실험동물품질관리
실험동물품질관리의중요성 실험동물 살아있는시약 사육환경 ( 온습도, 차압, 감염 ) 동물복지 & 실험의신뢰성 재현성에영향 일상적사육관리 실험의일부
품질관리항목 미생물모니터링 ( 연구 & 생산기관 ) - 반입시검사 - 정기검사 유전모니터링 - 일반동물 ( 생산기관 ) - 형질전환동물 ( 연구 & 생산기관 ) 환경모니터링 - 온습도, 차압, 낙하균, 수질검사, 조도, 암모니아등 기타 - 동물오염 ( 감염 ) 및탈출방지를위한대책등
실험동물반입절차 외부개인반입 - 반입신청서 + 1.5 년의미생물모니터링성적서제출 (FELASA 규정근거 ) 외부공급동물의검수및환경순화실시 (1 주일 ) * 설치류의경우 SPF 동물만반입
형질전환동물탈출방지턱설치 칸막이높이 : 45cm
애완동물소유자출입절차 다음의애완동물소유연구자 - 출입금지, 다른연구자로교체설치류 ( 햄스터, 기니피그, 저빌, 랫드, 마우스, 다람쥐등 ) 파충류 ( 뱀, 거북이 ) 양서류 ( 개구리 ) 상기이외의애완동물소유연구자 - 실험동물실출입금지요망, but, 부득이한경우애완동물소유확인서작성 출입허용
외부방문인출입절차 실험동물실출입은허가된사람에한하여허용 외부인의방문및견학시최소 1주전에외부방문인출입요청서제출 출입자방문구역방문목적 출입조건 7 일경과 1 일경과샤워수술내의덧가운 5 층 SPF 구역 ( 제한 ) 실험 O O O O 실험장비 A/S O (O) O O 외부 5 층 semi-barrier 구역 단순견학 ( 전실허용 ) O (O) O 실험장비 A/S O O O 4 층 단순견학 ( 전실허용 ) O O
구역간연구자동선 연구자가복수의실험을동일한시기에수행시오염확산최소화를위하여다음의순서에따라출입 4-5층동시출입의경우 - 5층 4층 : 당일가능 - 4 층 5 층 : 당일불가 semi-barrier 구역출입 : 24시간후허용 SPF-barrier 구역출입 : 24시간 /1주일후허용 5 층 4 층
구역간이동안내 5 층 SPF 구역 Semi-barrier 구역 4 층 Clean 구역소동물실 ( 랫드 / 마우스특수동물실, 재반입실 ) Clean 구역중대동물실 ( 토끼 돼지 개 ) 감염동물실험실
실험동물관찰포인트 항목전신피모눈귀구강항문생식기행동사료및물배설물 관찰포인트말랐다, 살쪘다, 외상털결, 광택, 탈모, 더러워짐눈물, 충혈, 안검폐쇄, 안구이상외상, 충혈침흘림, 출혈, 치아의이상더러워짐, 출혈기형, 외상, 분비물, 출혈활동성, 횡와, 선회, 경련소비량의급변형태, 색, 경도, 양의변화
아산생명과학연구소실험동물연구실헬스모니터링항목 분류 항목 Mouse Rat Sendai virus (HVJ) Mouse Hepatitis Virus (MHV) Hantaan virus Viral infections Ectromelia virus (Mouse pox) Lymphocytic choriomeningitis (LCM) virus Rat coronavirus (Sialodacryoadenitis virus) H-1 virus Murine Norovirus (MNV) Mycoplasma pulmonis Clostridium piliforme (Tyzzer's disease) Pseudomonas aeruginosa ( ) ( ) Bordetella bronchiseptica Bacterial infections Salmonella spp. Corynebacterium kutscheri Streptococcus pneumoniae Pasteurella pneumotropica Staphylococcus aureus ( ) ( ) Parasitological infections Citrobacter rodentium (E. coli O115 a, c : K (B)) Intestinal Eimeria spp. 등 Protozoa Roundworms Syphacia spp.(pinworm) / Tapeworms Ectoparacite 기타 s
소동물 (Mouse, Rat)
주요감염병 Sendai virus (HVJ) Mouse hepatitis virus (MHV) Rat coronavirus (SDA) Mycoplasma pulmonis Clostridium piliforme (Tyzzer's disease) Parasites - Syphacia spp. (Pinworm), protozoa 등 Murine Norovirus
Sendai virus Basics - RNA virus, family Paramyxoviridae - Natural hosts : mice, rats, hamsters - Spread by infective nasal secretions
Sendai virus Susceptibility (Mice) - Most susceptible 129/J, Swiss nude(s-nu/nu), DBA/1J, DBA/2J strains - Moderately susceptible A/HeJ, C57BL/10Sn, BALB/c - Less susceptible SJL/J, C57BL/6J and S
Sendai virus Infection patterns (Mice) - Asymptomatic in neonate, weanling, adult - Clinical signs chattering, mild respiratory distress, prolonged gestations, poor growth in weanlings, neonatal deaths Infection patterns (Rat) - Asymptomatic - But, can cause reductions in litter size & growth rates
Sendai virus Gross findings - Lung few gross lesions can be focally reddened atelectatic with serous fluid visible in pleural & pericardial cavities
Sendai virus Response - If detected in a colony, elimination of infected animals - essential - Depopulation of the entire colony - usually required - Cesarean rederivation - not justifiable in most circumstances
Sendai virus Effects on research - lifelong lymphocyte abnormalities, increased NK cell-mediated cytotoxicity, altered host responses to transplantable tumors - this interference - unpredictable - So, best to not conduct research with Sendai-infected rodents
Mouse hepatitis virus (MHV) Basics - RNA virus, family Coronaviridae - MHV & sialodacryoadenitis virus (SDAV) - coronaviruses of mice & rats respectively - share cross-reacting antigens (typical for coronaviruses) Note the club-shaped projections typical of coronaviruses emanating from the viral surfaces.
Mouse hepatitis virus (MHV) Basics MHV strains - Prototype strains : MHV-1, MHV-2, MHV-3, JHM (MHV-4), A59, S - Different strains have different visceral tropisms. some primarily infect the GI system; some the respiratory tract; some the brain
Mouse hepatitis virus (MHV) Basics - Natural hosts : mice only - Contagious with prevalence rates >80% in outbreaks - Active infection lasts 2-3 weeks, shedding the virus in GI & respiratory excretions - Viral transmission - direct contact with shedding mice, contaminated cell lines, fomites, airborne particles - Transplacental transmission - not important - Clearance & recovery from infection - dependent on an intact cell-mediated immunity - In immune-competent mice, an inapparent carrier state does not develop.
Mouse hepatitis virus (MHV) Clinical Signs - In naive, juvenile mice, - can produce nonspecific clinical signs (runting, failure to thrive) - Usually subclinical But, virulent enterotropic MHV strains - can produce diarrhea with mortality up to 100% Runted C57B1
Mouse hepatitis virus (MHV) Clinical Signs Nude mouse - In immune-compromised mice, - an emaciation slowly progressing to death (typical) - diarrhea or jaundice seen - In affected mice with encephalotropic strains - encephalitis developed - Can not clear themselves of MHV infection, Excrete the virus indefinitely An athymic mouse (nu/nu) with wasting & cachexia
Mouse hepatitis virus (MHV) Gross findings Acute lesions - Gross morphologic lesions (an acute MHV infection) last only 7-10 days nonspecific & subtle - Lesions depending on the strain of virus mild olfactory mucosal necrosis, neuronal necrosis and vacuolation of the brain, multifocal interstitial pneumonia, ulcerative enteritis, multifocal necrotizing hepatitis
Mouse hepatitis virus (MHV) Gross findings Nude mouse liver - Large coalescing cream-colored friable foci (arrow) of necrosis, resulted when acute multifocal hepatitis progresses to chronic active hepatitis - Immune-incompetent mice : more generalized & progressive than in immune-competent mice Nude mouse liver
Mouse hepatitis virus (MHV) Control - If detected, the affected population - promptly removed from the facility (depopulate) - or, quarantined to prevent further transmission to non-affected areas - Possible to let an MHV outbreak burn it self out - If the closed, non-breeding population completely recovers & is no longer shedding the virus, it should be safe to lift the quarantine - Cesarean rederivation - also useful
Mouse hepatitis virus (MHV) Effects on research MHV infection - shown to confound tumor-growth studies - induce α-fetoprotein production, anemia, thrombocytopenia, leukopenia - activate NK cells, alter interferon responses, IgM & IgG responses to sheep RBCs - increase an animal's susceptibility to other viral infections by interfering with IL-2 secretion
Mycoplasma pulmonis Basics - cause of murine respiratory mycoplasmosis - parasites surface of respiratory epithelium - initially colonizes nasal passages & middle ears - natural hosts - rats, & mice (less common) - transmitted directly or via aerosol from dam to sucklings
Mycoplasma pulmonis Clinical signs - begin in the upper airways, with variable progression to bronchi & lungs - usually subclinical, but, if advanced disease weight loss, nasal discharge, increased respiratory sounds, dyspnea, head tilt
Mycoplasma pulmonis Gross findings - Usually none - Exudate in middle ears - Dilation of airways or Bronchiectasis In extreme cases bronchial abscesses Bronchiectasis & bronchial abscesses (rat)
Mycoplasma pulmonis Control - primarily by cesarean derivation & barrier maintenance - but, can be vertically transmitted, not seroconvert until several months old in rats - So, intensive monitoring for long periods - required
Mycoplasma pulmonis Effects on research - altered respiratory function & carcinogenesis, - contaminate cell lines & transplantable tumors in or from mice - cause arthritis in mice injected with contaminated cells
Syphacia spp. Basics - Mice: Syphacia obvelata Rat: S. muris - Life cycle - completed in 11-15 days - Eggs - deposited on the perianal region Embryonate Become infective in 5-24 hrs - Embryonated eggs - ingested from perianal region by grooming, or on contaminated materials from the environment
Syphacia spp. Adult pinworms - Shows adult pinworms found in the cecum & large intestine of a mouse - Young mice (3-4 wks old) - more susceptible
Syphacia spp. Diagnosis - eggs - a distinctive banana shape - by demonstrating eggs in perianal area using cellophane tape test by finding adult worms in cecum & large intestine during necropsy - Fecal examination - not a reliable method Research complications - host immune responses 혼란 & 장애
Syphacia spp.
Aspiculuris tetraptera Egg type: ellipsoid Eggs NOT detected with cellophane tape test Adults - residing in the proximal colon of the mouse Easily distinguished from Syphacia spp. by its oval esophageal bulb Do not migrate to deposit eggs on the perianal area Eggs detected antemortem by fecal flotation & by direct smear examin. of feces
Murine Norovirus (MNV) Basics - In 2003, first reported by Karst et al (Science 299:1575-1578) isolated from immune deficient mice (RAG/STAT-/-) - non-enveloped, RNA virus, family Caliciviridae - Spread by fecal-oral transmission 외부환경에서수주간감염력지속전파속도매우빠름 - Isotypes MNV-1 MNV-2, -3, -4: 보고 (56 th AALAS meeting, 2005)
Murine Norovirus (MNV) Susceptibility & Research complications - Lethal infection in mice deficient in innate immunity STAT1-/- and IFNαβγR-/- mice RAG/STAT-/- mice encephalitis, pneumonia, meningitis, hepatitis - In immunocompetent mice (MNV-1) no clinical signs or tissue pathology transient infection - In mice deficient in T & B lymphocytes (MNV-1) persistent infection but no clinical signs
Murine Norovirus (MNV) MNV infections are prevalent in research mice MNV-1 - At 5 wks, fecal PCR negative but, spleen, jejunum, mesenteric lymph node (MLN) positive MNV-2, -3, -4 cause persistent infections for at least 12 wks - Persistently shed in feces - Persist in tissues MLN > jejunum > spleen MNV is efficiently transferred to sentinel mice by exposure to dirty bedding ( 항체형성시기 : 5-6wks) MNV-infected mice can be detected - Serology, RT-PCR (feces or MLN)
Murine Norovirus (MNV) Response - Many facilities (22.1% in USA) - contaminated with MNV-1 (vs 2.5% for mouse parvovirus, 2 nd rank) - to control or eliminate the virus - 1:10 dilution of bleach, autoclaving : disinfectant to kill the virus - 4급암모늄 : ineffective - Rederivation - an effective means for exclusion of MNV-1 Norovirus AALAS2006
토끼 (Rabbit)
질병관리 Signs of Disease - Snuffles - Head tilt - Diarrhea - Hair loss - Ear mite
질병관리 Signs of Disease - Failure to move about - Failure to eat; weight loss - Cloudy or red eye - Rough hair coat - Ulcerative legions, swellings - Bloody discharge - Infertility or stillborn pups
피부증상 1) 탈모증 : 둥지를만들거나영양불량, 외부기생충감염 ( 얼굴, 등 ) 2) 피부및피하부종 : Pasteurella multocida 3) 피부병 : ear mite, fur mite
Ear mite
Fur mite - Usually NO clinical disease But, severe infections hair loss, oily scale
호흡기증상 (by Pasteurella multocida) - nasal discharge, dyspnea
Mandibular prognathism (or malocclusion) - Inherited condition
소화기증상 - 세균성설사, 식욕부진
Hair mat
안구증상 : 결막염 (Pasteurella multocida) 사산 신경근육이상 : 사경 (Pasteurella multocida 에의한내이염으로발생 )
개 (Dog)
질병관리 피부증상 : 탈모, 비듬, 발진, itching 원인 - 세균, 진균, 기생충, 내분비장애등 소화기증상 : 설사, 구토, 식욕부진원인 - 소화불량, 세균, 바이러스등 호흡기증상 : nasal discharge, cough, dyspnea, tachypnea 호흡기병 - 전염성기관지염, 폐렴, 기관허탈, 심장사상충, 심장병등 안구증상 : ocular discharge, 각 결막염, corneal opacity 원인 - 외상, 바이러스등 귀증상 : 부종, 귀지, 비듬, itching 등원인 - 외상, 기생충, 세균등
Basic Examination of Incoming Animals Examining hair coat - exam for general appearance, ectoparasites such as fleas, areas of hair loss
Basic Examination of Incoming Animals Ear examination - exam for abnormalities(secretions or odors) - eyes, anus, nostrils, penis, or vagina - also exam for abnormalities
Basic Examination of Incoming Animals Oral examination - Teeth & gums - exam for any atypical conditions
Basic Examination of Incoming Animals Auscultation - to auscultate heart & lungs for abnormal sounds Body temperature - measured
돼지 (Pig)
질병관리 임상증상 : 머리와귀가쳐져있고, 호흡곤란, 설사, 파행등 주요질병 - 전신성질병 : 돼지콜레라, 돈단독, 오제스키등 - 호흡기병 : 1 바이러스성 : Influenza 2 세균성 : 위축성비염, 유행성폐렴, 흉막폐렴등 - 설사병 : 자돈에서문제시 ( 바이러스성, 세균성, 기생충성 ) - 기생충성질병 : 옴, 콕시디움증
악성고열증 (malignant hyperthermia) 돼지는심한스트레스로인하여폐사가능 Malignant hyperthermia = Porcine stress syndrome 증후 : 빈호흡, 근육긴장, 빈맥, 피부의청색증, 체온의급격한상승 (42-45 ) 유발원인 : 부적절한보정, 과도한운동, 특정약제의투여 ( 특히 halothane, succinylcholine, chloroform) 예방 : 증후를유발시키는특정약제, 거친보정, 과도한운동을하지않도록주의
구제역 ( 口蹄疫, Foot-and-mouth disease, FMD) 악성전염병 본래는소에서볼수있는전염성질병 돼지, 양, 사슴, 코끼리등과같이발굽이둘로갈라진동물 ( 우제류, 偶蹄類, cloven-hoofed animals) 에서도발생 증상 : 체온의급격한상승입 혀 발굽 젖꼭지등에물집 ( 수포 ) 식욕이떨어져심하게앓거나폐사
... 농장법안은동물의인도적인처리에관한법률을포함한다. 이법안의주된요점은동물들이실험과시험에사용됨으로써겪는고통과괴로움을최소화하는것이다. 이렇게함으로써, 생물의학적연구는정확성과윤리성이높아질것이다. 우리는실험동물에게많은것을빚지고있으며, 그빚은좋은대우와고통스런실험을최소화함으로써갚을수있다. Robert Dole 상원의원 1985 년 12 월 17 일하원에서동물복지법에관한 1985 수정안이어떤것인지를설명하면서. ( 미연방의회의사록 )