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Proceedings of the 4th National Symposium on Biosafety

Biohazards in Research Involving Large Animals

Fred Quimby, VMD, PhD
College of Veterinary Medicine
Department of Pathology
Cornell University
Ithaca, New York 14853-6401
607-253-3510

Introduction

I will review categories of agents, the level of risk or concern, and how we deal with these issues as it relates to research involving large mammals.

Although many types of agents may be hazardous in large animal research facilities (1), I will concentrate on microbial agents and restrict my statements to zoonotic agents and the products of recombinant DNA research. However, it should be noted that many of the special considerations employed to reduce risk of exposure to infectious agents when working with larger animals ie, ponies, horses and cattle, are applicable to reducing risks associated with hazardous chemicals, toxins and radioisotopes.

At the heart of any program designed to ensure the safe use of biohazards is the concept of Risk Assessment. Once a full assessment of risk(s) are in hand, the institution can begin to develop mechanisms to reduce risks which should incorporate occupational health and safety and risk monitoring.

Because the literature on this subject is sparse, I will draw from the program at Cornell University for examples of various principles.

Differences Between Conventional Laboratory and Large Farm Mammals Used in Research

Unlike most purpose bred laboratory animals, the larger farm mammals used in biomedical research are commonly not specific pathogen free (although there are exceptions to this which I will discuss later). Due to their size and temperament these animals generally spend much of their lives as social groups on pastures or housed in enclosures with access to paddock or loafing areas.

Having opportunities to acquire zoonotic agents from the time of birth (from their mothers) and then continuously from both conspecifics and wildlife in the field, the numbers and types of zoonotic agents are both large and diverse (respectively).

By wildlife I refer not only to small mammals which cross fence lines and contaminate pastures and paddocks, but also birds and bats that cohabit enclosures, rodent and other mammalian vermin which may contaminate feeds and bedding materials, as well as deer and larger mammals which can disseminate their infectious agents across fencelines by the aerosol route of exposure. While a vermin control program is essential, even the most intensive use of todays pest control technology cannot guarantee a risk free farm animal.

Unless the development and maintenance of truly SPF farm animals is available, the institution must rely on standard tools to control disease in farm animals:

Despite our best efforts, farm animals, at least in New York, usually do harbor a number of zoonotic agents; cattle herds frequently have endemic infections with Cryptosporidium, parvum, Coxiella burnetti and Trichophyton verrucosum.Equine herds frequently harbor Salmonella sp. and Leptospira pomona.

The incidence of these infections varies geographically across the U.S. and clinical signs, while usually absent, may be obvious only in the young (Cryptosporidium) or during certain seasons of the year (ringworm in winter and early spring in Northern areas).

Occasionally human infectious agents are the subject of research in large animals (although in most cases smaller laboratory animal models are used). In our experience the evaluation of recombinant DNA vaccines for safety and efficacy has been the research agent of concern. Current NIH regulations require that animals, inoculated with the products of recombinant DNA in which at least two-thirds of the viral genome (serving as the vector) remains intact, must be maintained using facilities, equipment and practices consistent with ABSL-2 (2). Common vectors used include members of the poxvirus, adenovirus and herpesvirus families as well as Salmonella sp.

Categories of Agents

There are multiple categories of agents with the potential to harm humans. For the sake of risk evaluation, we have referred to these agents as being either intrinsic (naturally harbored by the farm animal) or extrinsic (introduced as part of the research).

From a practical standpoint we have much more control over extrinsic agents since we can review research plans, properly prepare in advance of infection, precisely quantitate the dose and develop sensitive assays to monitor for the presence and absence of the agent. Intrinsic agents are often more problematic for all of the above reasons as well as their impact on the occupational health program.

Regardless of whether the agent is intrinsic or extrinsic, protocols involving farm animals are carefully evaluated by risk assessment. (3) We recognize that each agent varies according to the actual risk of a human acquiring the infection from an animal and the concern we have for the disease in humans once acquired. Thus, while farm animals are quite unlikely to be a source of human infection by rabies virus (low risk) the concern is very high. Both risk and concern are involved in establishing the final ranking of our protocols . (Table 1)

Risk Assessment and Occupational Health & Safety

Protocols are ranked Negligible, Low, Moderate or High Risk after taking into consideration the following factors:

Initially we rank protocols based on exposure of a normal adult person. All persons with direct animal contact or indirect contact (animal excretions, sharps, etc.) in protocols ranked Moderate or High Risk must be enrolled in the Occupational Health Program (PI's, research technicians, students, animal care staff) and based on their individual medical histories, a medical surveillance program will be created. Thus, factors such as pregnancy, heart abnormalities, or immunodepression discovered through the annual medical history will dictate our final level of concern and subsequent medical surveillance. Recent guidelines can assist institutions in development of Occupational Health and Safety programs(4).

While the PI is ultimately responsible for hazard concerns involving extrinsic agents, commonly the attending veterinary staff identifies and establishes procedures involving intrinsic agents. At Cornell University these decisions are guided by an Occupational Health and Safety Advisory Committee of experts on risk assessment, zoonotic diseases and risk reduction (Table 2). All proposed uses of extrinsic agents must be reviewed and approved by a Biohazards or rDNA Committee (or both). Approvals from all committees are directed to Cornell Center for Research Animal Resources where critical information is logged into a computer database shared by the Animal Resource and University medical staff. Only after all review groups have furnished Memoranda of Understanding and Agreement does the PI receive approval to begin work. The IACUC veterinarian checks to be sure that the PI has identified an animal facility designed to contain infectious agents at the recommended ABSL.

Risk Reduction

Before each study a risk reduction plan is initiated. Critical components include:

  1. Engineering Controls - the facility, animal room and support spaces are designed and constructed to contain agents at the appropriate animal biosafety level. Agent specific biohazard signs are posted throughout.
  2. Personal Protection - all personnel identified with the project are equipped with appropriate Personnel Protective Equipment (PPE). This may require a fitted respirator (ABSL-3).
  3. Practices and Procedures - appointments are scheduled with the Occupational Health Nurse Practitioner for all individuals involved with moderate or high risk protocols, at this point agent-specific fact sheets are reviewed. For animal care, SOPs are developed jointly by the veterinarians and animal care staff. For research, SOPs are generated jointly with the veterinarian, PI and research staff. All procedures are reviewed especially the handling of sharps. Emergency personnel servicing the animal facility (Life Safety, Electrical, mechanical and control shops) are informed of the agents being used and special precautions that must be taken before entering the building.
  4. Education - Only specifically trained animal care staff are allowed to work in these areas. They have completed both AALAS and inhouse biohazard courses. Special training is conducted on class II biological safety cabinets. Audiovisuals are reviewed on specific agents, sanitation practices and personal protection (3,5).

We have facilities specifically designed to contain agents at ABSL-2 and 3 for horses and cattle(6). Air is directional from a central corridor through antechambers into separate animal holding rooms. All surfaces in animal rooms are smooth epoxy-latex paint over concrete allowing for foam or steam disinfection and formaldehyde or vaporized hydrogen peroxide sterilization. Adaptation of new technologies developed for other industries such as foam disinfection and vaporized H2O2 provide more effective treatment, less human contact and safer application(7,8). All exhaust air is HEPA filtered (99.97%). Because of the large volumes of excrement generated by farm mammals (Table 3) compared to laboratory animals (Table 4), bagging and autoclaving excrement is impractical. To aid in sanitation and containment, all plumbing from the floor drains lead to a waste water treatment plant which combines hyperchlorination with high pressure and temperature inactivation before discharge of excrement into the municipal sewer.

Rooms are designed for 1 or 2 animals to move freely (120 or 144 SF). The animal can be cross tied for temporary restraint. A permanent 3 foot high gate inside each room provides a staging area and escape route for personnel. The building is equipped with exit showers, autoclaves and gas sterilization rooms. Room use can also be modified to accommodate survival surgery or necropsy.

One of the most perplexing problems associated with the biocontainment of large mammals involves carcass disposal. While autoclaving coupled with safe transport to an approved incinerator are usually employed with small animals, the safe transport of a 1600 pound cow or even 600 pound pony is a challene. Recently a new technology has been developed which may address this concern. The WR2 processor is a tissue digester which utilizes sodium hydroxide to hydrolyze all proteins, nucleic acids, lipids and most carbohydrates. This technology can be constructed and installed in most facilities, can accommodate from 10 to 2,000 pounds of tissue, requires no carcass pre treatment and converts solid material to liquid waste in 18 hrs(9). It is safe, inexpensive and produces waste which can be discharged into a municipal sewer. Preliminary tests demonstrate high efficacy of this process in the destruction of microorganisms (Table 5). It can also be used for the disposal of carcasses containing low level radioactivity.

Agent Monitoring

Because of our interest in evaluating equine vaccines we have established a herd of ponies vaccinated only for tetanus and rabies and serologically free of antibodies to other equine viruses as well as common zoonotic bacteria (Leptospirosis and Salmonella). This herd is geographically isolated from other horses and routinely surveyed serologically. We also have ABSL-2 and 3 facilities for poultry and maintain flocks of SPF chickens and ducks for vaccine testing(10). This facility has also been designed according to CDC recommendations(6).

Non SPF herds are monitored for intrinsic agents using a combination of serodiagnostic tests coupled with clinical examination and the submission of specimens for agent isolation. Sporadic introduction of disease agents generally results in the quarantine and treatment of the animal or culling the animal from the colony followed by monitoring other members of the herd for the agent. Endemic infections require constant vigilance by personnel handling animals, or their excrement and biological fluids.

Monitoring for extrinsic agents is more controlled, with appropriate tests established in advance for agent isolation or seroconversion. Control animals are maintained individually in biosafety facilities and monitored regularly to insure agent containment. All employees (including faculty and students) are enrolled in the Occupational Health and Safety program. They must be cognizant of the agent and SOPs designed to contain the agent. Employees are themselves monitored by written history and, in some instances, periodic testing.

In certain rDNA studies, when the best scientific information suggests no shedding of the organism, conspecifics are held together in one room with inoculated animals and monitored for horizontal transmission of the agent.

Conclusion

The strategies employed for the containment and safe handling of infectious hazards in large farm mammals are similar to those employed for other animals, although special considerations must be incorporated. While the literature dealing with this specific subject is sparse, general information regarding facility design (11), hazard reduction (12) and hazard disposal (13) are all applicable. And excellent texts are available as references to guid safety committees on issues of zoonotic disease in all animals (14, 15, 16). As with all other applications of biohazardous agents in research settings, it is prudent to incorporate four principal components in the institution's strategy to ensure human health and safety: Risk Assessment, Risk Reduction, an Occupational Health and Safety and Exposure Monitoring. Programs which fail to incorporate all four components are vulnerable. Every attempt should be made to include individuals with diverse knowledge and experience in every stage of program development. And appropriate education of personnel at all levels is mandatory.

References:

  1. Richmond, J.Y. Hazard Reduction in Animal Research. Lab Animal 20:23-29, 1991.
  2. National Institutes of Health. Guidelines for Research Involving Recombinant DNA Molecules. Federal Register (60 FR 20726), April 27, 1995.
  3. Richmond, J.Y., Arambub, P. and A. Ruiz. Protection From Risk in Animal Studies. Lab Animal 22:36-40, 1993.
  4. National Research Council. Occupational Health and Safety in the Care and Use of Research Animals, National Academy Press; Washington, In Press, 1996.
  5. Richmond, J.Y. Responsibilities in Animal Research, Lab Animal 20(4):41-46, 1991.
  6. Centers for Disease Control. Biosafety in Microbiological and Biomedical Laboratories, 3rd edition, HHS Pub. (CDC) 93-83, 95, U.S. Government Printing Office. Washington, 1993, 177 pp.
  7. Power Foamer, Pharmacal Research Laboratories, Inc., 33 Great Hill Road, Naugatuck, CT 06770.
  8. Vaporized H2O2, Advanced Barrier Concepts, Inc., 115 Disraeli Drive, Cary, North Carolina, 27513.
  9. U.S. Patent 5, 332, 532, Waste Reduction, Inc., 212 Pinewoods Ave., Troy, NY 12180-7244.
  10. Quimby, F.W., Schat, K.A., Lucio-Martinez, B., Ludders, J. and A. van Tienhoven, Poultry in, The Experimental Animal in Biomedical Research, vol. 2, Rollin, B.E. and M.L. Kesel, eds., CRC Press: Boca Raton. 1995, pp 195-248.
  11. Ruys, T. Handbook of Facilities Planning. vol. 2, Laboratory Animal Facilities , van Nostrand Reinhold: New York, 1991.
  12. Fleming, D.O., Richardson, J.Y., Tulis, J.J. and O. Vesley (eds.) Laboratory Safety, 2nd edition, ASM Press: Washington, 1995.
  13. National Research Council. Biosafety in the Laboratory: Prudent Practices for Handling and Disposal of Infectious Materials. National Academy Press: Washington, 1989.
  14. Acha, P.N. and B. Szyfres (eds). Zoonoses and Communicable Diseases Common to Man and Animals, 2nd edition, Pan American Health Organization Press: Washington, 1987.
  15. Weinberg, A.N. and D.J. Weber, Animal-associated human infections. Infectious Disease Clinic of North America 5:1-181, 1991.
  16. Steele, J.H. (ed), CRC Handbook Series on Zoonoses, vol. 1 and 2, CRC Press: Boca Raton, 1994.

TABLES

Farm Animal Zoonoses

Species Common Name /Organism Risk Concern
Sheep, cattle, goats Q-fever
Coxiella burnetti		 Moderate-High
Moderate
All Salmonellosis
Salmonella spp. 		Low
Moderate
Cattle Tuberculosis
Mycobacterium tuberculosis, var.bovis		 Low
High
Cattle, swine,sheep, goats Brucellosis
Brucella sp.		 Low
High
Sheep Contagious ecthyma
Orf virus 		Moderate
Low
Cattle Ringworm
Trichophytum verrucosum 		Low-Moderate
Moderate
Cattle Cryptosporidiosis
Cryptosporidia parvum		 Low-Moderate
Moderate
Equine Leptospirosis
Leptospira interrogans (Pomona) 		Low
High
All Toxoplasmosis
Toxoplasma gondii		 Low
Moderate-High
All Rabies
Rabies virus 		Low-High
High

Table 1

 

 

Occupational Health and Safety Advisory Committee
  • Director, Environ. Health and Safety
  • Director, Gannett Health Center
  • Program Veterinarians
  • Outside DVM, MPH
  • College Safety Officers

Table 2

 

 

Wastes
  Urine excreted Feces excreted
Species (kg) ml/kg/da per animal (L) kg/d
Horse (400) 3-18 7.2 15-20
Cattle (500) 17-45 22.5 25-45
Swine (100) 5-30 3.0 1-2.5
Sheep (60) 10-40 2.4 0.5-1
Goats (60) 10-40 2.4 0.5-1

Table 3

 

 

Average Urine Output
Species ml/day
Mouse 0.5-2.5
Rat 5-23
Cat 50-100
Rabbit 20-350
Dog 600 ± 125

Table 4

 

 

Efficacy of the WR2 Process in Microbial Decontamination
    Decontamination
  Pre Post Positive
Control
1. S. Aureus
ATCC 6538 		4.0 x 109 cfu/ml <100 cfu/ml growth
2. B. Subtilis
ATCC 6633 		6.0 x 108 cfu/ml <100 cfu/ml growth
3. C. Albicans
ATCC 14053 		1.4 x 108 cfu/ml <100 cfu/ml growth
4. M. Fortuitum
(NYSDH Lab Stain) 		6.1 x 107 cfu/ml <100 cfu/ml growth
5. Giardia cysts** 2.0 x 105 cysts/ml <101 cfu/ml cysts observed

Table 5

* Viability check of control suspension not processed but stored for 24 hours at 40oC during processing time.
** Pre counts were determined by hemocytometer and post samples were concentrated by centrifugation and sediment was observed microscopically.

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