4
Biosecurity
Biosecurity, for the purposes of this report, is defined as the policies and measures taken to minimize the risk of introducing an infectious pathogen into the human, agricultural animal, and research animal populations. Animals have long been recognized as hosts of zoonoses (infectious diseases that can be transmitted to humans or other species of animals). Biosecurity should be a consideration when transporting research animals because of the close contact that can occur between research animals and human handlers or other transported animals. This creates the potential for unintentional or intentional transmission of a zoonosis into the human or agricultural animal populations. Many zoonoses, including potential (agro)bioterrorism agents, are difficult to detect in an infected animal because they cause asymptomatic disease in the host species. However, the effect of zoonoses can be significant in humans and agricultural animals, causing severe disability or death and negatively affecting the capacity of the agricultural sector.
Another biosecurity concern is transmission of an infectious pathogen to a research animal during transportation and introduction of the pathogen into the colony that receives the animal. Infectious pathogens can negatively affect the health of the research animal and colonies, confounding research utilizing the infected animals.
PROTECTING PUBLIC HEALTH AND AGRICULTURAL RESOURCES
Minimizing Risks Associated with Transporting Research Animals with Experimentally Introduced Zoonoses
Increased efforts to improve the biosecurity of human populations and the agricultural sector have resulted from passage of the USA Patriot Act (2001), the Bioterrorism Preparedness and Response Act (2002), and enforcement of three parts of the Code of Federal Regulations (42 CFR 73, 7 CFR 331, and 9 CFR 121). These regulations establish lists of agents and toxins that have been deemed threats to humans, animals, and plants (see Table 4-1). The regulations require research laboratories that possesses any of the aforementioned agents to register its facility with the Centers for Disease Control and Prevention (CDC), designate a responsible official, perform background checks of persons who have access to the agents (conducted by the Department of Justice), and have a security plan for containment of the infectious agent. When infected animals must be transported, a plan for secure transportation must be in place. That plan would normally require:
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close communication between shipper and recipient;
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presence of responsible officials at the originating and receiving institutions;
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transfer of health records and assurances;
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identification of a carrier registered by the US Department of Agriculture;
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documentation of safety and security training of animal care personnel;
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notification of the appropriate institutional or CDC officials in case of emergency, loss, or theft;
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existence of emergency procedures (see Table 4-2); and
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good record maintenance.
Institutions are also required to have the appropriate level of laboratory biocontainment as outlined by CDC and the National Institutes of Health in the Biosafety in Microbial and Biomedical Laboratories Manual (BMBL). Although many shippers meet some of the requirements for laboratory biocontainment, not all meet all of the requirements, the result of which is a lack of uniformity in biosecurity during transportation. The characteristics of a good shipper are outlined in Table 4-3. Further, bio-containment requirements for transportation of infected animals (Appendix C, BMBL) are not as clearly defined as laboratory biocontainment
TABLE 4-1 Agents and Toxins That Require Registration of the Facility with CDC
Bacteria Bacillus anthracis Botulinum neurotoxin producing species of Clostridium Brucella abortus, Brucella melitensis, Brucella suis Burkholderia mallei, Burkholderia pseudomallei Cowdria ruminantium (Heartwater) Coxiella burnetii Francisella tularensis Mycoplasma capricolum/M.F38/M. mucoides capri(contagious caprine pleuropneumonia), Mycoplasma mycoides mycoides (contagious bovine pleuropneumonia) Rickettsia prowazekii, Rickettsia rickettsii Yersinia pestis |
Fungi Coccidioides immitis, Coccidioides posadasii |
Toxins Abrin Botulinum neurotoxins Clostridium perfringens epsilon toxin Conotoxins Diacetoxyscirpenol Ricin Saxitoxin Shigatoxin and Shiga-like ribosome inactivating proteins Staphlococcal enterotoxins Tetrodotoxin T-2 toxin |
Prions Bovine spongiform encephalopathy agent |
Viruses African horse sickness virus African swine fever virus Akabane virus Avian influenza virus (highly pathogenic) Bluetongue virus (exotic) Camel pox virus Cercopithecine herpesvirus 1 (Herpes B virus) Classical swine fever virus Crimean-Congo haemorrhagic fever virus Eastern Equine encephalitis virus Ebola virus Foot-and-mouth disease virus Goat pox virus Hendra virus Influenza virus (reconstructed replication competent forms of the 1918 pandemic influenza virus containing any portion of the coding regions of all eight gene segments) Japanese encephalitis virus Lassa virus Lumpy skin disease virus Malignant catarrhal fever virus (Alcelaphine herpesvirus type 1) Marburg virus Menangle virus Monkeypox virus Newcastle disease virus (velogenic) Nipah virus Peste des petits ruminants virus Rift Valley fever virus Rinderpest virus Sheep pox virus South American haemorrhagic fever viruses (Junin, Machupo, Sabia, Flexal, Guanarito) Swine vesicular disease virus Tick-borne encephalitis (flavi) viruses (Central European tick-born encephalitis, Far Eastern tick-borne encephalitis [Russian spring and summer encephalitis], Kyasanur Forest disease, Omsk hemorrhagic fever) Variola major virus (Smallpox virus) Variola minor virus (Alastrim) Venezuelan equine encephalitis virus Vesicular stomatis viruses (exotic) |
TABLE 4-2 Elements of an Emergency Plan
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TABLE 4-3 Characteristics of a Good Shippera
requirements. However, Appendix C of the BMBL, as well as Chapter 2 of this report, contains comprehensive information on which agencies must be contacted when transporting animals with human and agricultural animal zoonoses and can provide assistance in development of appropriate biocontainment plans for transportation.
Minimizing Risks Associated with Transporting Research Animals with Unknown Zoonoses
Though research animals may be experimentally infected with agents, many zoonoses of concern are endemic in research animals or may be naturally acquired. A list of zoonotic diseases communicable from research animals to humans is presented in Table 4-4. Because many of those zoonoses cause asymptomatic disease in animals, it may not be apparent that the animal is infected. The challenge is to identify the zoonoses that animals potentially harbor, consider the likelihood that the animal is a carrier and potential for exposure, evaluate the likely severity of an adverse event, and take steps to mitigate the risk.
TABLE 4-4 Examples of Zoonotic Diseases Transmissible from Research Animals to Humans
Disease |
Potential Animal Vectors |
Potential Route of Transmission |
Bacterial |
||
Anthrax |
Contaminated herbivores |
Cutaneous, inhalation |
Brucellosis |
Cattle, goats, swine, dogs |
Inhalation, ingestion, direct contact |
Leptospirosis |
Cattle, dogs, horses, swine, rodents, reptiles, amphibians |
Inhalation of contaminated fluids, direct contact |
Salmonellosis |
Birds, swine, reptiles, turtles, tortoises |
Direct contact, fecal-oral |
Tuberculosis |
Domestic and wild animals |
Droplets |
Q fever |
Cattle, sheep, goats |
Inhalation, direct contact with infected animals, their birth products, or infected materials such as bedding |
Viral |
||
Influenza |
Birds, horses, swine |
Aerosol, physical contact |
Hantaviruses |
Rodents |
Aerosol, direct contact with mucous membranes, animal bites |
Ebola |
Unknown |
Direct contact with infected materials, possibly droplets |
Monkey B virus |
Old World monkeys |
Animal bites, direct contact with mucus membranes |
Monkeypox |
Ground squirrels, gambian rats |
Droplets |
Rabies |
Dogs, cats, wild carnivores, bats, foxes, raccoons |
Animal bite, possibly airborne |
Fungal |
||
Ringworm |
Bovine, birds |
Direct contact |
There are reports of zoonotic disease transmission from pet hamsters, rabbits, and rodents to humans (CDC, 2001, 2005a, 2005b). Since companion animals are often transported in unfiltered containers and are transported along with research animals (particularly during air transport), the potential for cross contamination during transport must also be considered.
In general, contact between animals and people during transportation should be restricted to prevent exposure to or transfer of zoonoses. When possible, human contact should be limited to trained animal handlers who are knowledgeable of good sanitation practices, biosafety and biocontainment, and precautions for protection against zoonoses.
Special Considerations When Transporting Nonhuman Primates
The transportation of nonhuman primates requires special consideration because the risk of zoonotic disease transmission is greater with non-human primates than any other species of research animal due to the close phylogenetic relationship between humans and nonhuman primates (NRC, 2003b). Macaques imported for research have been implicated in the transmission of B virus and Ebola virus to laboratory workers, both potentially fatal diseases in humans (Cohen et al., 2002; Palmer, 1987). B virus (also known as Herpesvirus simiae) is of particular concern as it is endemic in some populations of macaques and infected animals are generally asymptomatic. B virus and Ebola virus can be transmitted through aerosols, animal bites, scratches, contact with body fluids or tissue material, or equipment that has been contaminated with body fluids (NRC, 2003b).
Due to the risks associated with zoonotic diseases transmitted from nonhuman primates, a common standard for personal protective equipment (PPE) has been established for workers who come into contact with nonhuman primates or equipment that has been exposed to nonhuman primates (NRC, 2003b). This standard recommends that dedicated clothing, gloves, and masks be utilized when in contact with nonhuman primates and that eye and face protection be mandatory for individuals who come into contact with macaques. Eye and face protection are also highly recommended for individuals who come into contact with other Old World monkeys.
Ensuring public safety and maintaining public confidence in the shipping process should be concerns of both regulatory agencies and carriers. Public confidence is difficult to maintain when airline passengers observe transportation workers wearing PPE boarding their plane. However, the development and use of overshippers (a closed, environmentally controlled container into which a standard primary enclosure would be
loaded in order to prevent a zoonotic exposure) would mitigate the need for some types of PPE (please refer to Chapter 5 for further discussion).
Special Considerations When Transporting Specimens and Tissues
Diagnostic specimens and tissues that are used for research are usually isolated from animals that are suspected of having an infectious disease or that are from an endemic area, and they should be treated as potentially infectious and hazardous materials. Those materials should be handled according to guidelines in BMBL (5th edition, Section VI and Appendix C). All diagnostic and tissue samples should be packaged according to IATA regulations as dangerous goods (Chapter 2). Depending on the suspect sample, it may be necessary for only persons who have IATA training to handle the sample. Access to the packages should be limited. If the sample potentially contains a select agent, both the shipper and the recipient must have all pertinent clearances and permits required by CDC and USDA and must have notified all appropriate agencies.
PROTECTING THE BIOLOGICAL INTEGRITY OF RESEARCH ANIMALS AND COLONIES
In recent years, greater attention has been paid to maintaining the microbial status of research animals and animal colonies. Scientists have rapidly expanded the use of immunocompromised rodents, such as nude mice and transgenic animals with immune deficits. Preventing exposure to infectious agents is necessary to maintain the health of these animals. In addition, scientists have discovered infectious agents, such as mouse and rat parvoviruses and Helicobacter species, that cause subclinical infections but can significantly alter research results (Jacoby and Lindsey, 1998). A list of viral, bacterial, and parasitic organisms found in commonly used species of research animals is presented in Table 4-5. Many of these organisms can infect multiple species, increasing the potential for intraspecies and interspecies disease transfer.
The most common routes of disease transmission between animals are infectious aerosols, close contact, and fomite (an inanimate object, such as clothing, capable of transmitting infectious organisms) transmission. Each of these routes poses a risk during shipping; however, there are methods to prevent the transmission of diseases among research animals, including barrier containment, specific-pathogen diagnosis, disinfection of vehicles and shipping containers, use of personal protective equipment (PPE), and segregation of animals. Though research animal vendors generally have well-established procedures to minimize biosecurity concerns, the typical researcher may need guidance in addressing biosecurity concerns
TABLE 4-5 Infectious Agents and the Susceptible Species of Research Animals
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Mice |
Rats |
Viruses |
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Sendai |
X |
X |
PVM (Pneumonia virus of mice) |
X |
X |
MHV (Mouse hepatitis virus) |
X |
|
MVM (Minute virus of mice) |
X |
|
GD-VII (Theiler’s murine encephalomyelitis virus strain) |
X |
|
REO 3 (Reovirus type 3) |
X |
X |
EDIM (Group A rotavirus) |
X |
|
Lymphocytic choriomeningitis vVirus |
X |
X |
Polyoma |
X |
|
MCMV (Murine cytomegalovirus) |
X |
|
Ectromelia |
X |
|
MPV (OPV) (Mouse parvovirus – Orphan parvovirus) |
X |
|
MAD (Mouse adenovirus) |
X |
X |
K virus |
X |
|
MTLV (Mouse thymic virus) |
X |
|
Hantavirus |
||
Adenovirus |
||
Parainfluenza |
||
Rotavirus |
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PI-1 (Parainfluenza-1) |
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PI-2 (Parainfluenza-2) |
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RHD (Rabbit haemorrhagic disease) |
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LCM (Lymphocytic choriomeningitis) |
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H1 (Toolan’s H1 virus) |
X |
|
KRV (Kilham Rat virus) |
X |
|
SDA/RCV (Sialodacryoadentitis virus/Rat corona virus) |
X |
|
HANT (Hantaan) |
X |
|
RRV (Ross River virus) |
X |
|
Foamy virus |
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Dengue |
||
Yellow fever |
||
Pox viruses |
||
Ebola |
||
SIV (Simian immunodeficiency virus) |
||
B virus |
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Rat picornarvirus |
X |
|
RPV (OPV) (Rat parvo virus – Orphan parvo virus) |
X |
|
Mice |
Rats |
Bacteria |
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Streptococcus zooepidemicus |
||
Pasteurella multocida |
||
Pasteurella spp. |
||
Treponema cuniculi |
||
Bordetella bronchiseptica |
X |
X |
Citrobacter rodentium |
X |
|
Corynebacterium kutscheri |
X |
X |
Clostridium piliforme |
X |
X |
Salmonella spp. |
X |
X |
Mycoplasma pulmonis |
X |
X |
Streptobacillus moniliformis |
X |
X |
Helicobacter hepaticus |
X |
X |
Campylobacter spp. |
||
Yersinia spp. |
||
Mycobacterium spp. |
||
Burkholdria |
||
CAR bacillus |
X |
X |
Parasites |
||
Ectoparasites |
X |
X |
Gastrointestinal helminths |
X |
X |
Gastrointestinal protozoa and sporozoans |
X |
X |
Encephalitozoon cuniculi |
X |
X |
Hepatic coccidia |
||
Metazoa |
||
Intestinal coccidia |
||
Other protozoa |
||
Demodex |
TABLE 4-6 Recommendations for Shipment of Research Animals Between Institutions
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when shipping animals to a colleague. Recommendations for shipment of animals between research institutions can be found in Table 4-6.
Barrier Containment
Most small-animal vendors have designed shipping containers that incorporate spun polypropylene filters to provide a physical barrier to the transfer of microbial contaminants into or out of each container, thus protecting research animals, colonies, and animal handlers from pathogen exposure during transportation. For gnotobiotic animals (animals whose microfauna and microflora are known in their entirety) and immuno-compromised animals, microisolation shipping containers are also available. Although the sturdy construction of the vendor containers may tempt researchers to reuse them to transfer research animals to other researchers, this practice is not recommended. Most vendors sterilize or disinfect the animal containers, food, and water before loading animals. Once a container has been opened at the recipient’s facility, its sterility has been compromised. Some facilities autoclave shipping containers for reuse; however, this may increase the air resistance of the polypropylene filters, restricting air flow (White, 2004). Until it can be established that autoclaving does not restrict air flow below acceptable levels, the committee suggests that the prudent course of action is to avoid autoclaving shipping containers for reuse. To ensure the biosafety of their animals,
individual researchers who wish to transfer animals to other researchers should purchase presterilized shipping containers from animal vendors.
Specific Pathogen Diagnosis
Normally, when major vendors communicate with clients, they identify the pathogens for which their animals have been tested to assure recipients that the animals sent to them are free of pathogens that could disrupt their colonies or experiments (for example, introduction of a respiratory infection would disrupt and invalidate an experiment on respiration). People shipping a few animals to colleagues do not always know the pathogen status of their animals and therefore cannot assure colony supervisors that the imported animals can be introduced safely into their colonies. To avoid inadvertent introductions of diseases, some colony supervisors quarantine incoming animals until they can gather the data required to ensure the health and safety of incoming animals (Otto and Tolwani, 2002). If facilities do not have the space and testing facilities required for that precaution, then it may be necessary for testing to be conducted at the institution of origin. Unfortunately, testing in such instances does not monitor for disease transmission during transport. Arrangements for testing and provision of assurances before introduction of shipped animals into a new colony require communication between responsible and knowledgeable officials of the institutions involved. Such officials can help to arrange safe and secure shipment of animals and arrange for the most efficient assurance of colony security.
Disinfection
Transportation protocols should have standardized procedures for disinfection of animal cages, transportation vehicles, and holding areas that conform to IATA, USDA, DOT, and CDC standards. Disinfection prevents transmission of pathogens from one shipment of animals to the next shipment transported in the same vehicle. Bedding, food, and water may be sterilized by autoclaving or gamma irradiation before and after shipment to prevent contamination of research animals and the receiving colony. Disinfection by sterilization or irradiation is not feasible for such items as transfer-vehicle cargo holds, large cages, and transportation-company holding areas. In these cases, however, chemical disinfection should be conducted after each transfer event. To ensure maximal efficiency of the disinfection process, disinfection should be applied using concentrations of chemical disinfectants and application times should be optimized according to manufacturers’ instructions.
Disinfection of the outside of the shipping containers should also be considered. As discussed above, companion animals are often transported in unfiltered containers and may be transported along with research animals. Therefore, the potential for cross contamination of shipping containers is present and must be considered. Another situation that may result in cross contamination is the entry of wild mice infected with lymphocytic choriomeningitis virus or mouse hepatitis virus into an animal holding area along the transportation route, such as an airport or cargo transfer station. The infected wild mouse can shed virus, thus contaminating the outside of the shipping container. Transmission of pathogens to research animals or colonies can then occur if the container is brought into a facility or animals are removed from the container without disinfection of the outside surfaces. Since such infections occur during transportation, diagnostic testing by the source provider does not ensure the biosecurity of either the animals or the receiving colony.
Personal Protective Equipment
The appropriate use of PPE can also protect research animals from human pathogens and cross contamination from other animals. For example, macaques are susceptible to human infections such as measles and tuberculosis. The use of PPE will not only prevent the transmission of B virus from a macaque to a human, but also can prevent the inadvertent transmission of measles or tuberculosis to the macaque. People who handle animals should cover their street clothing and exposed body surfaces with PPE to reduce the risk of pathogen introduction through direct contact or aerosol. In some instances, it may be appropriate to provide handlers with a shower-based entry system. The appropriate disposal of PPE is also necessary so that the PPE does not act as a fomite for transmitting pathogens. For example, if PPE is worn while disinfecting incoming shipping containers, the PPE should be disposed of before moving on to other tasks.
Segregation of Animals
Separation of different shipments of animals is also a method for preventing intra- and interspecies transmission of pathogens presented in Table 4-4. The committee suggests close adherence to the recommendations in the LARs regarding segregation of species and separation of animals of the same species of different origins (LARs Sections 5.3 and 10.3.2). Briefly, these regulations state that:
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animals in quarantine must be segregated from those which are not;
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animals known to be for laboratory use must not be stored adjacent to other animals in order to reduce any risk of cross-infection or contamination;
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nonhuman primates from different continents must be isolated from each other in aircraft holds, airport cargo warehouses, animal holding facilities, and during all phases of ground transportation; and
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animals that are natural enemies, e.g., cats and dogs, may be loaded in the same hold provided they are not in sight of one another.
Situations in which some aspects of these recommendations are not feasible may arise. For example, an airport may not have containment facilities to separate nonhuman primate species. In these cases, other measures must be employed to prevent disease transmission. An effective means of overcoming this problem would be the development of self-contained overshippers, as recommended in Chapter 5.