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Animal Transport Between Buildings
Experiments Involving Biohazards
Guidelines for Asceptic Surgery on Rodents
Media Coverage
Monoclonal Antibody Production
Occupational Health Program for Personnel with Substantial Laboratory Animal Contact
Pathogen Testing of Rodent Biologics
Personal Protective Equipment Program for Workers with Macaque Monkeys
Pets
Prolonged Restraint
Protection of the Animal Facility From Outbreaks of Adventitious Pathogens
Quarantine and Stabilization of Animals
Rederivation
Required Post operative Care Documentation
Standard Immunization Methodology/Practices
Survival Surgical Procedures
Animal Transport Between Buildings
Animals should not be transported from one research building to another. Exceptional circumstances require approval by Veterinary Resources. Under no circumstances should animals other than rodents and rabbits be transported in a public elevator (freight elevators should be used). All animals that are transported should be covered in a manner to obscure public view and prevent exposure of others to animal allergens.
Experiments Involving Biohazards
No hazardous agents (infectious, oncogenic, radioactive or chemical) may be used in any animal facility without being cleared by the IACUC. The use of radioactive materials requires additional approval by Radiation Safety (x6-6281). If special caging and care are required, Veterinary Resources must be contacted well in advance (prior to submitting an animal protocol is recommended).
The veterinary faculty and facilities personnel of Veterinary Resources are available for consultation and advice on matters relevant to animal housing, care and use when biohazard agents are proposed.
At the campus level, The Office of Environmental Health and Safety (x6-7055) and the UM,B Biohazard Committee oversee all experimentation and use of biohazardous agents. See the EHS handbook for further information at www.ehs.umaryland.edu/ .
If radioactive substances, infectious organisms, toxic chemicals, or chemical carcinogens are to be used in-vivo, the following points must be addressed in your protocol to the IACUC:
* ABL level (1, 2, or 3) for infectious agents.
* Concentration
* Route of administration
* Duration of exposure
* Length of time animals will be kept following exposure
* Room location where agent is administered
* Location of animal housing post exposure
* Method of animal disposal
We must protect the health of our employees and others. Those who attend to the care and housing of your animals may not be familiar with the nature of hazards being used. Therefore, we require the necessary information on the agent to best safeguard those who work with the animals.
Guidelines for Aseptic Surgery on Rodents
The following guidelines are consistent with our accrediting organization’s (AAALAC International) interpretation of the Animal Welfare Act regulations and PHS guidelines which provide satisfactory aseptic conditions as indicated below.
* Surgery should be conducted on a clean, uncluttered lab bench or table surface. The surface should be wiped with a disinfectant before and after use and/or covered with a clean drape.
* Hair should be removed from the surgical site with clippers or a depilatory. The surgical site should be treated first with an antiseptic scrub (chlorhexidine or povidone iodine scrub) and rinsed with alcohol.
* All instruments should be sterilized, but the method of choice may be determined by the surgical instruments or devices being used. Steam sterilization may be accomplished by autoclaving at 121 degrees Celsius for 20 minutes. Gas sterilization with ethylene oxide is an alternative for items which will not withstand high temperature. Acceptable techniques for cold sterilization include soaking in 2% glutaraldehyde for ten hours, in 8% paraformaldehyde for 18 hours, or in 6% stabilized hydrogen peroxide for six hours.
* The surgeon should wash his/her hands with an antiseptic surgical scrub preparation and then aseptically put on sterile gloves. If working alone, the surgeon should have the animal anesthetized and positioned and have the first layer of the double-wrapped instrument pack opened before putting on sterile gloves.
* The surgeon should wear a surgical face mask. A surgical cap and gown are recommended, but not required.
* Multiple surgeries present special problems. After the first surgery, the sterilized instruments may be kept in a cold sterilant. Hot bead sterilizers are also acceptable to use between animals. Sterile gloves should be changed between surgeries if the surgeon touches nonsterile surfaces.
* The abdominal or thoracic body wall should be closed with absorbable suture material. The skin should be closed with staples or with a nonabsorbable suture material in a simple interrupted pattern. Skin sutures or staples should be removed 7 to 10 days after surgery.
* Rodents should be recovered from anesthesia in a warmed environment. Because of the rodent's high surface area to volume ratio, they are particularly susceptible to heat loss (and overheating). Prevention of heat loss is therefore most desirable and may be accomplished by the placement of a dry cloth or pad under the animal during surgery. Warm water circulating heating pads are ideal for providing supplementary heat, if needed. Electrical heating pads should not be used as they can cause thermal burns and subsequent dermal necrosis. Heat lamps may be useful, but a thermometer should be placed near the animal to determine appropriate temperatures. Recovery on clean, dry bedding is sufficient for short procedures.
* Antibiotics are not given routinely after surgery unless justified by the investigator. Antibiotics should not be used as a substitute for proper aseptic technique.
* Analgesics should be used with all surgeries and whenever the potential for pain exists. Pain in rodents may be identified by observing the animal’s reluctance to move about, eat or drink and vocalization with handling. It is best not to wait for evidence of pain before giving analgesics. Buprenorphine 0.05-0.1 mg/kg SQ is a safe and effective analgesic for rodents and should be given at the time of surgery and then every 8-12 hours. Lidocaine applied topically to the skin incision provides preemptive analgesia and may be sufficient for minor surgical procedures.
Questions about aseptic or surgical technique may be addressed to Veterinary Resources staff by calling 706-3540
Media Coverage Policy for Research Utilizing Animals
It is understood that publicity for research is important at the University of Maryland, Baltimore, School of Medicine and therefore should be supported and encouraged. Media coverage of animal-based research must be conducted in a socially sensitive fashion and meet all federal regulations and guidelines regarding the humane care and use of animals. It is important that media coverage be presented in a positive light and not prone to misrepresentation. Also, it should be determined if it is truly necessary to exhibit the animals or demonstrate their use or care.
Any investigator who is contacted by the media to provide information on any animal-based research should contact the Office of Media Relations (X6-7820) without delay. If it is anticipated that animals will be exhibited to the media or if a portion of the media coverage includes specific use and/or care of animals, then the Office of Media Relations will contact the Chief of Veterinary Resources and the IACUC Chair for their recommendations.
The media event should not proceed until these persons (or their designees) are satisfied that all has been done to ensure that media coverage will be conducted appropriately.
A representative from Veterinary Resources and/or the IACUC may be present during the event and have authority to make any necessary changes to protect the investigator and University from misrepresentation.
Monoclonal Antibody Production Guidelines
* Monoclonal antibodies should be produced in tissue culture. Ascites production should be done only if tissue culture methods cannot be used or are inadequate. IACUC requires justification for ascites production.
* Tissue culture support for the production of monoclonal antibodies is available through Veterinary Resources. This is provided as a fee-for-service activity and includes production of monoclonal supernatants in standard or serum - free media, roller bottle culture, or hollow - fiber generation for larger quantities of antibodies.
* All hybridomas must be Mouse Antibody Production (MAP) tested for adventitious pathogens (e.g. murine viruses, mycoplasma) that could interfere with experiments or cause morbidity/mortality in individual rodents and/or place the animal facility at risk. All testing is provided by Veterinary Resources free of charge.
Occupational Health Program for Personnel with Substantial Laboratory Animal Contact
Please follow the link to a PDF file for further information.
Pathogen Testing of Rodent Biologics
Rodent origin cell lines, tumor lines or any other biologics (e.g., body fluids) passaged through live rodents have the potential to transmit a wide range of rodent pathogens. One example is mouse hepatitis virus (MHV) which can be devastating even as a subclinical infection as it can confound experimental results, especially in immunological studies. Another example is lymphocytic choriomeningitis virus (LCM) which will interfere with experimental data and result in rodent morbidity and mortality and is also zoonotic (i.e., may cause disease in personnel).
All animal biologics (any tissues, serum, cells, tumor, or other animal passaged materials) must be adventitious pathogen free prior to introduction into any animal. Veterinary Resources will test these biologics at no cost to the investigator.
This usually involves a serological evaluation (e.g. rodent cell line injected into a naive mouse which is subsequently assayed for antibodies to a panel of murine viral antigens).
Personal Protective Equipment Program for Workers with Macaque Monkeys
Training is required on the use of protective equipment for all who work with macaques or macaque tissues. This training is provided by the Veterinary Resources (VR) veterinarians.
Macaque monkeys present a risk to employees from natural infection with Herpes B virus (other terms used Cercopithecine herpes virus 1, Herpes virus simiae, and B-virus). This latent infection in macaques is commonly fatal to humans that become infected. Humans have been infected from bites, scratches, splashes to the eye and from tissues of macaque monkeys.
It is the investigator’s responsibility to make sure that each person under their employ receives this training and to notify Veterinary Resources of new employees who need training.
Pets
Research animals are not pets and should not be removed from the facilities. Concerns have to do with public perception of an animal removed from a research setting, the fact that these animals have generally been procured with grant monies, liability of the University, and our need to document the disposition of research animals in accordance with federal law.
Conversely, personal pets should not be brought onto campus for treatment, or otherwise. Veterinary Resources does not operate a clinic or provide service for pet animals. In addition, certain pet animals (especially mammals) can harbor and spread infectious diseases to animals within the research facility. Only pet fish are exempted from this policy.
Prolonged Restraint Policy
Brief physical restraint of animals for examination, collection of samples, and a variety of other clinical and experimental manipulations can be accomplished manually or with devices such as restraint stocks or squeeze cages. It is important that such devices be suitable in size and design for the animal being held and operated properly to minimize stress and avoid injury to the animal.
Prolonged restraint of any animal, including the chairing of non-human primates, should be avoided unless essential to research objectives. Less restrictive systems, such as the tether system or the pole and collar system, should be used when compatible with research objectives. The following are important guidelines for the use of restraint equipment:
* Animals to be placed in restraint equipment should be conditioned to such equipment prior to initiation of the research.
* The period of restraint should be the minimum required to accomplish the research objectives. Prolonged restraint for any reason must be approved by the IACUC committee.
* Restraint chairs or similar devices are not to be considered "normal" methods of housing, although they may be required for specific research objectives.
* Restraint chairs or similar devices must not be used simply as a convenience to investigators in handling or managing animals. When such devices are used, their use must be specifically approved by the IACUC committee.
* Attention must be paid to the possible development of lesions or illnesses associated with restraint, including contusions, decubital ulcers, dependent edema, and weight loss. If these or other problems occur, veterinary care must be provided to treat the animal, which if necessary must be temporarily or permanently removed from the restraint device.
Protection of the Animal Facility from Outbreaks of Adventitious Pathogens
The inadvertent introduction of pathogens and parasites into an animal facility can cause a significant disruption of research in terms of wasted time and dollars and unwanted effects on experimental systems. Therefore, it is imperative that all investigators carefully consider the source of research animals and biologic materials introduced to the animals. All research animals at UM,B are ordered from vendors that have been approved by Veterinary Resources. Vendors are approved based on their ability to provide animals that are reliably free of pathogens that can spread to other animals in the facility and hence adversely affect ongoing research projects. Other sources of animals, including other universities, pharmaceutical companies, or transgenic laboratories that are not currently on the approved list must be individually contacted by Veterinary Resources to provide health status and related information on the proposed animals to be delivered to our campus before any animals from those sources are shipped (includes UM,B, BVAMC or IHV/MBC.)
All animal procurement is done by Veterinary Resources. If animals are not from the approved vendor list, Veterinary Resources will contact the source to obtain health status information. Normally only animals believed to be pathogen-free are accepted. Non-approved vendor animals go into quarantine in any case. If animals are not pathogen-free, we make recommendations to rectify this with the source/investigator. Absolutely no animals may be placed in the facility without prior approval from Veterinary Resources. Any animals delivered to the School of Medicine without prior approval of Veterinary Resources will be rejected on arrival.
It is equally important that all animal biologics (any tissues, serum, cells, tumors or animal passaged materials) be adventitious pathogen-free prior to their introduction into any of the animals housed in our facilities. Veterinary Resources will test these biologics for adventitious pathogens at no cost to the investigator. This usually involves a serological evaluation (e.g. rodent cell line injected into a naive mouse which is subsequently assayed for antibodies to a panel of murine viral antigens).
Any investigator planning to administer animal biologics to a research animal must contact Veterinary Resources for approval prior to the use of such biologics. Testing of biologic materials normally requires 4-6 weeks to complete. Previous data on adventitious pathogen testing can be reported to Veterinary Resources for review and approval.
The forms for animal ordering and testing of biologics (Technical Services) are included in Appendix 6. Any questions regarding animal ordering should be directed to extension 6-3540, Veterinary Resources. Questions regarding testing of biologics should be directed to Veterinary Diagnostics (extension 6-3540).
In addition, investigators should limit their animal work to a single animal facility in order to decrease the chances of fomite transfer of infectious agents. All members of a single investigator’s laboratory should only need access to one of the following facilities - MSTF, BRB/HH, HSF, BVAMC, MPRC, Dental School, School of Pharmacy or IHV/MBC. Any exceptions must be approved in advance by the Chief, Veterinary Resources. Whenever possible, investigators should also limit their activities to areas in which their animals are housed or to common procedural areas.
This document has been prepared by the IACUC Subcommittee on Pathogens. The committee has established that any failure of the investigator’s laboratory to abide strictly by the above directions could lead to a suspension or permanent loss of ability to utilize UM,B, BVAMC or IHV/MBC animal facilities. Willful disregard of these rules will result in permanent loss.
Quarantine and Stabilization of Animals
All newly received animals must be allowed a stabilization period of at least 48 hours prior to their use. This permits the animals to adapt to their surroundings resulting in a more stable physiological and behavioral state. Studies indicate that mice have altered immune functions and elevated corticosterone levels for 48 hours following shipment.
Quarantine for Primates - ALL primates entering the facility must undergo a quarantine period of 13 weeks. Quarantine space is allocated on a first come, first serve basis.
Quarantine for Rodents - Rodents received from other than approved sources are required to be quarantined and tested prior to release to investigators. Quarantine period is 6 to 8 weeks.
Rederivation
Rodents that are positive for specific pathogens that may interfere with research are rederived by Veterinary Resources using embryo transfer techniques in flexible-film isolator containment.
Required Postoperative Care Documentation
Large Animal Types (to include Rabbits, ferrets, dogs, swine, sheep/ruminants, and non-human primates) Require:
1) Anesthesia Report
2) Post Procedure Form
3) Copy of experimental record documenting postoperative care.
To be submitted to Submit to VR, including lab notes, preferably within 24 hours post-surgery
Small Animal Types (to include rats, mice, other rodents, birds, amphibians)
1) The primary record-keeping tool for the small animal investigator is a logbook or bound notebook. The investigator for each rodent procedure or surgery must keep a logbook record. For batches of rodent procedures, a 12 group logbook can be maintained, but for high-risk procedures or procedures with high complication rates (consult with a Veterinary Resources veterinarian), individual information must be maintained for each rodent in the logbook or a separate animal record can be maintained. The PI must have their logbook available during normal work hours for review.
2) Cage cards: must be maintained on all animal cages, and must be completely filled out, noting the date of arrival, strain, source, investigator's name and protocol number. Veterinary Resources fills out the cage cards upon arrival but the PI is responsible to check that all information is correctly completed and updated. Contact the Veterinary Resources' facility supervisor if there are any questions or problems.
To place card on cage and have notes in lab preferably within 24 hours postsurgery
Standard Immunization Methodology/Practices
Veterinary Resources - School of Medicine University of Maryland, Baltimore
RABBIT (polyclonal antibody production):
Initial Injection
* Shave site area
* Antiseptic prep, alcohol or betadine
* Complete Freund's Adjuvant (CFA) + protein 1:1 (total 1 ml volume)
* 0.1 ml SQ or IM per site
* 0.05 ml ID per site
* maximum 10 sites, at least one inch separation of sites
Foot pad injections are not acceptable for any species.
Boost(s)
* Incomplete Freund's Adjuvant + protein -- same regimen as above.
* Blood sampling: maximum amount 1% of body weight
(i.e., 3 kg rabbit - 30 ml) at minimum 2 week intervals.
* Methods - either marginal ear vein or central artery, by venipuncture or cut down.
* Droperidol (2.5 mg/kg) is recommended as a vasodilator + tranquilizer. Topical xylene is acceptable, but requires thorough washing afterward.
* Terminal Exsanguination requires complete anesthesia (ketamine and xylazine at 50 mg/kg and 5 mg/kg respectively IP, recommended) and may be performed through ear vein/artery or by cardiac puncture.
* Approved method of euthanasia should follow, such as pentobarbital, 100 mg/kg.
MICE - monoclonal antibody production:
If Pristane priming is used, the recommended amount is 0.2 ml. intraperitoneal.
CFA should never be given intraperitoneally in any species.
Mice should be observed daily from the onset of ascites and tapped when appropriate.
They should be tapped only twice (the first tap will usually yield the most antibody). The second tap should be a terminal procedure and the mouse should be euthanized immediately prior to the tap.
Mice found moribund or in respiratory distress should be euthanized immediately by approved methods.
Blood sampling: maximum 1% of body weight (i.e., 30 g mouse -- 0.3 ml) at minimum 2 week intervals.
- Tail vein/artery venipuncture or laceration
- Retro-orbital bleeding (requires sedation of mouse)
Inhalation anesthesia with Isoflurane used with a precision vaporizor provides appropriate short term anesthesia but must be used in fume hood or with other scavenging provisions.
Cardiac puncture - only under deep anesthesia and as a terminal procedure.
Approved methods for euthanasia of mice include CO2 asphyxiation, followed by cervical dislocation, overdose of anesthesia or anesthesia followed by physical means.
Survival Surgical Procedures
The Institutional Animal Care and Use Committees have set minimum standards for animal operating rooms and laboratories in which surgery is performed. The standards are based on the NIH Guide for the Care and Use of Laboratory Animals. Adherence to these guidelines is necessary for compliance with the standards of accreditation by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The standards are meant to ensure that surgical procedures are performed in an appropriate environment using good surgical techniques.
These standards apply to all mammalian species, including rabbits, but excluding rodents. Aseptic technique (e.g., surgical gloves, mask and sterile instruments) should be used for surgical procedures on rodents such as rats and mice; however, the standards for the surgical facility are not as rigid. Rodent surgical areas may be a separate room or portion of a room. The area should be clean and orderly and should not be used for any other purpose during the time of the surgical activity. Animal housing areas may not be used for surgical procedures.
For all rabbits and higher species, designated survival surgical areas are required and should be used only for that purpose. Use of the area for other purposes such as office space and equipment and supply storage, except for surgical and research equipment, is not acceptable.
Non-survival surgical procedures may be performed in general purpose laboratories provided the rules outlined below are followed:
There must be no eating, drinking or smoking in the laboratory during the surgical procedure.
All equipment and surfaces within the room must be kept clean.
The surgical area should be free of non-essential equipment and supplies.
The use of a survival surgical area for non-survival surgery is satisfactory, provided the rules regarding survival surgery are followed, and the room is properly sanitized following the procedure. Any other laboratory outside of those designated for survival surgery CANNOT be used for survival surgical procedures in animals other than rodents. Approved surgical areas are in the Medical School Teaching Facility and the Bressler Research Building.
Aseptic Technique:
Aseptic technique, including aseptic preparation of the skin, sterilization of instruments, and wearing sterile gloves and masks (gowns and caps when appropriate), is necessary when performing survival surgery on all animals. Operating suites should not be used for eating, drinking or smoking. Personnel performing the surgery should have formal training in operative procedures and aseptic technique or have acceptable career experience approved by the IACUC.
Anesthetics and Analgesics:
Information concerning types, dosages and routes of administration of anesthetics and analgesics is available from members of the Program. Also see the Guide's Appendix.
Anesthetic techniques and use of post-operative analgesia should be in accordance with current methods in the literature and approved by the IACUC. Inquiries may be directed to Veterinary Resources. Records must be maintained of the anesthetics used, amounts, dates, procedures and animal species. If volatile anesthetic gases are used, a gas evacuation and scavenging system is necessary.
Analgesic usage - rule of thumb - if any particular procedure performed on animals would cause pain or discomfort if performed on a human then the animal should receive similar consideration in the form of analgesics.
Multiple Major Surgical Procedure:
Major surgery is defined as surgery which penetrates and exposes a body cavity or produces substantial impairment of physical or physiologic function.
Multiple major survival surgery is described as more than one major survival surgical procedure performed on a single animal.
Multiple major survival surgical procedures may be performed on the same animal only if they are related components of a project and have been reviewed and approved by the IACUC. Multiple major survival surgery on animals for economic purposes alone is not acceptable according to the NIH Guide.
Euthanasia:
Proper methods of euthanasia (see AVMA Panel on Euthanasia) should be used at the termination of the experiment. The methods used should be documented. A record must be kept of the amounts, date, and animal species when restricted drugs such as sodium pentobarbital are used for euthanasia.
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