Mouse Husbandry. Date Issued: June 15, Ed.: T Davis, D. Larsen

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1 Mouse Husbandry Date Issued: June 15, 1998 Date Revised: July 26, 2012; Dec 2011; July 2011; March Comparative Medicine Resources Ed.: T Davis, D. Larsen Purpose: The purpose of this standard operating procedure is to outline husbandry procedures for mice at the Central, Cancer Center and Transgenic RAFs. Introduction: Husbandry practices are designed to protect against contamination and ensure consistent husbandry practices that minimize experimental variables. Filtered caging, wearing protective personal equipment (PPE), using forceps when handling or transferring mice and using chemical sterilant and disinfectants are the best techniques to maintain a pathogen-free colony. Before entering any rodent room, personnel must wear the appropriate PPE, posted on the door and follow entry order. Shoe Covers, hair bonnets, gowns, gloves and masks are required to enter mouse rooms. Also see SOPs: Rodent Cage Space Requirements Sentinel Surveillance Procedures and Dirty Bedding Transfer Rodent Enrichment Contents: Introduction: A. Cage Change Procedure 1 B. Singly-housed Mice 5 C. Mouse & Room Checks 5 D. Mouse & Room Checks on Fridays 7 E. Room Breakdown Procedures 7 F. Room Sanitation 8 G. Receiving from Vendors 8 H. Animal Transfers 9 I. Monthly Animal Census 10 J. Use of Cage Cards: event reporting. 11 K. Nestlets 13 L. Feed on Cage Floor 13 Every Caretaker assigned to a particular rodent room or covering for another caretaker is responsible for checking the rooms in a uniform manner. A. Cage Changing Procedures Barrier 1 Conventional 1. Always Follow Room Handling Order and Research Animal Facility (RAF) Handling Order 2. Every Monday (or every 7 days) Vimoba 2 spray bottles are prepared. Every room must contain two round cylindrical stainless steel containers mixed with Vimoba Solution. Discard old Vimoba from the bottle. Place one 1.5g tablet into 1 pint (about 500 ml) of water in a well ventilated area. Date and initial the label. Let sit for 10 minutes, and mix before using. 3. Spray the cage change surface until the entire surface is wet with Vimoba. 4. All cages are changed under a Biological Safety Cabinet or cage-changing station(hood). Turn Blower on. It must remain on for a minimum of 10 minutes before changing the first cage and must remain on while changing Changing cages in a conventional room is done on a clean cart. Wipe down cart or hood surface with alcohol. 1 Barrier cages must be opened in BS Cabinet or changing station (hood) only. Never open them outside approved hood. 2 Vimoba always has ten minute contact time. Alcohol does not have a specified contact time. 1

2 subsequent cages. Wipe down cart or hood surface with alcohol. NOTE: ALWAYS CHANGE ROOMS IN PROPER ROOM HANDLING ORDER SEQUENCE. Only the Attending Veterinarian, Supervisor or Facility Manager can grant permission to alter change of cages out of sequence. 5. Soiled ventilated cages are changed every 14 days unless otherwise instructed by the Attending Veterinarian, Principal Investigator or Facility Manager. 6. Unless otherwise labeled, all animals are to receive Reverse Osmosis (RO) water. Unless otherwise labeled, all animals are to receive Reverse Osmosis (RO) water. Barrier housed animals receive autoclaved RO water once every two weeks. 7. Observe water bottles for any deficiencies e.g. cracked bottle, leakage from brittle stoppers. Remove water bottles and replace with a new water bottle. Before giving each cage a water bottle, hold the sipper tube downwards and eliminate air bubbles by shaking out some water to assure it works. Observe cages for cracks too. Set aside damaged bottles or cages for supervisor to examine. The supervisor determines if the cage or bottle can be used. For conventional caging, sipper tubes are pushed all the way through the neoprene stopper to prevent creation of air bubbles. 8. Cages with breeding cards receive autoclaved breeder chow 5021 Conventional mice receive standard chow (5001). Cages with regular cage cards receive autoclaved regular chow (5010) i Quarantine animals are placed on fenbendazole food.ad lib. 1 If the investigator request, food can be changed, Place round sticker indicating which food to use for that cage ( 5010, 5021) Any changes to the food and water of individual cages or a room of cages must be labeled at the cage level and be reviewed by supervisors or veterinarians. 9. Inside the hood place a clean cage alongside the dirty cage in addition to the sentinel cage or autoclaved paper cup. On the cart place the clean cage alongside the dirty cages in addition to the sentinel cage or autoclaved paper cup. 2

3 Only one cage is changed at a time. Only one cage is changed at a time. 10. Remove the cage card(s) from the dirty cage and transfer it to the clean cage card holder in the sequential order that it appears on the dirty cage card holder. Always check each cage for any special accommodations before changing. IF A CAGE IS MARKED WITH SPECIAL FEED, WATER OR BEDDING CHANGE, CONSULT WITH YOUR IMMEDIATE SUPERVISOR IF ANY DOUBT OF APPROPRIATE HUSBANDRY REQUIREMENTS. 11. a. To transfer the mice from the dirty cage to the clean cage, use forceps immersed in Vimoba prior to mice transfers. Blot forceps on sterile paper towel to remove excess Vimoba liquid. b. Pick up the mouse from the dirty cage by the base of the tail using the forceps, and place the mouse in the clean cage. Check that mice are the same size. If any mice appear small or weak complete a Rodent Report. DO NOT PICK UP MORE THAN ONE MOUSE AT A TIME USING THE FORCEPS. 12. Alternate forceps between cages of mice: place forceps into Vimoba solution between each cage of mouse transfers. 13. ALWAYS CHANGE GLOVES: a. b. Between cages of mice belonging to different Principal Investigators. Between every rack (same or different PI) 14. PUPS: Neonatal Mice Day 0-to Weaning. a. Day 0 (newborn pups) to Day 3. Do not change cage under any circumstances. If cage is wet consult the vet tech assigned to the area. b. Day 4 to Day 15. Cup your hand and gently scoop the pups with immediate bedding material from the dirty cage and place that nest with pups in the clean cage. Avoid direct contact with pups. c. Pups 16 or more days of age. Pups are generally weaned at Day 21. However, in some transgenic strains, pups must be weaned at Day 28 and is only permitted when the delayed weaned date is described in an approved IACUC protocol. Transfer pups with forceps. After all of the apparent mice and pups are transferred, GENTLY move around the loose bedding in the dirty cage to check for any hidden pups or dead/ sick/ small mice. Discard gloves and replace with clean gloves before proceeding to the next cage. Pups in Conventional Mouse Rooms. Mouse breeding is not permitted in conventional rooms. Promptly report discovery of litter in a conventional room to the husbandry supervisor to contact research laboratory. 3

4 15. Place a clean sterile cage or cup for sentinel bedding alongside dirty cages for soiled bedding sampling to transfer a defined amount of soiled bedding sample. Refer to Sentinel Surveillance Procedures for the specific details for complete procedure. 16. After the mice are transferred from the dirty cage to the clean cages, check the food and water. Ensure that the stopper is completely secure and the sipper tube length and shape is appropriate for that cage. 17. Place the clean cage back on the rack in the same location from where the cage was originally taken. With a small dust broom sweep off dust, debris or bedding off of the rack. Place the clean cage back on the rack in the same location from where the cage was originally taken. 18. Repeat this procedure until all of the cages on a rack are changed. 19. After the cages are changed, turn off the blower and lift up the floor of the hood and remove the front floor cover 3. Spray the interior of the hood floor and clean the inside of the hood with Vimoba spray. Let sit for 10 minutes and wipe with a sterile paper towel.. After the cages are changed clean the cart with Vimoba spray. Let sit for 10 minutes before wiping dry. Replace all pieces. 20. On the room sheet behind the room door complete and initial all tasks completed in the room. For example, the caretaker would check temperature, feed, water, sweep and mopping, sentinel surveillance, water sippers, and hood cleaning. Wipe surfaces with alcohol Transfer all dirty cages to cage wash or designated area. 21. If you must enter any rooms higher on the room handling sheet, get permission from your supervisor Change gloves between each room. If you must enter any rooms higher on the room handling sheet, get permission from your supervisor Change all PPE between each room. 22. Double-check your work. When finished changing a rack, double-check each cage and make sure each cage has full water bottles and sufficient food. 3 There are some differences in placement of pre-filters and how hoods are taken apart for cleaning. If you have any questions about a particular hood ask your supervisor 4

5 B. Singly-Housed Mice Singly-housed mice require environmental enrichment. See Rodent Enrichment SOP C. Mouse & Room Checks Frequency Barrier Conventional 1. Evaluate each mouse cage for: Daily Overcrowding: adults and/or pups Sick Dead Fighting: look for bite wounds and blood in cage Flooded cages Newborn pups. If new pups are found note DOB on the cage card. If you can count the pups without disturbing the nest. Add that information to the cage card too. 2. Submit a completed health report for all reportable events: sick, dead, incomplete cage card, overcrowded, flooded cages. Give one copy to the Vet Tech and another copy to the husbandry supervisor when done with room where the event was observed. However, when an emergency (life or health-threatening to staff or mice) the Caretaker must immediately contact a veterinary staff member or any other Supervisory personnel. Any abnormalities regarding animal health are reported on a Rodent Report on the same day of observation to the area veterinary technician. Any abnormalities with the facility (flood, fire, room temperature) must be reported to husbandry supervisor, the senor person on duty. 3. Check for food and water every day for every cage for: Sufficient volume of each Fungal or abnormal growth. Food and Water are required on every animal cage unless otherwise indicated by cage cards for Special Diet. 4. Sweep each room in your care on weekdays. Mop each room on day that cages are changed in and all other rooms are mopped as needed. On weekends, the on-duty caretaker is responsible for sweeping all the rooms and rooms are mopped as needed. 5. Carboys are not present: not Top the water level in carboys with 5

6 applicable reverse osmosis water each day. Label and date 6. Check room sheet posted on the back of the door or on the wall. Room Temperature should be 70 degrees, plus or minus 2 degrees. 7. Empty trash Frequency Barrier Conventional Weekly Food is replaced at time of cage change Note: Rodent food must be used with 180 days of the milling date. Rotate your stock to use the oldest food first. Restock supplies: Food is replaced once per week in conventional cages. Food barrels must be properly labeled (manufacturer ID. number and milling date) and stored in a covered container. Note: Rodent food must be used with 180 days of the milling date. Rotate your stock to use the oldest food first. Gloves Paper towels, Flag cage cards (Orange, green) Regular cage cards Cage card holders Purple dots for sentinel surveillance Black euthanasia tape. Extra shoe box cages for mouse transfers when ordered Black bags for mice transfer. and for dead animals. NOTE: Depending on room additional cards may need to be stocked. If it is a breeding room stock breeding cards. If there are mice on special diets or special water, stock the appropriate cage card. Barrier only Autoclaved Nestlets Extra autoclaved water bottles Autoclaved Paper towels Autoclaved paper cups for sentinel procedures Conventional only Extra water bottles Reverse osmosis water carboy is completely drained, washed in cage wash and refilled Label and date. Autoclaved Paper towels Autoclaved paper cups for sentinel procedures 6

7 Frequency Barrier Conventional Once every two weeks Not Applicable: yellow food bins are not in barrier rooms. Yellow food bins are lined with black trash bags and replaced every two weeks or more frequently as needed. Feed bags are placed in the bin with the mill date at the top of the bag. Frequency Barrier Conventional 1. Door sheets are checked and replaced each month. Monthly 2. Replace any stained or ripped door signs 3. Caretaker does monthly animal and cage census Note: Vet Techs check LAMBS and animal rooms for expired Protocols. Vet Tech changes protocol to new number or places animals on holding protocol. D. Mouse & Room Checks on Fridays 1. Each animal caretaker is responsible to ensure that all cages have enough water and food to last over the weekend. Any water bottle with visible dirty water, damaged water bottle, or water bottle less than ½ full is replaced. Top off all other bottles. 2. Caretaker must know who is on call and have pager numbers available. Information is on the weekend sheet. E. Room Breakdown Following Depopulation 1. Empty out the room completely and place the racks in a clean room. 2. Remove all litter from the floors, walls and surfaces of the breakdown room. 3. Thoroughly clean all room surfaces with detergent Diversey Stride SC Neutral detergent- Accumix. Use a 1:256 dilution or 1 spout per 2 gallon of water. 4. Close off the room. Mix eight 1.5-gram tablets or two 6-gram tablets of Vimoba into one gallon of water making 200 ppm solution of chlorine dioxide Mix solution in a well ventilated area. 5. Fill fogger with solution. 6. Cover the ventilation system completely and tape over the seam of the door with tape to contain the fogging in the room. Place the fogger in the center of the room or insert the nozzle of the fogger through a suitable opening in the room. 7. With the flow rate setting on HIGH output, apply the fog for 15 minutes for each approximately 3000 cubic feet of space in the room thoroughly wetting all surfaces. 7

8 NOTE: The fog generated is irritating to the eyes, skin and mucous membranes. Do not allow people to enter treated room until ten air exchanges or two hours of mechanical ventilation is completed. 8. Calculate time when room is completely fogged (treated). WAIT AN ADDITIONAL 2 Hours. Then enter room to unplug the fogger and mop up the wet floor with a dry mop. Wipe down any excess water. The room is ready for repopulation. 9. Mark the room with an orange sticker with the date that the room is broken down. Place sticker on acrylic sign holder that holds and the room door sheet on the back of the door. 10. All barrels and containers for animal rooms are cleaned with detergent (see number 5), then sprayed with Vimoba after 10 minutes, and dated with a green sticker marking the day it was cleaned. Alternatively, they are run through the rack washer. The method is determined by the supervisor. 11. Wash floor: Mix Stride Accumix Neutral Cleaner in designated bucket with water. F. Monthly Room Sanitation When Populated 1. Remove all litter from the floors, walls and surfaces of the breakdown room. 2. Thoroughly clean all room surfaces with detergent** and rinse with water. 3. All barrels for animal rooms are cleaned with soap and water**, then sprayed with Vimoba. Dry or remove excess after 10 minutes. Label and date with a green sticker or run through rack washer, label and date. 4. Wash floor: Mix Stride Accumix Neutral Cleaner (**1Spout/2gallons of water) in designated bucket with water. Wash floor. 5. Mark on the room door sheet G. Receiving Animals from Vendors or Transfers: 1. The Caretaker is responsible for receiving animals from a commercial vendor. The caretaker ensures that all animals that are expected to arrive on that date are present. If any animals are missing by 12 noon, please seek your immediate facility Supervisor or Manager for instructions on how to proceed with this matter. 2. When receiving animals from a commercial vendor please ensure that ALL information from the receiving sheet is on the box or crate. IF ANY INFORMATION DEVIATES FROM THE RECEIVING SHEET, SPEAK TO THE SUPERVISOR OR FACILITY MANAGER FOR CLARIFICATION. 3. Remove the enclosed packing slip information from the front of the box and give it to the Head Clerk. Taconic does not have a packing slip on the box. Jackson and Harlan have packing slips in front of boxes. 8

9 THIS ROO 4. Spray down all surfaces, especially the bottom, of shipping boxes with Vimoba and let them sit for ten minutes before boxes can leave the receiving area. 5. When animal caretakers are putting animals away in housing rooms, VERIFY that all information is printed and correct on the cage card: Date of arrival Size/weight Sex (verify) Number of animals. Caretaker adds Date of birth information from packing slip or shipping box to the cage card. 6. All animals must receive food and water after the animals are housed properly in their cages, find the census sheet for the protocol and record: The number of animals and the number of cages on the date received and initial. ADDED TO THIS ROOM REMOVED F DAY FROM VENDOR OR OTHER INSTITUTION WEANED, SEPARATED OR TRANSFERRED No. of ANIMALS No. of CAGES No. of ANIMALS No. of CAGES INITI AL No. of ANIMALS ewr 4 6 Document that you placed animals in the designated room by printing your initials on the receiving sheet. Document animal placement, after each room. H. Animal Transfers (Room to Room or Facility to Facility including ICPH RAF) 1. All Animal Transfer orders shall take no longer than 72 hours for full completion. If the animal transfer needs to be expedited, this must be approved by the Facility Manager or Supervisor. 9

10 2. The Facility Manager or Supervisor must authorize in writing all animal transfers. Transferring animals by husbandry staff or research staff without prior authorization from the Facility Manager or Supervisor will result in disciplinary actions. 3. On the Animal Transfer Form there are four important areas that must be reviewed: Animal source (Protocol Number) Animal recipient ( Protocol number) Animals for transfer include numbers, strain and transgene Relocation from room to room or facility to facility 4. Investigator must label each cage to be transferred with neon yellow Transfer Cards to ensure proper transfer FROM and TO rooms. Transfer only animal cages with designated Transfer Cards. 5. Once a transfer card is in place, count the number of mice and cages. This ensures that the correct number of cages and animals transferred are recorded on the transfer sheet. Check original cage cards on cages. 6. Once the correct number of animals is verified, document the number of animals and cages that is deducted from the room in the appropriate place in the room census book. 7. Place cages in a black plastic bag on a cart labeled with the desired destination i.e., Central, Cancer Center. For ICPH RAF transfers, sterile crates are used for transfers 8. The animal caretaker at the source is responsible for properly securing the food and water bottles on the cage. This includes removal of water bottles to prevent excess water from dripping or completely spilling out into the cage during the cage transfer process. Alternatively animals are placed in shoebox cages with food and hydrogel for transfer to another facility. 9. The recipient animal caretaker ensures that the number of animals and cages match the transfer sheet. If the number of animals and cages received does not match the Animal Transfer Form, the recipient caretaker must promptly inform the facility Supervisor or Manager of the discrepancy. 10. The recipient caretaker must ensure that water bottles are placed back on the cage once the cage is transferred. 11. Once the Caretaker has the correct number of animals, he/she records the number of cages and mice listed on the correct Principal Investigator s protocol in the correct Row and Column in the room census book. 12. Animals are transferred to new cages. 13. Once the Caretaker has received the cages and the information is verified, the Caretaker must sign the bottom of the Animal Transfer Form documenting the transfer. This sheet is given to the Supervisor for filing. 14. Research staff places cards. The cards remain on the cage after the move to enable research staff to find the cages. Caretakers remove all transfer cards when you do your monthly cage count. 10

11 I. Monthly Animal Census 1. Every Caretaker is responsible for ensuring an accurate manual cage count at the end of every month. 2. The Caretaker must obtain a monthly census sheet from the Facility Manager or Supervisor, and on the monthly census sheet document the following information in the table: Investigator Protocol Number Room Number Cage Count Date of count Species Your signature or initials 3. The Caretaker is responsible to count each cage by specific protocol number. 4. Cages of the same protocol number are frequently scattered among several locations in the same room. Use great care when adding cage numbers! 5. After all information is gathered and written down on the form, the Caretaker takes the census sheets along with the completed monthly census sheet to the Program Assistant no later than 3 p.m. on the last weekday of each month. J. Use of Cage Cards: event- reporting procedures When changing cages, if there is ever any doubt whether the identity of individual mice were mixed between cages or cage cards are missing, always contact a Supervisor to resolve the problem. Refer to the table below for event identification. Event Who can use card Caretaker responsibility Vet Tech responsibility Overcrowding (OC) (See SOP on Rodent Space requirements) Card color Green Caretakers and Vet techs Place card on overcrowded cage, complete a Rodent Report and turn into vet tech. Inform staff. PI has 72 hours to correct. If at that time they do comply, separate the mice and Vet techs charge them. UNDER OBSERVATION Caretaker Vet Tech Vet Place cards when a mouse is found sick in a cage. This would include but not limited to masses, malocclusion, hunched posture, trauma, or discharge from eyes or nose. Caretaker completes a Rodent Report sheet and notifies the Vet Techs. Vet tech receives the report and checks the animal(s) in the cage and reports the problem to the investigative staff and/or veterinarian. The card can be removed when the animal is euthanized or removed from the cage. Otherwise, the card is replaced with a turquoise card if the mice are to remain under observation 11

12 Card Color Orange staff If any part of card is blank, report to Vet Techs Card must have complete information with a start and end date. Example would be to watch animals for 1 to 2 weeks after surgery UNDER VETERNARY Observation Card color Aqua Vet Techs Vets Monitor the cage and do not report the same problem. If a new problem arises or if additional mice in the cage become sick. Complete and file another Rodent Report Vet techs place Aqua card. Assess the problem(s) with the cage and triage the future observation and or treatment of the cage. Consult Vet as needed. See additional information in Appendix A. Rodent report sheet Dead mouse White Sticker Caretakers Vet Techs When caretakers find dead mice: 1) they complete the sheet; 2) notify the Vet Tech 4 ; 3). label the cage with the number of animals found dead on that date; 4). date and initial; 5) place in black garbage bag per cage found and label with dead sticker/ label; 6) deduct the animal or cage from the census sheet. When the Vet tech receives the report they notify the investigative staff. Vet techs must also notify the veterinarians in cases of unexpected animal death. Found Pregnant : Card color Pink staff If pups born in conventional housing rooms, notify supervisor and or vet staff immediately. If investigator placed this card on a cage do not change the cage until you consult your supervisor or investigator staff. If animals are born in a conventional room it may be an accident or an unapproved breeding. Notify Veterinarian ASAP. Animals are euthanized or isolated Investigators can order timed pregnant animals or moms with litters. This is allowed. Please give litters a nestlet. Euthanize animal: Black tape staff Investigators may label cages of animals for euthanasia. Only euthanize the animals you are sure the intention is clear. When in doubt, consult your supervisor or a vet tech before euthanizing animals. Vet techs can euthanize investigators animals with written requests in the form of or Vet Tech request. See euthanasia SOP 4 Vet tech notifies the research staff and carcasses are stored in necropsy for three days. 12

13 Transfer Cards More detailed information in section H. staff Only move labeled cages Caretakers remove all transfer cards when you do your monthly cage count. Research staff places cards. The cards remain on the cage after the move to enable research staff to find the cages. Export Cards Vet tech/ Staff None Research staff places cards Vet tech removes cards when they pack the animals for shipment Special Diet Beige Card Special Water Teal Card Fasting Staff staff staff When cages are labeled with special food or water cards, check for instructions and contact information. Contact supervisor if food or water runs out. staff hangs and removes the cards. There a start and end time on the card. If information missing from the card or the card is still on the cage after the end time notify vet tech or supervisor immediately. Animals marked for euthanasia need confirmation by the Vet Tech, Facility Manager or Supervisor prior to euthanasia. Move animals to designated area in shoebox cages and make sure they have food and water. K. Nestlets 1. Nestlets are autoclaved inside a microisolator cage. Seal the cage bottom and lid with autoclave tape and document the autoclave date on the tape with a pen. Alternatively, place into each cages prior to autoclaving. 2. Give females with litters nestlets after the pups are born Females with litters from 0-14 days of age get a nestlet. 3. UNDER NO CIRCUMSTANCES ARE NESTLETS PLACED INSIDE ROUND CAROUSEL RACKS. (Optimice cages) Plastic or cardboard huts may be used instead. 4. Provide Nestlets, red huts, or cardboard huts to singly-housed mice unless contraindicated in IACUC protocol. L. Feed On the Cage Floor Mice unable to reach feeders are written up on a Rodent Report, and an orange card is placed by Caretaker for veterinary staff attention. Only upon veterinary approval is feed placed on the cage floor below the feed container. D Mal 13

14 Appendix A: Veterinary Observation Aqua Card: procedures and options. Vet tech places an Aqua card in response to a Rodent Report from a Caretaker, a report from an investigator or finds a health problem during rounds. Upper left corner is to indicate subsequent observation or treatment frequency e.g. D=daily; W=weekly; MWF=Monday, Wednesday and Friday. Upper Right Corner to indicate symptom or treatment (other) Use 2-3 letter code Mal Malocclusion (check once a week) Bar Barbering (check once a week) Tu: Animals have a mass or a tumor (decide on follow-up frequency) Pro: Prolapse TX Animal(s) on treatment i If the investigator request food can be changed, Place round sticker indicating which food to use for that cage ( 50010, 5021) 14