Animal Handling in Microbiology - Mackie & McCartney

1. Purpose of Laboratory Animals in Microbiology

• Isolation of fastidious organisms that cannot be cultured on artificial media (e.g., Treponema pallidum, Mycobacterium leprae)
• Virulence testing - distinguishing virulent from avirulent strains
• Pathogenicity testing of suspected organisms
• Production of antisera and vaccines
• Toxin detection (e.g., botulinum toxin, Clostridial toxins)
• Sensitivity testing for certain infections (e.g., clostridial myonecrosis)
• Maintenance of stock cultures of organisms that lose properties on artificial media

2. Animals Commonly Used

3. General Principles of Animal Handling

3.1 Restraint and Handling

• Grasped by the base (not tip) of the tail with one hand
• Placed on a rough surface; the scruff of the neck is grasped firmly between thumb and forefinger
• The tail is held between the little finger and the palm of the same hand
• This immobilizes the animal for injection

• Picked up by placing one hand under the thorax and the other supporting the hindquarters
• Never lifted by the scruff or limbs
• Should be handled gently - prone to shock

• Grasped by the scruff of the neck with one hand while the other hand supports the body weight from below
• Never lifted by the ears
• The hindquarters must be controlled to prevent kicking - a powerful kick can fracture the rabbit's own spine or injure the handler

4. Routes of Inoculation

4.1 Subcutaneous (SC)

• Common route for mice and guinea pigs
• Site: loose skin over the flanks or back
• A fold of skin is raised and a fine needle (25-26G) introduced parallel to the body wall
• Volume: up to 0.5 mL in mice, 1-2 mL in guinea pigs

4.2 Intraperitoneal (IP)

• Most common route in mice for primary isolation and virulence testing
• Site: lower right quadrant of the abdomen
• Mouse held head-downward so viscera fall away from injection site
• Needle directed cranially at 45° through the abdominal wall
• Volume: up to 0.5-1.0 mL in mice

4.3 Intracerebral (IC)

• Used for neurotropic viruses (e.g., herpes simplex, arboviral encephalitis, rabies diagnosis)
• Mouse lightly anaesthetized or not, depending on protocol
• Site: midpoint between ear and eye on the side of the skull
• Fine needle (26G) inserted 2-3 mm; 0.03 mL (30 µL) maximum volume
• Highly specialized - risk of fatal haemorrhage if done incorrectly

4.4 Intravenous (IV)

• Mice: tail vein - tail warmed in warm water (40-45°C) to dilate veins; needle directed toward the body at a shallow angle
• Rabbits: marginal ear vein
• Used for bacteraemia studies, toxin assays

4.5 Intradermal (ID)

• Used in skin sensitivity tests (e.g., Dick test, Schick test in guinea pigs)
• Site: shaved flank or ear pinna
• Very small volumes (0.1 mL); forms a visible bleb

4.6 Intramuscular (IM)

• Thigh muscle (quadriceps) of rabbits and guinea pigs
• Less common in routine diagnostic work

4.7 Intratesticular

• Rabbits: for cultivation of Treponema pallidum (syphilis) - organisms maintain virulence through serial testicular passage

4.8 Intranasal

• Mice, ferrets: for respiratory viruses, M. tuberculosis aerosol challenge
• Animal lightly anaesthetized; small volume dropped onto external nares and inhaled

5. Specific Diagnostic Tests Using Animals

5.1 Virulence Testing for Corynebacterium diphtheriae

• Guinea pig subcutaneous test: Two guinea pigs used - one protected with antitoxin (1000 units IP, 1-4 hrs before), the other unprotected
• 0.2 mL of 18-24 hr broth culture inoculated SC into both
• Toxigenic strain kills unprotected animal in 1-4 days; protected animal survives
• Signs: local oedema, then necrosis at injection site, haemorrhagic adrenal glands at autopsy

5.2 Tuberculosis - Guinea Pig Inoculation

• Guinea pig is highly susceptible to M. tuberculosis (human and bovine types)
• Clinical material (sputum, urine, CSF) injected SC into the groin
• Animal observed for 4-8 weeks; progressive inguinal lymphadenopathy, then generalized TB with caseous lesions in lungs, spleen, liver
• Now largely replaced by culture on Löwenstein-Jensen medium and PCR

5.3 Mouse Protection Test (Pneumococcal Typing)

• Historically used to type Streptococcus pneumoniae by Quellung reaction
• Mice inoculated with pneumococcal cultures; type-specific antiserum used for protection

5.4 Botulinum Toxin Detection

• Mice (IP) - 0.5 mL of suspected material injected
• Death within 24-48 hours confirms presence of toxin
• Confirmatory test: neutralization with specific monovalent antitoxin (types A, B, E, F)
• Protected mice survive; confirms toxin type

5.5 Clostridial Toxin (Gas Gangrene)

• Mice (IM into thigh) - Nagler reaction, gas gangrene tissue extracts
• Local muscle necrosis confirms presence of alpha toxin

5.6 Leptospira Isolation

• Young guinea pigs (IP) - blood or urine from suspected cases
• Dark-field examination of peritoneal fluid and blood after 5-14 days

6. Safety Precautions in the Animal House

• All work with infected animals must be done at the appropriate Containment Level (1-4) per ACDP guidelines
• Gloves must always be worn; heavy-duty gloves for rabbits
• Protective clothing: lab coat, face mask, and eye protection where aerosols are possible
• Needle-stick injuries: never recap needles; use puncture-resistant containers
• Bites and scratches must be reported, wound washed immediately with soap and water, then disinfected
• All animal carcasses and bedding must be disposed of by incineration or autoclaving before disposal
• The animal house must be kept under negative pressure relative to corridors when working with hazardous organisms
• Adequate ventilation (10-15 air changes/hour) to prevent build-up of allergens and aerosols
• Animals must be kept in appropriate cages with secure lids; overcrowding increases stress, aggression, and infection

7. Post-Inoculation Observation and Autopsy

• Animals observed twice daily for signs of illness: weight loss, ruffled fur, hunched posture, laboured breathing, neurological signs
• Humane endpoints must be defined in advance (UK Animals (Scientific Procedures) Act 1986 - 3Rs: Replacement, Reduction, Refinement)
• At necropsy: examine for gross lesions - enlarged lymph nodes, splenic enlargement (splenomegaly), caseous foci, haemorrhagic adrenals (diphtheria), oedema
• Samples collected: blood (cardiac puncture), spleen, liver, lymph nodes, lungs for culture and histology
• All carcasses handled as infectious material and incinerated

8. The 3Rs Principle (as discussed in Mackie & McCartney)

• Replacement - use cell cultures, serology, PCR where possible instead of animals
• Reduction - minimize animal numbers; use statistical justification
• Refinement - minimize suffering; use appropriate anaesthesia and analgesia

• ELISA and other serological assays
• Nucleic acid amplification techniques (PCR, RT-PCR)
• Cell culture systems for virus isolation
• Advanced culture media (e.g., BACTEC for mycobacteria)