee health status, and participating in postexposure man-agement. However, as with vaccination, the implementationof medical surveillance may vary by institution. In the ar-thropod vector laboratory, this must be combined with regularmonitoring for escaped arthropods, for example, inventory ofinfected arthropods, an effective arthropod trapping program,and regular inspection of the facilities for disrepair that couldresult in escape.

Risk assessment must also include an evaluation of theexperience and skill level of at-risk personnel such as la-boratorians, maintenance, housekeeping, and animal carepersonnel. Additional education may be necessary to ensurethe safety of persons working at each BSL.

Arthropods containing unknown infectious agentsor whose status is uncertain: diagnostic samples

The challenge here is to establish the most appropriatecontainment level with the limited information available.Some questions that may help in this risk assessment includethe following:

� Why is an infectious agent suspected?� What route of transmission is indicated?� Are agents that the arthropod transmits transferred

horizontally?� Are there reasons to believe that a novel or unknown

agent is present?� What epidemiologic data are available?� What is the morbidity or mortality rate associated with

the agent?Bringing field-collected arthropods into a laboratory may

be associated with the possibility that personnel who wouldnot otherwise be exposed to any risk because they do notwork in field sites might be placed at risk. Researchersworking in field sites often handle arthropods of unknowninfection status under conditions that do not allow im-plementation of typical laboratory precautions, but they are inthe field and understand that their actions may expose them toassociated risks. Answers to the questions above will assistresearchers in determining potential risks and reasonablesolutions.

Although the most conservative approach would be toconsider all field-collected arthropods as potentially infec-tious, it should be recognized that the prevalence of infectionfor most vector/pathogen relationships is small. With manyarboviruses, a prevalence of 1 in 1000 may be typical. In-fection may not be viable, or the quantum of infection (po-tential dose) may be below that required to produceproductive infection. There may be strains or populations ofinfectious agents that are avirulent. Delivery of an agent bycontamination (exposure to aerosol or arthropod tissues) maybe significantly less efficient in producing infection than bybite, in which immunosuppressive and anti-inflammatorymolecules in salivary secretions may promote infection. Forthose agents with complex arthropod developmental cycles,the stages that are present may not be capable of productiveinfection. There are thus many factors involved in assessingrisk associated with unknown or diagnostic samples, and aconservative approach may not be warranted.

156 ARTHROPOD CONTAINMENT GUIDELINES, VERSION 3.2

The recent guidelines for clinical laboratories (Centers forDisease Control and Prevention [CDC] 2012) provide anexcellent approach to risk assessment that may be applied todiagnostic or unknown arthropod samples. Diagnostic clini-cal samples (humans or animals) are mainly handled at BSL-2, although specific scenarios (suspected viral hemorrhagicfever, tuberculosis, generation of aerosols, larger volumes)may require additional safety practices and procedures. Thispublication provides particularly detailed and nuanced dis-cussion of risk assessment in the clinical diagnostic settingthat places into context a risk assessment that allows for otherthan the very conservative recommendation that if an ar-thropod is suspected to contain a certain agent, the corre-sponding BSL should be applied.

Vector arthropods containing recombinantDNA molecules

The purpose of this section is to present principles of riskassessment of vector arthropods that have been geneticallymodified, typically via recombinant DNA technology. Thisincludes both vector arthropods that contain modified mi-crobes and which themselves are genetically modified.These principles primarily address the public health signif-icance of the modified organisms rather than environmentalconcerns. These technologies continue to evolve rapidly, andexperimental procedures designed to derive novel modi-fied symbionts and recombinant arthropods are becomingcommonplace.

The NIH Guidelines (2016) are a key reference in estab-lishing an appropriate BSL for work involving recombinantorganisms, including microorganisms for use in arthropodsand genetically modified arthropods themselves. In selectingan appropriate ACL for such work, the greatest challenge is toevaluate the potential biohazard change resulting from aparticular genetic modification relative to the unmodifiedarthropod. In the context of public health, the selection of anappropriate level begins by establishing the phenotypicchange in the arthropod and/or microorganism due to DNAmanipulation, and potential impact of escaped arthropodscontaining the modification.

Among the points to consider in work with recombinantarthropod vectors and those containing recombinant mi-crobes are the following:

� Does the inserted gene encode a product known orlikely to alter the vector capacity or competence forpathogens it is known to transmit?

� Does the inserted gene cause phenotypic changes thatcould significantly affect the ability to control the ar-thropod if there were an accidental escape, for exam-ple, an insecticide resistance marker?

� Does the modification have the potential to alter therange or seasonal abundance of the arthropod?

� If so, would the new range increase the likelihood thatthe vector could transmit new pathogens?

� Is the modified strain disabled in a way that viabilityafter escape would be limited (e.g., eye-color mutants,cold-sensitive)?

� Does the modification have the potential to increase thereproductive capacity of the arthropod that carries it?

� Is the phenotype conferred by the modification, in-cluding its marker and other expressed genes, if any,

consistently expressed after numerous generations ofpropagation?� Is the modification undergoing rearrangement or other

mutations at a measurable rate?� Can the DNA transgene vector be mobilized in natural

populations?� Is the host range of the symbiont known?� Would the modified symbiont pose increased risk to

immunocompromised persons relative to the nativesymbiont?� Is the entire sequence of the DNA insertion known, and

are the coding sequences defined?� Is horizontal transfer of the transgene to other microbes

with which the modified microbe is likely to come intocontact possible?� Is the original insertion site known so that stability can

be assessed later?This list of questions is not meant to be exhaustive; expert

discussions and guidelines are now available for this partic-ular aspect of vector biology (Booij 2013, Benedict 2014,WHO/TDR and FNIH 2014). Rather, the list illustrates theinformation needed to provide an accurate and conservativeassessment of risk to judge the appropriate containment level.Since in many cases the answers to the above questions willnot be definitive, it is important that the organization has aproperly constituted and informed IBC, as outlined in theNIH Guidelines, to evaluate risk assessment and provideprudent adherence to appropriate safety guidelines for theassigned risk.

Arthropod Containment Levels

The Arthropod Containment Level (ACL) guidelines werefirst published over a decade ago (American Committee ofMedical Entomology and American Society of TropicalMedicine and Hygiene 2003) (see Appendix 2). In the in-terim, new pathogens have been described, BSL-3 laborato-ries have proliferated, and the Select Agent Rule has evolvedto include thorough biosafety assessment in addition to bio-security. This revision of the guidelines was initiated with theobjective of clarifying and modifying or strengthening rec-ommendations to ensure the safety of researchers as well asthe community without hindering the pursuit of knowledgeand development of vector control methods and life-savingsolutions to vector-borne disease issues. We particularlyemphasize the absolute need for local risk assessments tocomplement the regulatory framework as opposed to achecklist approach based on these guidelines. Where appli-cable, we point out critical aspects of the Select Agent Rule.

When arthropods are used in research, facilities, trainedstaff, and established practices must be in place to ensureappropriate safety, and the protection of health and well-being of workers and the environment. This article providesguidelines for laboratory work with hematophagous arthro-pods and arthropod vectors of pathogenic agents, and wasoriginally drafted in response to concerns related to theconsequences of an accidental release of arthropods. Theseconsequences are basically answering the question ‘‘Whathappens if the arthropod escapes?’’ and the suggested con-tainment levels address the question ‘‘How do we preventescape?’’ When working with a vector in a particular set ofcircumstances (Table 1), a certain containment level may be

ARTHROPOD CONTAINMENT GUIDELINES, VERSION 3.2 157

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158

recommended. It should be noted, though, that the local IBCis an essential component in establishing the appropriateACL. It is responsible for reviewing a research protocol anddecides at what level of containment the experiments must beperformed. Accordingly, a ‘‘one-size-fits-all’’ checklistbased on these guidelines is not appropriate for risk assess-ment; local IBCs may, for example, allow research to proceedeven if it is not possible to adhere to all recommendedguidelines, such as those of the BMBL. In such cases, alter-native means of mitigating risk would be evaluated in thesite-specific risk assessment (Cauchetaux and Mathot 2005).

One significant practice emphasized as a result of theoriginal ACL guidelines is that when an arthropod is infectedwith an agent, the containment level required is automaticallyincreased to at least that required for the agent, regardless offactors such as the competence of that arthropod for theparticular pathogen. Containment levels for most infectiousagents, the rationale for categorizing them at such contain-ment levels, and recommendations for their safe manipula-tion are specified in the BMBL. An example is the use of malemosquitoes to propagate dengue viruses. Although theycannot transmit by bite, the presence of the viable agent re-quires that they be held at BSL-2. Injection of male mos-quitoes with dengue virus or homogenizing them afterward toassay for viral replication might expose personnel to infec-tion. Even if the actual infection status of each individualmale mosquito inoculated with dengue virus is not known,the standard of practice with diagnostic materials that maycontain an infectious agent is to manipulate them at BSL-2.

Another practical example of the utility of the ACLguidelines relates to the use of exotic arthropods. Biologicalintroductions are to be prevented by all reasonable means,and handling exotic arthropods at ACL-2 or higher greatlyreduces that risk. At any rate, even before the formal ACLguidelines, USDA import and possession permits wouldstipulate the equivalent of the ACL-2 recommendations foruse of most exotic arthropods. It is impossible to prescribeuniversal levels of containment for a particular species sincethe risks associated with accidental release from a laboratoryare determined by several factors, for example, the climateat the facility and history of transmission in that location. Forexample, the accidental release of an uninfected anthro-pophilic tropical vector species, during the winter in Wis-consin, should be considered significantly less of a risk thanthe release of the same species in a tropical area in which itcould become permanently established and act as a bridgevector of an established zoonotic pathogen to humans. Thus,one of a number of possible approaches to reduce the risk of arelease might be to perform relatively high-risk experimentsduring the winter when any escaped arthropods wouldquickly be killed by adverse environmental conditions; or arainforest species could be used in a laboratory located in axeric environment. IBCs might even use such biologicalconsiderations to ‘‘downgrade’’ a particular protocol fromACL-3 to ACL-2, providing that experiments are performedduring a particular period or in a particular site. On thecontrary, the possibility of zoonotic transmission or pro-moting a risk to domestic animal production means that wehave to consider in risk analysis those pathogens that arepredominantly an animal health issue; USDA Guidelinesmay need to be considered when assigning a containmentlevel to a particular vector species.

The current research and applied focus on geneticallymodified arthropods also mandate recommendations thatreduce the risk of accidental environmental release. Althoughthe ACL guidelines can be used as one basis for risk as-sessment and mitigation, the science of transgenic vectors isan evolving one and the reader is directed to specific docu-ments that provide experience-based guidance on such ex-periments (Centers for Disease Control and Prevention[CDC] 1985, Benenson 1995).

Although specific recommendations are not made hereinbecause they depend on site-specific details, all IBC proto-cols should outline an emergency response procedure that isappropriate in case of an accidental release. The ideal re-sponse would be one in which all released arthropods arekilled almost immediately after the escape. This may beimpossible if the escaped arthropods get outside of the lab-oratory, and hence, redundant facility and practice barriersare recommended to maximize the opportunities for locationand destruction of the escapees.

Arthropod Containment Level 1

ACL-1 is suitable for work with uninfected arthropodvectors or those infected with a nonpathogen, including (1)arthropods that are already present in the local geographicregion regardless of whether there is active vector-bornedisease transmission in the locale and (2) exotic arthropodsthat on escape would be nonviable or become only tempo-rarily established in areas not having active vector-bornedisease transmission. This category would include most ed-ucational use of arthropod vectors. A summary of the con-tainment levels is provided in Table 1.

Standard practices

Location of arthropods. Furniture and incubators con-taining arthropods are located in such a way that accidentalcontact and release are minimized. This may be achieved bylocating arthropods out of the flow of general traffic, avoidinghallways, or placing them in closets.

Supply storage. The area is maintained to allow detec-tion of escaped arthropods. For example, materials unrelatedto arthropod rearing and experimentation (e.g., plants, unusedcontainers, clutter) that provide breeding sites and harboragesare minimized.

General arthropod elimination. Accidental sources ofarthropods from within the insectary are eliminated. Thismay be accomplished by cleaning work surfaces after a spillof materials, including soil or water that might contain viableeggs. For example, personnel in mosquito laboratories shouldimmediately eliminate any standing water.

Primary container cleaning and disinfestation. Practicesshould be in place such that arthropods do not escape byinadvertent disposal in primary containers. Cages and otherculture containers are appropriately cleaned to prevent ar-thropod survival and escape (e.g., heated to, or chilled below,lethal temperature).

Primary container construction. Cages used to hold ar-thropods effectively prevent escape of all stages. Screened

ARTHROPOD CONTAINMENT GUIDELINES, VERSION 3.2 159

mesh, if used, is durable and of a size appropriate to preventescape. Nonbreakable cages are recommended. Bags, rearingtrays, and so on effectively prevent leakage and escape.

Disposal of arthropods. All life stages of arthropods mustbe killed before disposal. Arthropods may be killed with hotwater or freezing before flushing down drains or placed intotrash bags.

Primary container identification and labeling. Arthropodsare identified with descriptive labels to include the species,strain/origin, date of collection, responsible investigator, andso on; labels are firmly attached to the container (and cover ifremovable). Vessels containing stages with limited mobility(e.g., eggs, pupae, hibernating adults) are likewise labeledand (if applicable) housed or stored to prevent progression to,and escape of, a mobile life stage.

Prevention of accidental dispersal on persons or viasewer. Personnel take appropriate precautions to preventtransport or dissemination of live mobile arthropods from theinsectary by practicing appropriate disposal methods andpreventing escapees at every level of containment (primarycontainer, environmental chamber, laboratory, etc) to preventdispersal on persons.

Escaped arthropod monitoring. Investigators assesswhether escapes are occurring. An effective arthropod trap-ping program is recommended to monitor the escape pre-vention program.

Pest exclusion program. A program to prevent the en-trance of wild arthropods (e.g., houseflies, cockroaches,spiders) and rodents effectively precludes predation, con-tamination, and possible inadvertent infection.

Source and harborage reduction. Harborage and breed-ing areas are reduced as appropriate. Furniture and racks areminimized and can be easily moved to permit cleaning andlocation of escaped arthropods.

Notification and signage. Persons entering the area maybe made aware of the presence of arthropod vector species bysignage if recommended by an institutional research over-sight committee.

Special practices: vertebrate animal use

Institutional approval. Investigators should consult withtheir institutional research oversight office if vertebrate ani-mals will be used to feed hematophagous arthropods. Therequirement for IACUC and/or IBC review is an institutionaldecision, although highly recommended.

Housing of vertebrate animals. Animals used as hosts orblood sources should be housed according to institutionallaboratory animal guidelines. If necessary, vertebrate animalsmay be housed within the insectary but need to be adequatelyprotected from access by escaped arthropods. Animals notnecessary for maintaining arthropods should not be accessi-ble to hematophagous arthropods in the laboratory setting.

Containment during blood feeding. Special consider-ations should be taken when hematophagous arthropods are fedon host animals. The primary container must be sufficientlyrobust to prevent escape during feeding. When handling/re-moving vertebrate animals after exposure to arthropods, pre-cautions must be taken to prevent arthropod escape throughscreens, covers, and by flying. Host animals are inspectedclosely (e.g., concealment in fur, ears, axillae, or other possiblehiding places). Finally, all precautions should be taken toprevent arthropods fed on host animals from accidental transferto host cages and therefore dispersal outside of containment, ifanimals and their cages are returned to a holding room.

Blood source. The blood source should be considered apossible source of inadvertent arthropod infection andtransmission. Whenever feasible, use of sterile blood or bloodfrom sources known to be specific pathogen free is re-commended, whereas use of blood from animals or humanswhose disease status is uncertain should be avoided. In someinstances, a vector colony is specifically adapted to and willnot propagate without human blood acquired directly byfeeding on a volunteer. Such arthropods should not be fed asecond time on a different volunteer; those fed initially bymembrane on animal or human blood should not be allowedto subsequently feed on a human volunteer.

Safety equipment (primary barriers)

Gloves. Latex or nitrile gloves should be used whenhandling host animals or blood used to feed the arthropods,but local risk assessment and institutional policy may provideexceptions.

Torso apparel. White laboratory coats, gowns, and/oruniforms should be worn at all times in the insectary whenhandling blood and vertebrate animals, but local risk as-sessment and institutional policy may provide exceptions.

Arthropod-specific personal protective equipment. Per-sonal protective equipment is worn as appropriate, for ex-ample, respirators for arthropod-associated allergies, particlemasks, and head covers, but local risk assessment and insti-tutional policy may provide exceptions.

Facilities (secondary barriers)

Location of insectary. The insectary area is separated, ifpossible, from areas that are used for general traffic within thebuilding.

Insectary doors. Door openings, whether covered by ri-gid panels, glass, screens, plastic sheets, or cloth, minimizeescape and entrance of arthropods or pests.

Insectary windows. Windows, if present, effectivelyprevent escape of the smallest arthropods contained within aswell as prevent entry of wild arthropods and pests.

Lack of an insectary. Arthropods may be maintained atACL-1 in rooms other than those specifically designed asinsectaries. If the facility does not have secondary barriersthat would minimize escape or entry of pests, and is notseparated from general traffic, specific operating proceduresmust be developed and tested to mitigate such risks. For

160 ARTHROPOD CONTAINMENT GUIDELINES, VERSION 3.2

example, mosquitoes might be held by a ‘‘cage within alarger cage’’; removal of adult mosquitoes accomplished bythe aspirator manipulated through cage sleeves placed per-pendicular to each other and the sample container loadedentirely within the outer cage. Alternatively, entire mosquitocontainers may be chilled before aspirating individual mos-quitoes. Plexiglas glove boxes might also be used for ma-nipulations, particularly if exotic species are maintained.Nonflying species may be manipulated on designated tablesor benches in pans within moats of water, and housed in vialsor other containers held within a secondary storage containersuch as a lidded plastic food container.

Arthropod Containment Level 2

ACL-2 should be practiced if working with exotic andindigenous arthropods infected with BSL-2 agents associatedwith animal and/or human disease, or that are reasonablysuspected of being infected with such agents (diagnosticsamples). The PI must perform a risk assessment when de-ciding whether arthropods are reasonably suspected of beinginfected with a pathogen. For example, live mosquitoescollected during the course of a disease outbreak and main-tained in the laboratory would present more of a risk to lab-oratory personnel than those that are cold-immobilized orkilled before sorting and identifying them for standardsurveillance purposes. Uninfected genetically modified ar-thropod vectors also fall under this level provided the mod-ification has no or only negative effects on viability,survivorship, host range, or vector capacity. ACL-2 builds onthe practices, procedures, containment equipment, and fa-cility requirements of ACL-1. It is more stringent in physicalcontainment, disposal, and facility design requirements.Moreover, access is more restricted than ACL-1. The deci-sion to propagate infected exotic arthropods under ACL-2conditions in active transmission areas or in cases in whichestablishment is a possibility typically requires that measuresthat otherwise would only be recommended or preferred mustbe instituted as policy.

Standard practices

Location of arthropods. Furniture and incubators con-taining arthropods are located in such a way that accidentalcontact and release by laboratorians, custodians, and servicepersons are unlikely. This may be achieved by locating ar-thropods in dedicated rooms, closets, incubators located outof the traffic flow, or similar measures. Nonflying arthropodssuch as ticks are typically held in primary containers (vials)that are placed within an environmentally controlled con-tainer such as a desiccator or plastic food container; often, thisin turn is held within an environmental chamber. Although adedicated space is recommended for long-term storage ofticks, appropriate risk assessment by the local IBC or otherinstitutional entities, informed by the PI or other experts, mayallow for the housing of ticks in noninsectary settings.

Supply storage. The area is designed and maintained toenhance detection of escaped arthropods. Equipment andsupplies not required for operation of the insectary should notbe located in the insectary. All supplies for insect mainte-nance that must be kept within the insectary are located in adesignated area and not on open shelves. It is recommended

that a closed storage room, cabinets with tight-fitting doors ordrawers, be used. Doors and drawers are opened only foraccess. Insect diet should be kept in sealed containers.

Primary container cleaning and disinfestation. In addi-tion to cleaning cages and culture containers to prevent ar-thropod escape as in ACL-1, containers are disinfectedchemically and/or autoclaved if used for infected material,according to an IBC-approved protocol and/or laboratorystandard operating procedures. To reduce the risk of mixingup uncontaminated with potentially contaminated waste byhaving different methods for disposal, a laboratory may wantto consider routine autoclaving, incineration, or other ap-propriate decontamination of all primary containers.

Primary container construction. Cages used to hold ar-thropods are shatter-proof and screened with mesh of a size toprevent escape. Containers are preferably autoclavable ordisposable. Openings designed to prevent escape during re-moval and introduction of arthropods are recommended.

Disposal of arthropods. All life stages of arthropods mustbe killed before disposal by freezing or other suitable meth-ods. Infected arthropods should be autoclaved, or deconta-minated with chemical disinfectants such as 10% bleach or70% ethanol based on an agent-specific risk assessment. Thelack of an autoclave or means of incineration should beevaluated by local risk assessment and appropriate substitutessought.

Isolation of uninfected arthropods. Spread of agents touninfected arthropods is usually a low risk, given that mostinfections occur via hematophagy. Containers must beclearly marked to easily distinguish infected from uninfectedarthropods. It is good practice to separate infected arthropodsin a separate room, if possible, to prevent them from beingmistaken as being uninfected.

Primary container identification and labeling. As perACL-1.

Prevention of accidental dispersal via sewer or on per-sons. Before leaving the insectary and after handling cul-tures and infected arthropods, personnel wash their hands.Care should be taken to not disperse viable life stages into thedrainage system. No infected material is disposed through thesewer unless it is decontaminated. Physical barriers (over-lapping sheets and screens) or air curtains are recommendedas appropriate; personal protective equipment that is reused(laboratory coats, gowns) should be checked for infestationbefore exiting the insectary.

Pest exclusion program. As per ACL-1.

Escaped arthropod monitoring. Investigators assesswhether escapes are occurring by instituting an effective ar-thropod trapping program to monitor the escape preventionprogram. Oviposition traps, ground-level flea traps, oil-filledchannels surrounding tick colonies, light traps for mosqui-toes, and so on are recommended. Particularly in the casewhen exotic arthropods are used, exterior monitoring is re-commended. Records of exterior captures are maintained.

ARTHROPOD CONTAINMENT GUIDELINES, VERSION 3.2 161

Any evidence of escape should trigger a review of practicesand procedures before resuming work.

Source and harborage reduction. Harborage and breed-ing areas are eliminated. Furniture and racks are minimizedand can be easily moved to permit cleaning and location ofescaped arthropods. Equipment in which water is stored ormight accumulate (e.g., humidifiers) is screened to preventarthropod access, or contains chemicals to prevent arthropodsurvival.

Laboratory sharps. Disposable sharps should be dis-carded in puncture-proof containers or as mandated by in-stitutional policy. Forceps, dissecting probes, and othersharps that are reused should be frequently disinfected bychemical disinfection or flame sterilization.

Routine decontamination. Equipment and work surfacesin the insectary are routinely decontaminated with an effec-tive chemical disinfectant.

Notification and signage. Persons entering the areashould be made aware of the presence of BSL-2 agents inarthropod vectors, but institutions may vary in their policiesfor security or other reasons. If infected material is present,typically a BSL-2 biohazard sign is posted on the entrance tothe insectary, listing all species handled within and is updatedwhenever new species are introduced or pathogenic infec-tious agents are present. The hazard warning sign typicallyidentifies the arthropod species, agent(s) known or suspectedto be present, lists the name and telephone number of theresponsible person(s), and indicates any special requirementsfor entering the insectary (e.g., the need for immunizations orrespirators).

Procedure design. All procedures are carefully designedand performed to prevent arthropod escape.

Safety manual. A site-specific safety manual is prepared,approved by the IBC or other institutional review entities, andadopted. The manual contains emergency procedures, stan-dard operating procedures, waste disposal, and other infor-mation necessary to inform personnel of the methods for safemaintenance and operation of the insectary. If the institutiondoes not have formal review committees, the PI or departmenthead should develop laboratory-specific safety manuals.

Training. Laboratory personnel are advised of specialhazards and are required to follow instructions on practicesand procedures contained in the safety manual. Adherenceto established safety procedures and policies is made acondition of employment and is part of the annual perfor-mance review, if applicable, of every employee. Personnelreceive annual updates and additional training as necessaryfor procedural or policy changes. Records of all training aremaintained.

Medical surveillance. An appropriate medical surveil-lance program should be considered, although institutionalpolicy may vary with this requirement. At the minimum, allpersonnel should be educated by the PI about the risks as-sociated with the specific tasks and experiments, as well as

the signs and symptoms of any illness caused by the agent(s)under study. Specialty immunizations or a serum surveillancesystem may or may not be part of an institutional occupa-tional health program. In general, persons who may be atincreased risk of acquiring infection, or for whom infectionmay be unusually hazardous (e.g., immunocompromised),are not allowed in the insectary unless special personal pro-tection procedures are in place to eliminate extra risk.

Access restrictions. Routine access is limited to trainedpersons and accompanied guests. Service persons are madeaware of the hazards present and the consequences of ar-thropod release and contact with agents that may be present.

Special arthropod handling containers and areas. Infectedarthropods are prevented from release into the laboratoryarea. A dedicated area for handling infected material is re-commended. This is preferably a separate cubicle, walk-inincubator, or screen room. Additional physical barriers (e.g.,glove box, biosafety cabinet) or procedures (incapacitatedarthropods, e.g., removing a wing from a mosquito) may berequired depending on the local risk assessment. BMBL andother sources (Crampton et al. 1997) may provide specificrecommendations that can be adopted or modified accordingto the local risk assessment.

Safe transport in the laboratory. All infectious and po-tentially infectious samples are collected, labeled, trans-ported, and processed in a manner that contains and preventstransmission of the agent(s). Transfer of arthropods betweenmanipulation and holding areas is in nonbreakable securecontainers.

Special practices

IBC and IACUC approval: as for ACL-1. Microbial agentsclassified at BSL-2 require at the minimum registration withthe appropriate institutional entity (Biosafety Office) andmany institutions require IBC review and approval beforestarting any work. Work with recombinant organisms willusually need review and approval. If applicable at the insti-tution, IACUC review may also be required for work withvertebrate hosts. There may be some institutions, for exam-ple, in low- or middle-income countries, where these ad-ministrative entities are absent or less formally structured,and hence, this guidance may not easily be applied. In suchcases, PIs and department leaders benefit from the existenceof these guidelines and could institute site-specific policies oftheir own to ensure the safety of researchers and the sur-rounding community.

Housing of nonarthropod animals. Other animals are notaccessible to the arthropods. Animals used as hosts or bloodsources generally are not housed with arthropods. If present,they are adequately protected from access by escapedarthropods.

Containment during blood feeding. Recommendationsfor ACL-1 containment of arthropods during blood feedingare more stringently assured by special practices and con-tainer design, as recommended by the local risk assessment.

162 ARTHROPOD CONTAINMENT GUIDELINES, VERSION 3.2

Blood source: as per ACL-1. To prevent inadvertentcontamination of the clean colony, sources of infection, suchas a tube of infected blood, should not be stored in the samerefrigerator as a tube of uninfected blood for maintaininguninfected colonies by membrane feeding. Colony arthro-pods should be maintained in an ACL-1 area and transportedto an ACL-2 area for infection; there should be no transport ofliving arthropods from ACL-2 to ACL-1 without specificlocal risk assessment.

Escaped arthropod handling. Loose arthropods must bekilled and disposed, or recaptured and returned to the con-tainer from which they escaped. Infected arthropods must notbe killed with bare hands and must be manipulated usingfiltered mechanical or vacuum aspirators or other appropriatemeans (e.g., forceps, paintbrushes, gloved hands).

Accidental release reporting. A release procedure is de-veloped and posted. This includes contacts and immediatemitigating actions. Accidents that result in release of infectedarthropods from primary containment vessels or that result inovert exposure to infectious material must be reported im-mediately to the insectary director (PI) who is responsible forensuring that appropriate and documented action is taken tomitigate the release. The room where the incident occurred isclosed off, a warning sign indicating the location, number,and type of material released is prominently posted, and otherlaboratory personnel are informed until the source is elimi-nated. Follow-up medical evaluation, surveillance, and treat-ment are provided as directed by institutional policy and localrisk assessment, and written records are maintained.

Movement of equipment. All equipment must be appro-priately decontaminated and disinfested before transfer be-tween rooms within the insectary, and before removal fromthe insectary.

Safety equipment (primary barriers)

Personal protective equipment should be evaluated as partof the local risk assessment. It should be noted that very fewinfected arthropods are directly infectious by handling; vir-tually all require exoskeleton disruption or the act of feedingto be hazardous although there are exceptions (body liceexcrete feces that contain Rickettsia prowazekii or Bartonellaquintana; newly fed mosquitoes may diurese infectious vi-rus). Clothing (primary as well as safety) should conform toinstitutional policy, if any, and to the risk assessment by thelocal IBC. As an example, entering a room containing anenvironmental chamber holding plastic food containers withtick vials would not require personal protective equipment(PPE), unless the vials were opened and the ticks manip-ulated. The use of latex or nitrile gloves, although highlyrecommended, may not be required as a result of risk as-sessment by the local IBC or equivalent; manipulation ofarthropods within a glove box fitted with Hypalon gloves, forexample, would not necessarily require additional gloves.

Eye and face protection. Appropriate face/eye and re-spiratory protection is worn by all personnel entering theinsectary, if recommended by the local risk assessment.

Gloves. Gloves (latex or nitrile) are worn when handlingpotentially infected arthropods, blood, and associated equip-ment and when contact with potentially infectious material isunavoidable. Local risk assessment may provide for excep-tions, for example, the need for dexterity or tactile control(e.g., during the inoculation of suckling mice).

Torso apparel. White laboratory coats, gowns, and/oruniforms are typically worn at all times in the insectary whenhandling vertebrate animals and infected materials. Universalblood precautions (BMBL5) are recommended when blood ismanipulated.

Personal clothing. Clothing should minimize the area ofexposed skin (e.g., skirts, shorts, open-toed shoes, sandals,and tee shirts are inadvisable), since this can increase the riskof attracting and being bitten by a loose arthropod.

Arthropod-specific personal protective equipment. Otherequipment may be required as determined by the local riskassessment. Homogenization of infected arthropods, for ex-ample, may require an appropriate respiratory protectivedevice if the procedure is not performed within a biosafetycabinet or glove box.

Facilities (secondary barriers)

An insectary may simply be a room with a door that may beclosed tightly; it may or may not have environmental con-trols. Dedicated spaces to be used as insectaries are highlyrecommended, but resources may not exist to permit sucharrangements. The use of infected arthropods may be per-mitted after risk assessment by the local IBC even in theabsence of a dedicated space. Ticks, for example, may besafely manipulated within general BSL-2 laboratory settingsthat are otherwise not considered to be insectaries.

Location of insectary. The insectary is separated fromareas that are open to unrestricted personnel traffic within thebuilding. It is recommended that this be accomplished by atleast two self-closing doors that prevent passage of the ar-thropods. Increased levels of physical isolation are re-commended, for example, separate buildings, wings, andsuites. However, the lack of a dedicated insectary should notimply that infected arthropods may not be manipulated; site-specific risk assessments may provide mitigating alternativearrangements. For example, nonflying infected arthropodssuch as ticks or fleas may be safely manipulated in a dedi-cated area within a BSL-2 laboratory using a moat system(pan within a pan of water) and accounting for all specimens.

Insectary doors. Recommended entrance to the insectaryis via a double-door vestibule that prevents flying andcrawling arthropod escape. For example, the two contiguousdoors must not be opened simultaneously. Internal doorsmay open outward or be sliding, and are kept closed whenarthropods are present. Self-closing doors are highly re-commended. Additional barriers (e.g., screened partitions,hanging curtains) may be required by the local risk assessment.Alternative arrangements may be specified by local risk as-sessment in the absence of a dedicated insectary.

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Insectary windows. Windows are not recommended, but ifpresent cannot be opened and are well sealed. Windows shouldbe resistant to breakage (e.g., double paned or wire reinforced).

Vacuum systems. If a central vacuum system is installed,each service outlet is fitted with suitable barriers/filters toprevent arthropod escape. Filters are installed to permit de-contamination and servicing. Other vacuum devices are ap-propriately filtered to prevent transfer and exhausting ofarthropods.

Interior surfaces. The insectary is designed, constructed,and maintained to facilitate cleaning and housekeeping. Theinterior walls are preferably light colored so that a loose ar-thropod can be easily located, recaptured, or killed. Glossfinishes, ideally resistant to chemical disinfectants and fu-migants, are recommended. Light-colored floors are alsohighly recommended, smooth and uncovered. Ceilings are aslow as possible to simplify detection and capture of flyinginsects. Inability to conform to these recommendations maybe mitigated by other physical or procedural methods as in-dicated by the local risk assessment. A static glove box with alight-colored interior, for example, may be used to manipu-late infected arthropods where the color of walls and floorscannot be easily changed.

Floor drains. Floor drains are modified to prevent acci-dental release of arthropods and agents. If present, traps mustbe filled with an appropriate chemical treatment to preventsurvival of all arthropod stages (e.g., mosquito larvae).

Plumbing and electrical fixtures. Internal facility appur-tenances (e.g., light fixtures, pipes, ducting) are minimalsince these provide hiding places for loose arthropods. Pe-netrations of walls, floors, and ceilings are minimal andsealed/caulked. Ideally, light fixtures are flush with the ceil-ing, sealed, and accessed from above.

Heating, ventilation and air conditioning (HVAC). Ven-tilation is appropriate for arthropod maintenance, but does notcompromise containment of the agent or arthropod. Examplesinclude the following: exhaust air is discharged to the outsidewithout being recirculated to other rooms; appropriate fil-ter/barriers are installed to prevent escape of arthropods; thedirection of airflow in the insectary is inward; a progressivelynegative pressure gradient is maintained as distance from themain entrance increases; fans located in the vestibule and in-ternal corridor can be used to help prevent escape of flyingarthropods; and hanging or air curtains are located in vesti-bules and doorways. Local risk assessments may provide site-and task-specific alternatives to these recommendations, forexample, the use of a static glove box in which infected ar-thropods are manipulated may provide adequate security ifdirectional airflow is not possible.

Sterilization equipment. An autoclave is available, con-veniently located in rooms containing arthropods within theinsectary building. If an autoclave is not available, an appro-priate decontamination system or set of practices and proce-dures may be recommended by the local risk assessment.

Sink. The facility has a hand-washing sink with hot waterand with suitable plumbing to prevent arthropod escape.

Illumination. Illumination is appropriate for arthropodmaintenance, and does not compromise arthropod contain-ment, impede vision, or adversely influence the safety ofprocedures within the insectary. Lighted (or dark) openingsthat attract escaped arthropods are avoided.

Facility compliance monitoring. The facility should beevaluated annually for compliance to ACL-2. The PI or in-sectary director inspects the facility at least annually to en-sure that alterations and maintenance have not compromisedthe containment characteristics. Adequacy of the practicesand facility in view of changes in research protocols, agents,or arthropods is considered.

Arthropod Containment Level 3

ACL-3 involves practices suitable for work with potential orknown vectors that are or are likely to be infected with BSL-3agents associated with human disease. Arthropods that areinfected or potentially infected with BSL-3 pathogens maypose an additional hazard if the insectary is located in an areawhere the species is indigenous, or if alternative suitablevectors are present, as an escaped arthropod may introduce thepathogen into the local population. ACL-3 builds on thepractices, procedures, containment equipment, and facilityrequirements of ACL-2. It differs in that access is more re-stricted, and the microbiological containment takes a moreprominent role in determining the practices and facilities.

In the United States, the Select Agent Rule (www.selectagents.gov) restricts access to certain pathogens of hu-man or veterinary importance, all classified at BSL-3 or BSL-4.Many of these Select Agents are naturally maintained by ar-thropods. All possession and use of these restricted agents mustcomply with the biosecurity requirements promulgated by theUnited States Title 42 CFR Part 72. Violations are criminaloffenses. These revised guidelines do not review the require-ments of the Select Agent Rule other than those that may affectpractices and procedures needed for safe research with ar-thropods containing these restricted pathogens. Other countriesmay have similar restrictions and these should be kept in mindwhen planning for the development of an ACL-3 facility.

Comments

An aspect of working with BSL-3 pathogens that needsclarification is the use of biological safety cabinets. TheBMBL states that ‘‘All procedures involving the manipula-tion of infectious materials are conducted within biologicalsafety cabinets or other physical containment devices, or bypersonnel wearing appropriate protective clothing andequipment.’’ Most workers with BSL-3 agents utilize bio-safety cabinets and seem to regard this approach as thestandard. Many medical entomologists and vector biologistshave therefore been introduced into BSL-3 research under theimpression that they must perform all work involving BSL-3agents within a biosafety cabinet. Manipulating small ar-thropods in a biosafety cabinet can be extremely difficult. Theairflow can blow small arthropods around the cabinet, into thefilters, and into inaccessible locations. If working with cold-anesthetized mosquitoes on a chill table, for example, the

164 ARTHROPOD CONTAINMENT GUIDELINES, VERSION 3.2

arthropods can be blown from the table, recover, and then flyaround. The airflow may also disrupt the presentation of cuessuch as body temperature that stimulate host seeking andfeeding. The use of a biological cabinet can thus increase therisks associated with working with arthropod vectors.Whereas a cabinet might safely be used to prepare infectiousmaterial, the best option may be to perform infectious pro-cedures in a secure area (glove box) within a containmentroom and not exposed to strong air currents.

Hunt and Tabachnick (1996) recommend that ‘‘insects arenever manipulated on an open bench.’’ These workers provideplans to construct a purpose-designed glove box for such work.SALS (The Subcommittee on Arbovirus Laboratory Safety1980) stated ‘‘infection, anesthetization with carbon dioxide,and transfer of arthropods are done in such a manner that riskof infection of workers by aerosols is minimized. This can beaccomplished by use of (a) protective clothing and respira-tors/masks, (b) a biosafety cabinet, or (c) a plastic isolator withsleeve openings with or without an air exhaust.’’ There may bealternative approaches, such as the use of disposable plasticglove bags; or using an entire room as primary containmentwith enhanced PPE and multiple physical barriers (e.g., nestedmoats for ticks). Again, the local risk assessment may weighthe costs and benefits of alternatives to the accepted or typicalrecommendations.

To prevent arthropod escape, arthropod work is performed ina designated area, preferably small and self-contained within thelaboratory, for example, a cage-like room constructed of finemesh (see Arthropod Containment Levels 2, facilities (second-ary barriers)). In the event of escape, the search area is thereforesmall, and the chances of locating the escaped arthropod arecorrespondingly high. When manipulating arthropods that re-quire ACL-3, biosafety cabinets may be inappropriate becauseof the airflow and reduced humidity. Safe containment of thearthropods is thus achieved through the use of several levels ofcontainment (cages within incubators, and designated insectaryareas) within the BSL-3 laboratory, and appropriate procedures(traps, etc.) including those described below. It is recommendedthat where possible, the researcher take advantage of the safetyprovided by working within a biological safety cabinet. Proce-dures such as virus isolation from frozen mosquito pools can beeasily performed in a biosafety cabinet (BSL). Glove boxes mayalso be useful for manipulating small infected arthropods.

Field-collected arthropods from sites of active transmis-sion of BSL-3 agents do not necessarily require manipulationat ACL-3. Such material is considered diagnostic and may bemanipulated at BSL-2/ACL-2; identification of specificsamples containing viable agents would prompt transfer toBSL-3/ACL-3. Enhanced practices and procedures, however,should be instituted to prevent escape of living samples whilethey are assayed, or if aerosols may be generated.

Detection of Select Agents within arthropods, animals, andclinical samples requires prompt reporting of the detection tothe Select Agent Program and such samples, if viable (or thosecontaining a regulated positive strand RNA virus, which ispotentially directly infectious), require secure storage untiltheir disposition as mandated by the Select Agent Program.

Standard practices

Location of arthropods. Furniture and incubators con-taining arthropods are located in such a way that accidental

contact and release by laboratorians, custodians, and servicepersons do not occur. This is usually achieved by locatingarthropods in dedicated BSL-3 rooms, wings, or suites,preferably in incubators, which would serve as an additionallayer of containment. Ticks or other less mobile vector ar-thropods may be held within vials contained in desiccatorcabinets or other escape-proof secondary or tertiary housingother than a dedicated incubator. Site-specific risk assess-ment may provide for other practices.

Supply storage. Equipment and supplies not absolutelyrequired for ongoing ACL-3 work are removed from the in-sectary after appropriate decontamination or are stored in adesignated area and not on open shelves. It is recommended thata closed storage room, cabinets with tight-fitting doors ordrawers be used. Doors and drawers are open only during access.

General arthropod elimination. In addition to measuresfor general arthropod elimination within the insectary, ma-terials used to wipe or mop are autoclaved before disposal.Only persons trained to work with arthropods and BSL-3agents and equipped with appropriate PPE clean up spills.

Primary container cleaning and disinfestation. Care istaken to disinfest primary containers in a manner that doesnot create aerosols. All primary containers are autoclaved orincinerated for disposal, or as specified by the site-specificrisk assessment.

Primary container construction. Cages used to hold ar-thropods are nonbreakable and screened with mesh of a sizeto prevent escape. Containers are autoclavable or disposable.Openings are designed to prevent escape during removal andintroduction of arthropods. Disposable containers are re-commended.

Disposal of arthropods. In addition to ACL-2 disposalpractices, the outer surfaces of containers are decontaminatedbefore moving the material. All arthropod waste materials areautoclaved or incinerated. Living, infected arthropods shouldbe killed by freezing or other appropriate methods, followedby autoclaving or incineration; those not being used in anexperiment should not be allowed to persist alive. An ex-ception would be long-lived or developmentally delayed ar-thropods such as ticks, which may require extended durationsbefore they may be physiologically capable of feeding againand hence must be kept for weeks or months. The lack ofaccess to an autoclave or incineration device should behandled during local risk assessment to identify the bestsubstitute available.

Isolation of uninfected arthropods. Where possible, onlyarthropods requiring ACL-3 procedures are housed in theACL-3 insectary. If it is necessary to house ACL-2 or lowerarthropods in the ACL-3 insectary, all procedures and prac-tices must meet the ACL-3 standards.

Primary container identification and labeling. As perACL-1.

Prevention of accidental dispersal on persons or viasewer. Viable potentially infected arthropod life stages must

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not leave the laboratory. PPE is doffed as specified by the riskassessment and IBC protocol. Hands are washed on exit;showering out may or may not be required depending on therisk assessment. No material is disposed through the sewerunless appropriately decontaminated, for example, by treatmentwith 10% hypochlorite or by heat, autoclaving, or incineration.

Escaped arthropod monitoring. Ideally, risk assessmentsidentify practices, procedures, and equipment that will pre-vent escape of any infected arthropod. ‘‘Count-in, count-out’’procedures have been recommended for ACL-3 and shouldbe implemented if possible. Although this practice can beimplemented well for studies of winged adult insects in theabsence of a vertebrate host, there are a number of scenarioswhere such a practice is logistically difficult. For example,ticks, mites, fleas, and lice may be groomed by an infestedhost and eaten if they are not contained within a feedingcapsule. Feeding capsules themselves may partially detachand liberate some of the nonattached arthropods within andthus should not be considered as the only way to safelycontain a feeding nonflying arthropod. ‘‘Count-in, count-out’’ also poses difficulty for studies of transovarial trans-mission. Eggs may be laid but few hatch; larvae may start todevelop but then die and be consumed by the remainder.Pupae may develop but adults may not emerge. Finally,motile diminutive arthropods such as mites might be easilymiscounted and lead to a false conclusion that an escape hadoccurred. The risks that might be associated with not using a‘‘count-in count-out’’ protocol might be balanced by addi-tional barriers to escape, for example, one or more moatscontaining the cage with the infested animal, as provided by alocal risk assessment.

Strong suspicion of an escape by infected arthropods mayrequire facility shutdown and disinfection, or treatment withinsecticide. The ACL-3 PI is usually required to promptlynotify institutional authorities such as the biosafety officer ifan escape is likely. The appropriate response would be theoutcome of deliberations by institutional authorities advisedby the PI.

Additional measures are taken to measure the effective-ness of the arthropod surveillance program and these aredocumented. As part of the review and commissioning pro-cess of a new facility, the physical integrity and securitypractices might be tested by a simple release/recapture study.A known number of noninfected arthropods would be re-leased and then these would be recaptured to assess thephysical integrity of security barriers. Such an experiment isdescribed by Hunt and Tabachnick (1996). Exterior andwithin-building monitoring should be considered. Records ofexterior captures are maintained.

The Select Agent Rule poses a specific quandary and ad-ditional preparations during initial risk assessments as it re-lates to arthropod escape: the rule requires that all SelectAgents be accounted for. Even a simple miscounting maytrigger a federal investigation. Arthropods deliberately in-fected by Select Agents are themselves considered SelectAgents and they must be entered into a written inventory andrecord of disposition must be made for each individual in-fected arthropod. The failure to account for all infesting in-fected arthropods (e.g., 1 of 10 infecting ticks is not foundwhen recovering the replete ticks in a moat surrounding amouse cage) would be considered an environmental release

(or theft) and subject to investigation by the Select AgentProgram. It might reasonably be concluded that the host mayhave eaten the infecting arthropod and such a conclusionneeds to be recorded in writing and the reasoning or evidencepresented. If this contingency is not approved in advance bythe institutional oversight committee during the IBC protocolreview, the failure to recover all infecting arthropods wouldbe considered a release and an emergency response wouldlikely be triggered, including prompt notification to the SelectAgent Program. Hence, ACL-3 work with Select Agents canrepresent a particularly onerous and difficult scenario forwhich there is no easy solution.

Pest exclusion program. As per ACL-1.

Source and harborage reduction. As per ACL-2.

Microbiological and medical sharps. As per ACL-2.

Routine decontamination. As per ACL-2.

Notification and signage. As per ACL-2.

Procedure design. All procedures are carefully per-formed to prevent arthropod escape and the creation ofaerosols or splatters. Protocols are practiced with noninfectedarthropods/animals and modified before implementation.

Safety manual. As per ACL-2.

Training. The training required for laboratory personnelunder ACL-3 is more detailed and extensive, particularly ifthe pathogen in use also is a Select Agent. Specific training incontainment practices and procedures may be required byinstitutions and is highly recommended if personnel are notpreviously experienced. Such training, and annual refreshertraining, is a requirement of the Select Agent Program andmust be documented.

Medical surveillance. An institution with an ACL-3 fa-cility should have a medical surveillance or occupationalhealth program. This is a requirement of the Select AgentRule and is the standard of practice for many U.S. institu-tions. It is possible, though, that some institutions do not havea formal medical surveillance or occupational health plan andthis may apply to programs in resource-poor sites as well. Insuch cases, the PI should develop a medical response plan andarrange with local health care providers for education ofworkers or providing care in the event of an exposure. Wherethere is a formal occupational health program, institutionalpolicies and the specific (usually independent) program dic-tate the extent of surveillance, whether prophylaxis may berequired, and the appropriate response to exposure. Hence, aone-size-fits-all recommendation on surveillance or occupa-tional health is not advisable.

Access restrictions. The insectary director limits accessto the insectary to the fewest number of persons possible.Personnel who must enter the insectary for program or ser-vice purposes when work is in progress are accompanied bytrained laboratorians and are advised of the potential hazardto themselves, coworkers, and the potential consequences of

166 ARTHROPOD CONTAINMENT GUIDELINES, VERSION 3.2

arthropod release. Because of the increased risk to nontrainedpersonnel, laboratory staff should perform general cleaningactivities that would otherwise be performed by custodialstaff. The Select Agent Rule has specific requirements forpersonnel access to restricted agents, including backgroundchecks and suitability assessment. In addition, any laboratoryregistered to possess and use Select Agents must keep themsecure, with at least three physical barriers (locks or otherrestrictions) in place to deter theft.

Special arthropod handling containers and areas. Allwork is done within a primary barrier. Appropriate biologicalsafety cabinets, other physical containment devices, and/orpersonal protective equipment are used whenever conductingprocedures to infect arthropods with BSL-3 agents, or whenhandling arthropods. Appropriate designs will consider thelife history and behavior of the arthropod and may differ fromthat required by the agent alone. Such modifications shouldbe made in consultation with biosafety experts. Manipulationof arthropods and, for example, rearing of transovarially in-fected immature stages are performed in a designated area.SALS suggests ‘‘a separate room or double screened area thatis separated from the main insectary by rooms having twoscreened or solid doors that open inward and closing auto-matically.’’

Safe transport in the laboratory. As per ACL-2.

Special practices

IACUC and IBC approval: as per ACL-2. Note that theSelect Agent Program now requires that all experiments andactivities with Select Agents be outlined as part of the reg-istration process and will be reviewed by their administration.

Housing of nonarthropod animals. As per ACL-2.

Containment during blood feeding. Recommendationsfor ACL-1 containment of arthropods during blood feedingare strictly assured by special practices and container designsthat prevent escape of arthropods.

Blood source. As per ACL-1.

Escaped arthropod handling. Loose arthropods must bekilled and disposed, or recaptured and returned to the con-tainer from which they escaped. Infected arthropods are notkilled with hands and must be transferred using filtered me-chanical, vacuum aspirators, forceps, or other appropriatetools. Only personnel properly trained and equipped to workwith designated arthropods and BSL-3 infectious agents areto recover and/or kill escaped arthropods.

Accidental release reporting: as per ACL-2. Institutionsmay have specific reporting requirements. The Select AgentProgram must be notified by the institution of any accidentalrelease of a Select Agent.

Movement of equipment. As per ACL-2.

Inventory of arthropods. In addition to appropriate pri-mary containment cages, when possible, the number of ar-

thropods must be included on the label, and records aremaintained to account for all arthropods from the time oftransfer to the ACL-3 insectary to the time of termination.The Select Agent Rule has specific inventory requirementsfor arthropods infected by Select Agents. The naked nucleicacids of positive strand RNA Select Agent viruses are con-sidered by the Select Agent Program to comprise SelectAgents and must be stored and documented as required.

Safety equipment (primary barriers)

BMBL recommends enhanced PPE for BSL-3 studies.Laboratory surfaces and floors, however, should be consid-ered noninfectious because all procedures and manipulationsof infectious material must be within a biosafety cabinet orequivalent primary containment device (and hence any con-tamination of the laboratory should be considered a breach ofoperating procedures and trigger institutional review).Nonetheless, many institutions and authorities follow a strictpolicy that the entire BSL-3 facility is to be consideredhazardous and maximum PPE must be used for any and allentry. The cornerstone of biosafety is the site- and task-specific risk assessment and ideally this should guide therequirement for PPE. It should be noted that the maximal PPEmay impair dexterity that is essential for performing proce-dures such as the dissection of small arthropods. In suchcases, the local risk assessment should carefully weigh therisks presented and the benefits of using the recommendedprotective gear and identify other means of mitigating risk ifthe recommendations are modified. If enhanced PPE is re-quired, workers should rehearse procedures working withuninfected arthropods. Indeed, a good safety practice for allBSL-3 work is to practice all procedures within the BSL-3facility using a BSL-2 substitute agent before undertakingthem at BSL-3.

Some ACL-3 facilities may be designed so that the roomitself is the primary containment. Special practices and pro-cedures, including specific PPE, would be mandated by thelocal risk assessment. No recommendations are made hereinas to the details of such practices, procedures, or PPE becausethey would be site and task specific and should be developedas a result of a discussion of risk assessment by institutionalofficials and the PI. External biosafety consultants andmedical entomologists experienced with containment workmight be consulted to help develop such recommendations.

Eye and face protection. As per ACL-2.

Gloves. Personnel wear latex or nitrile gloves whenhandling infected arthropods or host animals and associatedequipment. Gloves are removed aseptically and are changedfrequently. Under specific circumstances, and as allowed byinstitutional review of practices and procedures specific tothe site and task, gloves may not be required, for example, inrestraining suckling mice for inoculation (tactile cues anddexterity are required for safe execution of this procedure, aswell as for humane purposes).

Torso apparel. Changing out of street clothes into scrubs,to be worn under PPE, is highly recommended, although such arequirement should be a result of local risk assessment. Whitelaboratory coats, gowns, or jumpsuits should be worn at all

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times by all personnel entering the insectary. Wraparound orsolid-front gowns are typically worn over this clothing. Front-button laboratory coats alone are unsuitable. The gowns areremoved and left in the insectary. Before leaving the insectary,scrub suits are removed and appropriately contained and de-contaminated before laundering or disposal.

Foot apparel. Boot, shoe covers, or other protectivefootwear and disinfectant foot baths (with appropriate anti-arthropod measures) are available and used where indicated.

Footwear dedicated for use in the ACL-3 facility is highlyrecommended.

Personal clothing. As per ACL-2.

Arthropod-specific personal protective equipment. Asper ACL-2.

Pesticide. Pesticide for emergency use is available inareas in which escape of arthropods is likely.

Facilities (secondary barriers)

Location of insectary. The insectary is strictly separatedfrom areas that are open to unauthorized, untrained personnelwithin the building by locked doors. These are opened, forexample, by key lock, proximity reader, or card key.

Insectary doors. Access to the facility is limited totrained, approved personnel by a self-closing and self-locking door. The external insectary entry doors are con-trolled by a key lock, card key, or proximity reader. Entry intothe insectary is via a double-door entry that includes a changeroom and shower(s). Showers are plumbed to prevent ar-thropod escape. An additional double-door access (air lock)or double-door autoclave may be provided for movement ofsupplies and wastes into and out of the facility, respectively.The two contiguous doors must never be opened simulta-neously. Internal doors may open outward or be sliding, butare self-closing, and are kept closed when arthropods arepresent. Additional barriers (e.g., hanging curtains) are re-commended.

Insectary windows. Windows are not recommended. Anywindows present are resistant to breakage (e.g., double panedor wire reinforced) and well sealed.

Vacuum systems. As per ACL-2.

Interior surfaces. In addition to the recommendations forACL-2, spaces around doors are sealed to facilitate decon-tamination. Troughs surrounding door frames may be in-stalled and filled with sticky or greasy material that will trapcrawling arthropods, depending on the species in use andlocal risk assessment.

Floor drains. Floor drains are not recommended. Ifpresent, traps must be filled with an appropriate treatment toprevent survival of any arthropod stage (e.g., mosquito lar-vae). Ideally, all drains are plumbed to a holding tank tofacilitate heat or chemical treatment to kill all stages of ar-thropod before disposal into the waste system.

Plumbing and electrical fixtures. As per ACL-2.

HVAC. Ventilation is appropriate for arthropod mainte-nance, but does not compromise containment. Exhaust air isdischarged to the outside without being re-circulated to otherrooms. Exhaust must be dispersed away from occupied areasand air intakes, or the exhaust must be high-efficiency par-ticulate air (HEPA)-filtered. The USDA requires that airsupply vents also be HEPA filtered if working with BSL-3veterinary agents. Appropriate filter/barriers are installed toprevent escape of arthropods. The direction of airflow in theinsectary is inward. A progressively negative pressure gra-dient is maintained as distance from the main entrance in-creases. Personnel must verify that the direction of the airflowis proper (a visual monitoring device/meter is recommendedto confirm directional inward airflow). Audible alarms alertpersonnel to system failure.

Sterilization equipment. An autoclave is available withinthe suite of rooms containing arthropods. If an autoclave is notavailable within the suite, local risk assessment may provide fora suitable alternative (heat treatment, tissue digester) or provideprotocols for transport of materials to a nearby autoclave.

Sink and shower. In addition to the ACL-2 recommen-dation, an appropriately plumbed shower is available withinthe insectary suite. If a shower is not available within the suite,local risk assessment may provide for a suitable alternative.

Illumination. As per ACL-2.

Biosafety cabinets. HEPA-fitted exhaust air from ClassII biological safety cabinets can be recirculated into the in-sectary provided it is certified annually. If Class III cabinetsare used they must be installed appropriately and also certi-fied annually. Static glove boxes for arthropod manipulation(Plexiglas or other more expensive materials) do not requirenegative air pressure, although accommodation for interiorair pressure may be needed (e.g., 0.22 lm filter vent).

Facility compliance monitoring. The completed ACL-3insectary design and operational procedures must be docu-mented by the PI and reviewed by the IBC. In some cir-cumstances, ACL-3 insectaries are not built de novo and mustbe retrofitted into existing space. The PI should be a memberof any design team and the space modified to reflect the likelyuses. If certain ACL-3 recommendations are not practical orcannot be implemented, the local risk assessment may pro-vide site- and program-specific mitigation by alternativephysical design or added procedural/practice stipulations.

The insectary must be tested for verification that the designand operational parameters have been met before operation.ACL-3 insectaries are reverified by the PI and by the insti-tutional biosafety representatives where applicable at leastannually against these procedures. Laboratory design, prac-tices, and procedures may need to be modified iteratively byoperational experience.

Arthropod Containment Level 4

ACL-4 safety guidelines are for the most dangerouspathogen-infected arthropods. All of the Standard Practicesof ACL-3 should be in place, with the additional caveats

168 ARTHROPOD CONTAINMENT GUIDELINES, VERSION 3.2

described here. No compromise is acceptable at this level ofwork. BSL-4 agents are associated with a high risk of in-fection from aerosol exposure, and cause life-threateningdisease. Certain other pathogens such as those listed as ‘‘re-stricted animal pathogens’’ may also necessitate BSL-4containment if used in vectors. For vector work, productionof aerosols is a potential risk when preparing infectious mealsor inocula, and can also result from analytical practices in-volved in virus isolation. If work with vectors must be per-formed in a BSL-4 facility, then BSL-4 requirements must bestrictly followed. As described below, vectors must be safelycontained at all times possibly by use of specially designedapparatus that is tested and approved before use.

Although arthropods collected from sites where BSL-4agents are known to occur should be handled with extremecare, they are by definition diagnostic samples and can behandled at ACL-2 in field laboratories. It would be excessiveto suggest, for example, that malaria entomology programswithin Ebola virus endemic sites be required to handle field-collected parous Anopheles spp. within ACL-4; of course,appropriate caution should be taken to prevent exposure toblood regardless of source (universal precautions). Anyidentification of a BSL-4 agent within such samples requiresthat the sample be transferred to an ACL-4 facility or treatedso that the agent is no longer viable. It should be noted thatRNA from a Select Agent that is a positive sense RNA virusis considered to be a Select Agent because of a theoreticalconsideration of infectivity (by transfection into cells). Be-cause BSL-4 agents are exotic, importation of field-derivedmaterials into the United States will require import permitsfrom CDC and USDA/APHIS, which will have specificstipulations, perhaps including the need for unpacking andmanipulating imported materials only within ACL-4 re-gardless of diagnostic status.

Of the 12 infections requiring BSL-4 containment in theUnited States, 5 are transmitted by arthropods: Congo/Crimean hemorrhagic fever, Kyasanur Forest disease, Omskhemorrhagic fever, Far Eastern tick-borne encephalitis, andSiberian tick-borne encephalitis. All of these viruses areSelect Agents. Only ticks have been implicated in their nat-ural transmission cycles, although other arthropods havebeen experimentally infected with BSL-4 agents (e.g., Aedesaegypti with Marburg, and mesostigmatid mites with Junin).With this information one might only consider measures andprotocols that safely contain species of ticks as relevant toBSL-4 research with arthropods. However, new infectionsmay emerge, and thus, it is necessary to consider other ar-thropods that might require high containment, particularlyflying insects. Furthermore, research on newly discoveredpathogens often requires experimental attempts to infect ar-thropods in an attempt to determine the life cycle.

As the number of BSL-4 laboratories is quite limited, thereader should refer to the appropriate sections of the BMBL. Forarthropod work, a simple, minimalist approach is adopted. Anarea designated for arthropod research is small, light colored,and contains only items required for the study. There are twotypes of BSL-4 laboratories: (1) the cabinet laboratory wherethe agent is handled in a Class III biological safety cabinet and(2) the suit laboratory. Personnel working in a BSL-4 suit fa-cility don one-piece positive pressure personnel suits ventilatedby a life support system. Construction of a BSL-4 facility, andrequired operating procedures, is sufficient to guarantee that no

life stage could survive, and escape from primary containmentis mitigated by efficient secondary containment.

Ideally, under ACL-4, an infected arthropod must never behandled outside of a primary containment barrier; for example,cages are opened only in an arthropod-secure glove box (Huntand Tabachnick 1996). Although a glove box is highly re-commended for all manipulations of live arthropods at ACL-4,wearing a positive pressure containment suit would make such arecommendation difficult in practice. Well-lit, stand-alonehandling tables incorporating a moat to prevent escape could beused in the open BSL-4 laboratory for manipulating ticks ormites. Flying insects would pose a containment difficulty andthe specific experiment may need to custom practices, proce-dures, and equipment to safely carry out the required tasks.

At ACL-4, every arthropod is counted and accounted forthroughout the experiment. No one enters or leaves the roomuntil all arthropods are accounted for and, if living, secured indouble-taped cages or vials and placed in secondary sealedholding trays. Infected arthropods will be discarded (killedand decontaminated), processed for analysis (preferably kil-led and held under conditions that inactivate the agent), orheld alive in primary and secondary secure containers. If onearthropod is missing and cannot be found, the facility is shutdown and treated with a pesticide.

The nature of this research and the protective equipmentrequired dictate that staff must be trained to the very highestlevel. Since working with arthropods often requires the use ofsmall instruments and hence considerable dexterity, it is re-commended that a specific person be designated for this workand be trained extensively using a space suit so that they arewell rehearsed before actual ACL-4 work. Equipment that isused for ACL-3 work will be specially adapted for ACL-4research, and such work would require extensive practice.

Transportation and Transfer of Biological Agentsand Arthropod Vectors

Transportation refers to the packaging and shipping ofmaterials by air, land, or sea, generally by a commercialconveyance. Transfer refers to the formal process of ex-changing these materials between facilities.

Biological agents include infectious agents of humans,plants, and animals, as well as the toxins that may be producedby microbes and by genetic materials potentially hazardous bythemselves or when introduced into a suitable gene deliveryagent. Etiologic agents and infectious substances are closelyrelated terms that are found in the transfer and transportationregulations. Biological agents may exist as purified and con-centrated cultures and may also be present in a variety ofmaterials such as body fluids, tissues, and soil samples. Ar-thropod vectors are organisms such as mosquitoes, ticks, andfleas that may transmit infectious agents to animals or humans.Biological agents and materials and vectors that are known orsuspected to contain them are recognized by federal and stategovernments as hazardous materials, and their transportationand transfer are subject to regulatory control. Transport andtransfer of live, uninfected vectors may also be subject tofederal and state regulatory control.

Transportation

Regulations on the transportation of biological agents andlive vectors are aimed at ensuring that the public and the

ARTHROPOD CONTAINMENT GUIDELINES, VERSION 3.2 169

workers in the transportation chain are protected from ex-posure to any agent that might be in the package, and that thepackage prevent escape of the agent or live vector. Protectionis achieved through (1) the requirements for rigorous pack-aging that will withstand rough handling and contain all liquidmaterial within the package without leakage to the outside;(2) appropriate labeling of the package with the biohazardsymbol and other labels to alert the workers in the transpor-tation chain to the hazardous contents of the package; (3)documentation of the hazardous contents of the packageshould such information be necessary in an emergency situ-ation; and (4) training of workers in the transportation chain tobe able to respond appropriately to emergency situations.Regardless, nonmotile forms such as eggs or nonflying stagesshould be shipped if possible.

Transportation Regulations

Public Health Service 42 CFR Part 72: Interstate Trans-portation of Etiologic Agents. Harmonizes with the otherU.S. and international regulations [see Federal Register64(208) p. 58022 at www.access.gpo.gov]. A copy of thecurrent regulation can be obtained from The Code of FederalRegulations (INTERSTATE SHIPMENT OF ETIOLOGICAGENTS 1, 1980).

Department of Transportation: 49 CFR Parts 171–178—Hazardous Materials Regulations. Applies to the shipmentof both biological agents and clinical specimens. Informationmay be obtained from the Internet: www.gpo.gov/fdsys/pkg/CFR-2012-title49-vol2/xml/CFR-2012-title49-vol2-subtitleB-chapI-subchapC.xml (accessed 12/8/2017).

United States Postal Service: 39 CFR Part 111—Mailabilityof Etiologic Agents. Codified in the Domestic Mail Manual124.38: Etiologic Agents Preparations (www.gpo.gov/fdsys/pkg/FR-2003-06-06/html/03-14185.htm accessed 12/8/2017).

Occupational exposure to blood-borne pathogens: Occu-pational Health and Safety Administration (OSHA)—29 CFRPart 1910.1030. Provides minimal packaging and labelingrequirements for transport of blood and body fluids within thelaboratory and outside of it (www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p_id=10051 accessed 12/8/2017).

Dangerous Goods Regulations: International Air TransportAssociation. These regulations provide packaging and la-beling requirements for infectious substances, materials,clinical specimens that have a low probability of containingan infectious substance, and live vectors. These are the reg-ulations followed by the airlines and are therefore of partic-ular relevance for express shipment of arthropods. Theseregulations are derived from the Committee of Experts on theTransport of Dangerous Goods, United Nations Secretariat,and the Technical Instructions for the Transport of DangerousGoods by air provided by the International Civil AviationOrganization (ICAO). A copy of the Dangerous GoodsRegulations (DGR) may be obtained by calling 1-800-716-6326 or through the Internet: www.iata.org/publications/pages/index.aspx (accessed 12/8/2017).

General packaging requirements for transportof live arthropod vectors

Transport of live arthropod vectors requires packaging thatprevents the escape of arthropods and agents, maintains theirviability, and protects personnel in the transportation chainfrom exposure to the contents. This is true regardless ofwhether or not the arthropods are infected. Fortunately, un-like many larger animals, most arthropod vectors do not re-quire large containers, ventilation, feeding, or added waterduring their transport. Most are shipped without free water sothe possibility of leaking is rare, and the container tempera-tures normally maintained during shipments are adequate.

This means that appropriate physical packaging of vectorarthropods is fairly simple and, for infected arthropods, canbe similar to that which is appropriate for the agents theycontain. The following section is intended to provide specificinstructions for determining the type of container and label-ing required for shipment of vector arthropods.

International Air Transport Association (IATA) Live An-imal Regulations (LAR) 42nd edition (available for purchaseat www.iata.org/publications/pages/index.aspx, accessed 12/8/2017) describes containers that are appropriate for theshipment of arthropods, including insects and arachnids. Thedesign of these, while not as demanding, is consistent withcontainers used to ship etiologic agents (see Container Re-quirement 62 of LARs). It is therefore possible to selectcontainers that satisfy the requirements of LARs, DOT 49CFR Part 173.196: Transportation of Etiologic Agents, andUSPHS 42 CFR Part 72—Interstate Shipment of EtiologicAgents.

According to the IATA DGR, a live, intentionally infectedanimal that is known to contain an infectious substancecannot be transported by air unless it cannot be transported byany other means. A specific exemption from DOT must beobtained. It should be noted that although infected vectorspose different kinds of risks than do infected vertebrates, anyhazard does not exceed that of the infectious agents that areallowed for transport under the DGR. Consulting withtransportation authorities or transport companies is stronglysuggested in advance of shipping of any infected arthropods.

Three packaging scenarios are considered:

� Arthropods free of infection by specific pathogens.� Domestic and exotic arthropods containing a nonselect

agent.� Domestic and exotic arthropods containing a select

agent.

Definitions. Domestic arthropods. Those that are extant inthe 49 continental United States. Note that this differs from thedefinition used in Risk Assessment and Containment Levels.

Exotic arthropods. All others.

Select agent. Etiological agents listed in 42 CFR Part 72.

Nonselect agent. Agents other than those above that areknown to cause disease in humans.

� Noninfected exotic and domestic arthropods that vectordisease are packaged consistently with the minimumpackaging requirements of 42 CFR 72.2. This requiresthat the container must prevent ‘‘leakage (i.e., escape,

170 ARTHROPOD CONTAINMENT GUIDELINES, VERSION 3.2

note added) of the contents, shocks, pressure changes,and other conditions incident to ordinary handling intransportation.’’ We recommend that this consists threelevels of containment, including a primary receptacleconsisting of a sealed plastic bag or tube surrounded bypadding, a secondary container such as an insulatedchest whose lid is sealed with tape, and a durable fi-berboard, wood, plastic, or wooden outer container.The container may bear the ‘‘live animal’’ label nam-ing the species within. If aquatic stages are shipped, thecontainer should also contain sufficient absorptivematerial to absorb and contain all of the water.

� Domestic and exotic arthropods containing a nonselectagent are packaged as above. The outer container bearsa ‘‘biohazard’’ label as described in CFR 72.3. Anitemized description of the contents is placed betweenthe outer and inner containers.

� Domestic and exotic arthropods containing a selectagent are packaged, labeled, and tracked as required forthe agent they are known or suspected to contain. Thisincludes all attendant regulations required for the agentalone, including notice of delivery and failure to re-ceive, and laboratory registration.

Transfer

Regulations on the transfer of biological agents and livevectors are aimed at ensuring that the change in possession ofbiological materials is within the best interests of the publicand nation. These regulations require documentation of thepersonnel, facilities, and justification of need for the bio-logical agent in the transfer process, and subsequent approvalof the transfer process by a federal authority. The followingregulations fit in this category.

Importation of Etiologic Agents of Human Diseaseand Live Vectors

42 CFR Part 71 Foreign Quarantine

Part 71.54 etiologic agents, hosts, and vectors. Thisregulation requires an import permit from the CDC for im-porting etiologic agents of human disease, any materials thatmay contain etiologic agents, including live animals and livevectors. This regulation also requires that an import permit beobtained by the recipient for transfer from the original per-mit holder of an imported etiologic agent or live vector withinthe United States. An application and information on im-portation permits may be obtained at: www.cdc.gov/od/eaipp/importapplication/agents.htm (accessed 12/8/2017).

Interstate transfer of biological agents and live vectors mayalso be restricted by state regulations. Shippers and recipientsof these materials may obtain additional information directlyfrom state health or agriculture departments.

Importation of Etiologic Agents of Livestock,Poultry, and Other Animal Diseases

9 CFR Parts 92, 94, 95 96, 122, and 130

These regulations require an import permit from the USDA,APHIS, and Veterinary Services to import or domesticallytransfer etiologic agents of livestock, poultry, other animals,and any materials that might contain these etiologic agents.

Information may be obtained at (301) 734-3277, or from theInternet: www.aphis.usda.gov/aphis/ourfocus/animalhealth/animal-and-animal-product-import-information/ct_organisms_and_vectors (accessed 12/8/2017).

Transfer of Select Biological Agentsof Human Disease

42 CFR Part 72.6 additional requirements for facilitiestransferring or receiving select agents

Facilities transferring or receiving Select Agents must beregistered with the CDC and each and every transfer of aSelect Agent must be approved by the Select Agent Program.Information may be obtained on the Internet: www.selectagents.gov (accessed 12/8/2017).

Export of Etiologic Agents of Humans, Animals,Plants, and Related Materials

Department of Commerce: 15 CFR Parts 730–799

This regulation requires that exporters of a wide variety ofetiologic agents of human, plant, and animal diseases, in-cluding genetic material, live vectors, and products that mightbe used for culture of large amounts of agents, must obtain anexport license. Information can be obtained by calling theDepartment of Commerce (DoC) Bureau of Export Adminis-tration at 202-482-4811 or through the Internet: www.bis.doc.gov/index.php/regulations/export-administration-regulations-ear (accessed 12/8/2017).

Acknowledgments

This version of the Arthropod Containment Guidelineswas updated by Sam R. Telford III (Department of InfectiousDiseases and Global Health, Cummings School of VeterinaryMedicine, Tufts University, North Grafton, MA), Michael J.Turell (Virology Division, United States Army Medical Re-search Institute of Infectious Diseases, Fort Detrick, MD),Kevin R. Macaluso (Vector-Borne Disease Laboratories,Department of Pathobiological Sciences, School of VeterinaryMedicine, Louisiana State University, Baton Rouge, LA),Philip M. Armstrong (Department of Environmental Sciences,Center for Vector Biology and Zoonotic Diseases, The Con-necticut Agricultural Experiment Station, New Haven, CT)and Lyric C. Bartholomay (Department of Pathobiolog-ical Sciences, School of Veterinary Medicine, University ofWisconsin-Madison, Madison, WI).

Author Disclosure Statement

No conflicting financial interests exist.

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Centers for Disease Control and Prevention (CDC). Biosafety inmicrobiological and biomedical laboratories. U.S. Departmentof Health and Human Services, Public Health Service. 2009.Available at www.cdc.gov/biosafety/publications/bmbl5 (ac-cessed January 22, 2019).

Centers for Disease Control and Prevention (CDC). Guidelines forsafe work practices in human and animal medical diagnosticlaboratories. MMWR. 2012. Available at www.cdc.gov/mmwr/preview/mmwrhtml/su6101a1.htm (accessed January 22, 2019).

Crampton JM, Beard CB, Louis C. The Molecular Biology ofInsect Disease Vectors: A Methods Manual. New York:Chapman & Hall, 1997.

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Hunt GJ, Tabachnick WJ. Handling small arbovirus vectorssafely during biosafety level 3 containment: Culicoides var-iipennis sonorensis (Diptera: Ceratopogonidae) and exoticbluetongue viruses. J Med Entomol 1996; 33:271–277.

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Appendix 1. Draft Committee Members v.3.1

*Kate Aultman, NIHAbdu Azad, U MD*Ben Beard, CDC, Atlanta, GA*Mark Benedict, CDC, Atlanta, GAJohn Beier, Tulane U.Al Handler, USDA-ARS, Gainesville, FL*Steve Higgs, UTMB, Galveston, TXPeter Jahrling, USAMRIDAnthony James, UC IrvineCynthia Lord, FMEL

Robert McKinney, NIH*Roger Nasci, CDC, Ft. Collins, COKen Olson, CSU, Ft. Collins, COJonathan Richmond, CDC, Atlanta, GA*Tom Scott, UC Davis*Dave Severson, U Notre DameWalter Tabachnick, FMEL, Vero Beach, FLNed Walker, Mich. St.*Dawn Wesson, Tulane U.* Lead drafting authors

Appendix 2. Drafting Process v.3.1

A subcommittee appointed by the American Society ofTropical Medicine and Hygiene (ASTMH)/American Com-mittee of Medical Entomology (ACME) at the 1999 meeting

in Washington, DC (above), consisted of persons selected orwho volunteered to serve on the committee. They werecharged with the task of formulating Draft Guidelines

172 ARTHROPOD CONTAINMENT GUIDELINES, VERSION 3.2

reflecting the containment principles of documents that arecurrently circulating both in the United States and interna-tionally. The document was intended to address the followingitems: scope and intent, principles of risk assessment, defi-nition of risk levels considering diseases vectored, phenotypeand genotype including that of transgenics, biological con-tainment, risk relative to that in existence due to accidentalescape, and containment facilities, practices, and shippingmethods appropriate to each risk level.

During the spring of 2000, the Draft Committee for-mulated a first draft and circulated it among the member-ship of the committee for comments and revision. Aftercomments were received and considered, a second draftwas written (v 2.1). This draft was circulated electron-ically in numerous places, including the Vector, Mosquito-

L, and Biosafety listservers, and was also posted onProMED. Additional copies were distributed electroni-cally to individuals identified by the Draft Committee asbeing influential and knowledgeable in the area. Severalpersons were identified to review and comment on theGuidelines to the Draft Committee at the 2000 ASTMHmeeting in Houston, TX.

That meeting was held as an open meeting both to presentthe guidelines and to receive comments.

National Institutes of Health (NIH) and Centers for Dis-ease Control and Prevention (CDC) Office of Health andSafety personnel and other biosafety experts examined thedocument and revised it before ACME approval. Theguidelines were published in June 2003 as a special issue ofVector Borne Zoonotic Diseases, volume 3, number 2.

Appendix 3. Description of Revisions

Draft 3.2

October 15, 2015

Noting that it has been nearly 15 years since the last revi-sion, at the 2013 American Committee of Medical En-tomology (ACME) Council meeting, a subcommittee wasformed to examine and update the guidelines. The subcom-mittee comprised Sam Telford (Tufts University), chair; Mi-chael Turell (USAMRIID); Kevin Macaluso (Louisiana StateUniversity); and Philip Armstrong (Connecticut AgriculturalExperiment Station). The entire document was examinedand the actual recommendations edited and modified toreflect the experience accumulated with operating underversion 3.1. Additional edits were made by Lyric Bartholomay(UW-Madison) and Philip Armstrong (Connecticut Agri-cultural Experiment Station) and were reviewed and approvedby the ACME taskforce on advocacy. The ACME ExecutiveCouncil unanimously endorsed its publication in 2018.

Changes since Draft 3.1

Minor editorial and grammatical changes were madethroughout the text. The major additions and deletions includethe following:

(1) The major effects of the Select Agent Rule on workwith arthropods were specifically incorporated.

(2) Recommendations were modified or added to betteraddress nonflying arthropod vectors (ticks, fleas,mites, fleas), a gap in the prior versions.

(3) Language has been added and grammar modified toreemphasize that the arthropod containment level(ACL) guidelines are recommendations and not reg-ulations and that site- and task-specific modificationsare based on a local risk assessment.

(4) Language has been added to address diagnostic sam-ples. The recent publication of guidance for clinicaldiagnostic laboratories is referenced because it con-tains excellent discussions of risk assessment andmitigation.

Draft 3.1

December 20, 2001: changes since Draft 3.0

Removed ACL-F designation and text. Moved field siteinformation to the Intent section. Removed section Im-portation of Plant Pests. Minor changes in grammar andwording. Removed citation to Sullivan, Songer and Estrem.Submitted to the ACME Executive Council for considerationregarding publication. Submitted to the Centers for DiseaseControl and Prevention (CDC) and National Institutes ofHealth (NIH) Offices of Health and Safety for considerationand comment. Resubmitted to Mary Bartlett (CDC/DPDeditor) for detailed editing.

Draft 3.0

February 15, 2001: changes since Draft 2.3

Added ACL-F designation and text. Added language inrisk assessment regarding autonomous transposable elementsin transgenic arthropod experiments. Minor grammatical er-rors corrected.

Draft 2.3

November 14, 2000: changes since Draft 2.2

Added URLs, minor wording changes. (See e-mail tocommittee of November 14, 2000, for details.)

ARTHROPOD CONTAINMENT GUIDELINES, VERSION 3.2 173