aahll aeRs als ! ||
`
`||
`
`may 2a 1990
`
`|ost
`
`UNIV. CHICAGO EX. 2070
`
`SS)
`ee
`Genome & Co. v. Univ. of Chicago
`N]/PGR2019-00002
`
`

`

`“Experimental Animals”
`EDITORIAL BOARD
`
`Shigeru SUGANO
`
`Editor-in-Chief, The University of Tokyo, Tokyo
`
`Jiro ADACHI
`
`Kunio DOI
`
`Chugai Pharmaceutical Co. LTD, Tokyo
`
`The University of Tokyo, Tokyo
`
`Tsuyoshi FURUYA
`
`National Institute of Hygienic Sciences, Tokyo
`
`Kazuyoshi MAEJIMA—Keio University, Tokyo
`
`Masaro NAKAGAWA_National Institute of Health, Tokyo
`
`Katsumoto UEDA
`
`The Institute of Public Health, Tokyo
`
`Junzo YAMADA
`
`Kyoto University, Kyoto
`
`Editorial Office | Department of Veterinary Medical Science, Faculty of
`Agriculture, The University of Tokvo, 1-1-1 VYaver Bunkyo-ku
`Tokvo 113. Japan
`
`
`
`“RM” i Re A
`
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`
`ME
`
`(BBL) WOESARE 1TLis
`
`£ if = rfWU DDH PEGE SET DeRy te
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`FR AOC Ade =i) dO Rea BREA)
`(03) 812-2111
`Fes. 439]
`
`

`

`EXPERIMENTAL ANIMALS Vol. 39,No. 2
`April 1990 ——
`
`Review:
`The Histery of the “dewtsche Maus" the Origin of the dd Mouse Group.
`Tanaka. S and Matsuzawa, A, ct bet neenn center ene neetnenews ae taetaugegtaegeeaeetereenseneeegsneeeereenauas 141
`
`Symposium ¢
`Role of Nonhuman Primates in Aging Research :
`Oshima, K., Terao, K., Kanazawa, L., lwai, E., ltoigawa, N.,
`Bowden, D. M., Honjo, S., and Yoshikawa, Yo ‘+++ HEE Senet Sets ea ES rien 15
`
`Originals :
`Pentatrichamonas hominis from Beagle Dogs — Detection Method, Characteristics and
`Route of Infection — :
`Fukushima, T., Mochizuki, K., Yamazaki, H., Watanabe, Y., Yamada, 5.,
`Aoyama, T., Sakurai, Y., Mori, H., and Nakazawa, Mo cette stresses ee ese ces cee bhseaneatna 187
`Changes in Progesterone Levels in Precnanty Block Caused by the
`Proximity of a Strange Male and by the Injectionof PMSG::
`Furudate, &., Yoshida, O., and Nakano, T.
`Evaluation of Separating X-and Y-Sperms by Percoll tabaity Gradient Centrifugation
`Using Sperms of Transgenic Mice Carrying a Transgene on Y-Chramosome !
`Toio. H.
`and Kubo, M.
`Sensitivity of Spontaneously Epileptic Rats to External Stimuli That Induce Seizures
`Asano, Y.. Okaniwa, A., Serikawa, T., and Yamada, J
`An Evaluation Method for Hematological and Clinice biochemical Values
`in Aged F 344 Rats on Chronic Toxicity Tests such as Long-term Inhalation
`Studies on the Effects of Diesel Kxhaust
`Maejima, K, and Nagase, 8.
`--trtssrc
`cert errr
`Virucidal Effect of Ozone Treatment of Dabedelbnty Amma! Viruses
`Sato, H., Watanabe, Y., and Miyata, H.
`Diurnal Rhythms of Body Temperature, Heart Rate, and Behaviour on
`Adult Shiba (ioats
`Matsui, K.
`Spontaneous Lesions in engl Dias thas4in Toxicity Stdas
`Morishima, H., Nonoyama, T., Sasaki, 5., and Miyajine, HH,
`HVJ Infection in Alveolar Macrophages of Various Laboratory Animals-
`Watanabe, Y., Miyata. F., and Satin, Hi.
`te<te-seesvasenetessspednnnttivn catisicniien.
`Blastogenic Response in the Rat Lymphocyte Using Glucose Consumption Test:
`Ikarashi, 1., Sunouchi, N,, Toyohara, 8., and Tauchi, K. +
`Comparison of the Intestinal Bacteria in Specific Pathogen
`Free Mice from Different Breeders :
`Hirayama, K., Endo, K,, Kawamura, S., and Mitsuoka, Ty tests-tesseereereeeeeeee eter entree er ees 203
`
`198
`
`(99
`
`207
`
`2\4
`
`22a
`
`the Stanehs ined
`
`23
`239
`
` uuebis
`
`249
`
`‘255
`
`Notes :
`Genetic Profile of Alloxan-induced Diabetes-susceptible
`Mice (ALS) and-resistant Mice (ALR) :
`and Ino, T
`Sekiguchi, F., Ishibashi, K., Katoh, H., Kawamoto, Y.
`Studies on Hepatocystis sp.
`in Rhesus Monkevs (rom Yunnan, | hina
`Takenaka, T., Bastiencth K.. Gaton, H., Matsumoto, 5
`and Nishikawa. T. ©:
`pu
`Aenes
`sh
`gin
`Composition of Milk and Blastiaghuvenie Analysis of Milk
`Casein in Herbivorous Voles (Microlus moniebedls)
`Sugawara, M., Nakamura, T., Ohizumi, T..
`and Oki. ¥.
`wes
`Peripheral Neuropathy Associated with Osteosarcoma ina: debe nese Monkey
`Yasuda. H.. Taniguchi. Y.. and Shigeta.
`Yo
`-+---+
`
`?69
`
`ert
`
`28
`
`285
`
`

`

`Diagnosis of Abortion and Fetal Death by the Ultrasonographical
`Device in Cynomolgus Monkeys under Indoor Individually-caged Conditions :
`
`Kohno, M., Suzuki, M. T., Ono, T., Ogawa, H., and Cho, Bl oveecerrsssrtstesssetsseerseesesscess 29]
`Breeding of Cataract Rat Strain (ICRF/Kmu//Yg) :
`
`Kato, S., Ohno, K., and Ihara, Ny cececcersescesssssentecetevtosnstasnvessssersssunssesseserareessnanigs 295
`Cryopreservation of Mouse Strains by Ultrarapid Freezing :
`Nakagata, N. Dee ea eR E eRe Tene eee E PEN CORT N TET EEE Petre Teena eee HEE EHS E OSES SSE SSSEHES ESS S EMER 299
`Cryopreservation of Unfertilized Mouse Oocytes from Inbred
`Strains by Ultrarapid Freezing :
`Nakagata, N, Tr eTIVerrererrircreriT Ter rey err rere rT re reT ee ee ee eetr 305
`Polymorphisms Detected in Actin-related Sequences of Rats (Rattus norvegicus) +
`Kunieda, T., Ikadai, H., Matsui, M., Nomura, N., Ishizaki, R
`and Imamichi,ee 30
`Miscellanea :
`JALAS News tala dich arse estab shea elenlnld ng dedctaba lal rete mia erlom Rae EE aap peas RaiRtd a oe ete See aie die tee MAT Rade RE LAO Beane 3L
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`289
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`70 + SERRESBHIES (ALS) ts LORIEHER (ALR) 9 20i877 9 Te:
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`- 291
`HY Bh + MACHA * APSEIG +UPS + Re estereecetnesneneencccneSEND ootesatins
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`HWE:(PIE de SP HAEFy b ICRF) Kmu/ ‘Yamti)im
`295
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`
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`
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`

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`MDF tH -cnrivivervennnerchibes verses seysisviatupin en tutes iuadaas baad gupbosacat Wlicuans Veet qiadeaommtislanie units 31
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`
`

`

`
`This material may be protected byCopyrightlaw(Title 17 U.S.Code}
`
`
`
`
`Avp Anon 92) Bee
`
`a0
`
`Comparison of the Intestinal Bacteria in Specific Pathogen
`Free Mice from Different Breeders
`
`Kazuhiro HIRAYAMA‘, Kimiko ENDO**, Seiji KAWAMURA***,
`and Tomotari MITSUOKA*, **, ***
`
`“Department of Veterinary Public Health, Faculty of Agricullure, The University of Tokyo,
`Bunkyo-ku, Tokyo 1138, ** Laboratory for Intestinal Flora, Frontier Researeh Program,
`RIKEN, Wako-shi, Sattame 351, and*** Department of Biomedical
`Setence, Faculiy of Agriculture, The Universiiy
`of Tokyo, Bunkyo-ku, Tokyo 113, Japan,
`
`(Received 7 November i989/ Accepted 15 December 1989)
`
`laboratory animal
`Specific pathogen free BALB/c mice fram 3 commercial
`breeders in Japan were compared on the composition of caecal flora revealed by
`selective and nonselective cultivation as well as direet microscopical observation on
`smears, andrelative caecal weight, Large differences were détected in viable counts
`of total bacteria and almost all bacterial groups, while direct microscopical counts
`which consisted mainly of fusifrom bacteria were almost equal,
`resulting in
`diverse recovery rates among 3 breeders.
`Fubaelerium and spiral
`shaped
`organisms were not detected from all breeders There also existed variations
`within breeders, especially those in the number of Enterobacteriaceae. Relative
`caecal weight also differed among breeders, suggesting the participation of variety
`of eaecal bacteria in determining this parameter, As
`these variations
`in
`bacteriological status of animals can influence experimental results. standardiza-
`tion of intestinal microbial flora is requ red.
`
`differences
`some
`are
`there
`Although
`(SPF)
`and
`free
`between specific pathogen
`conventional animals in physiolgy (2°. haema
`tology [4],
`lifespan and diseases | 3] and sus-
`ceptibility to pathogenic bacteria 8),
`SPF
`animalé
`are
`chosen
`in many
`studies
`as
`experimental animals.
`It
`is mainly because
`SPF animals
`are
`free
`of
`common
`and
`troublesome
`diseases
`such
`as
`infantile
`diarrhoea, ectramelia and chronic respiratory
`diseases, etc. However, SPF animals have been
`defined only as’ animals that are free of
`specified micro-organisms and parasites, but
`not necessarily free of the others not specified’
`[7], and
`therefore
`it
`is possible that
`the
`microbiological status of SPF animals differs
`from colony to colony.
`ined and ec mpared
`in this study, we deters
`the composition of
`intestinal
`flora 1 wiace
`from SPF colonies of 3 commercial breeders
`using selective and non-selective culture media,
`We
`alsa
`performed
`direcL muroscopicai
`observation on smears to enumerate fusifrom
`
`haeteria which are fastidious to cultivate but
`are
`considered
`as
`the most
`predominant
`bacteria [5],
`
`Materials and Methods
`
`Animals + SPF male BALB/e mice were
`purchased al 7 weeks of age from 3 commercial
`breeders (A, B, C)
`in the Tokyo area, Japan.
`They were kept
`in cages containing wood
`shavings autoclaved at 121°C for 30 min. and
`were given NMFdiet (Oriental Yeast Co, Ltd.,
`Tokyo) and tap water ad libitum for 1 week to
`eliminate influences of
`transportation and
`diet, The cages were placed in a laminar flow
`rack (LFR-A. 2. 'Tokiwa,
`‘Tokyo!
`in a
`raom
`under controlled lighting irom& 00a. m, to8
`uup. m. at 24+ 1 C with
`the
`relalive
`humidity of 552 5%. Seven mice from each
`breeder were used in one examination and the
`examinations were repeated twice in different
`periods.
`Bacteriological culture technique . Bacte-
`
`

`

`264
`
`riological procedures were essentially the same
`as described previously [13,14]. After mice
`were killed by cervical dislocation or carbon
`dioxide, caecal contents were collected, and
`homogenized in 20-fold anaerobic diluent (10°!
`dilution)
`and serial 10-fold dilutions were
`prepared with the same diluent. Amounts of
`0.05 ml of diluted samples were spread on the
`surfaces of selective and non-selective culture
`media. The media and cultural conditions used
`were summarized in Table 1. ES agar [9] was
`used instead of CS agar.
`Direct microscopical observation ‘| The
`10°" dilutions of caecal contents were smeared
`in 1 * 1-em square on slides and stained by
`Gram's method, Bacterial cells and spores
`were counted microscopically and the numbers
`of bacteria in the original
`samples were
`calculated, Recovery rates were designated as
`percentages of viable bacteria against direct
`microscopical count.
`Relative caecal weight(RCW) : Caeca were
`removed and weighed with their contents in
`the second examination, The weight was
`expressed as
`a percentage of
`total
`body
`weight.
`Student’s t-test was
`Statistical analysis :
`to determine
`used
`significant
`differences
`between breeders.
`
`Results
`
`the
`As there was no significant difference,
`data obtained from the 2 examinations were
`presented together in Tables 2 and 3. Results
`of statistical analysis were summarized in
`Table 4.
`counts
`in viable
`Apparent differences
`among 3 breeders were observed for almost all
`bacterial
`groups.
`The
`viable
`counts
`of
`Bacteroidaceae
`and
`Lactebacillus,
`the
`2
`predominant groups, were the highest in breeder
`A (10.07 and 9.10 logis count per gram caecal
`content, respectively), As a result, the number
`of total viable bacteria in breeder A (10.12)
`was higher
`than in the other 2 breeders (9.54
`and 9.37). On the other hand, direct micro-
`scopical counts of 3 breeders which consisted
`mainly of fusifrom bacteria had almost no
`variation (10.97 to 11.01).
`In consequence,
`mice from breeder A showed caecal flora with
`relatively high recovery rate compared with
`mice from the other 2 breeders.
`Hubacterium was only detected in 6 mice
`from breeder B.
`In
`direct microscopical
`examinations, spiral shaped organisms, which
`could not be cultivated on any media used,
`were observed in all mice from breeders B and
`C but were not in mice from breeder A.
`
`Culture media and conditions for incubation
`Table |.
`
`Medium
`
`EG agar
`BL agar
`NBGTagar
`BS agar
`ES agar
`VS agar
`NWN agar
`LBS agar
`
`TS agar
`DHL agar
`‘TATAC agar
`PEES agar
`PD agar
`
`Organisms usually
`
`enumerated Incubation method
`
`Anaerobes
`Anaerobes
`Bacteroidaceae
`Bifidobactertian
`Fubactertion
`Verllonella
`Clostridian
`Lactobactles
`
`Steel wool method
`37°C, 48h
`
`Aerobes
`Enterobacteriaceae
`Streplococens®
`Staphytococens
`Yeasts, Molds
`
`Aerobic
`37°C, 24-48h
`
`at Including Eyleracacces
`
`

`

`Table ¢.
`Composition at caecai Lora of SPF mice fram 3
`breeders
`=on=(4)
`
`abo
`
`breedér A
`W.12
`2 W.20"
`0.28
`G37 & 0,28
`9.p4
`Wio7 4
`Bacteroidaceae
`9.19 + O87
`O.47 + 0.40
`nh.
`Pubactertian
`NS. 0.
`1.78 2 0,69"
`Lactobacillus
`9.10 +
`4 22
`B.5G 2 0.27
`$.83 4+ O17
`
`
`Enlerobacteriacene 3.46 +|)AY
`4.fli
`&
`1,58!"
`173 £ 0.49
`Streplocaccis
`a.19 +
`0 Sl
`417 + 0.70
`5.08 2 0.49
`4.97 +
`(a0
`5.4)
`© 0.40
`Staphylocacers
`5.28 + 04
`
`breeder 1
`
`breeder C
`
`Bacterial groups
`Total bacteria
`
`DL
`
`ai Mean = 5. D. of logio count of viable bacteria per gram caecal content
`: Detected from 6 mice
`di De-
`when present
`b Nat detected
`tected from tb mice
`
`Table i
`Results of direct micrascopicai exmination (n=l) and relative
`caecal weight (n = 7) of SPF mice from3 breedersee
`
`breeder A
`breeder B
`breeder C
`
`Direet count
`Recovery rate (a)
`Spiral) shaped
`VSEAUNSINS
`Relative caecal
`weight (5)
`
`05
`ii,
`498
`13,96
`NW. Li
`
`0 04
`11 oo +
`iW + 2.48
`9 82 2013
`
`1,07 + 1 1
`2.93 + 1,46
`QAO +
`() 21
`
`249 + DIA
`
`2.08 +
`
`0.21
`
`goad
`
`alo
`
`a bi Refer to footnotes in Table 2
`
`
`
`Tabled Resuits of statistical analysis
`AB
`A
`B
`
`vaetersa
`Tota
`Bacternidaceae
`Enhaectortin
`Leactobactilus
`Enlerobacteriaceae
`Afhyplacaeces
`Staplivlncoccus
`Direct count
`Reenvery rate (%)
`Spiral shaped
`organisms
`Relative caecal
`weight ("5
`
`ae ee
`ak ok
`EE
`* ke
`*
`oe th ok
`
`He oR ak
`ok eo
`
`ee
`
`RH
`Kk
`
`Ak ok
`hak
`
`#
`xe
`oH
`
`oh Kk
`de
`
`eo ok
`ok a a
`
`+k
`
`$e pO),
`
`ee pool,
`
`#
`
`peo OA
`
`among
`the variations
`addition to
`inc
`breeders.
`relatively large variations we{hin
`breeders were shown
`for viable counts of
`faculLative anaerobic bacterial groups
`‘hal
`is,
`Enterobacteriaceae.
`Streplucoccus
`and
`Stapivlacaceus. Three mice from breeder B
`did net harbor detectable number of Enterobac-
`
`Leriaceae,
`PB
`RCW was low in mice from breeder
`compared with mice from the other 2 breeders
`
`Discussion
`
`SPF animals are considered to be suitable
`for almost all disciplines and likely to become
`standard laboratory grades [17]
`However,
`microbiological
`standardization
`of
`SPF
`animals is not fully achieved. O'Rourke ef al.
`[15]
`pointed out
`large differences in the
`numbers
`of
`facultative
`anaerobic Gram-
`negative bacteria in the gastrointestinal
`tract
`of mice from 3 major SPF units in Australia.
`In the present study, we revealed more extensive
`differences in viable counts ef anaerobic and
`facultative
`anaerobic
`bacteria
`direct
`micrascopiea| observations and RCW among
`mice from 8 Jaboratorv animal breeders. We
`also observed variations
`in gastrointestinal
`microflora of mice within breeders. especialy
`those in the number of Enterobacteriaceae of
`moce from breeder B in which some mice with
`a «detectable Enterobacteriaceae
`existed
`
`

`

`References
`
`266
`
`Compared with the other breeders, mice fram
`breeder B showed low RCW and low counts of
`Enterobacteriaceae, both of which are impo-
`rtant parameters to assess
`‘normalization’.
`It was
`suggested
`from experiments with
`germfree mice receiving pure cultures from
`"nhormal’ mouse intestine that ‘normalization’
`of germfree anismals requires a large variety
`of microorganims [9,12].
`In this study,
`the
`caecal flora of mice from breeder B contained
`both Kubacterium and spiral shaped organisms,
`and seemed to be more complicated than those
`of mice from the other breeders as judged by
`colony morphology on primary cultures, All
`these
`factors
`could
`contribute
`to rather
`‘normalized’ state of mice from breeder B.
`These differences were probably dependent
`on how the animals were established.
`In
`practice SPF animals are established from
`hysterectomy derived or germfree animals by
`allowing them to acquire microflora naturally
`contaminated in a barrier environment | 16),
`by association with pure cultures of micro-
`organisms (6|, by infecting them with diluted
`culture of intestinal contents [11], or by as-
`sociation with SPF microflora obtained from
`other animal
`species (10). SPF animals are
`also established by selective decontamination
`with antibiotics L18].
`of
`standardization
`Although
`complete
`microbial flora is not an absolute requirement
`in some fields of biomedical research, animals
`with a defined microflora are needed in fields
`of research concerned with oncology, dental
`research, microbiology,
`nutrition and
`im-
`munology. Experimental results can possibly be
`influenced by variation in pastrointestinal
`microflora of mice employed, e. ¢., survival of
`mice following total body irradiation varied
`among mice from different SPF units [15].
`Moreover, it is considered that composition of
`microbial flora could play a key role in the
`development of both humoral and cellular
`immunity of the hosts [11] .
`In the establishment of SPF animals,
`microbial standardization is
`required along
`with genetic and environmental standardiza-
`tion, On the other hand,
`it
`is necessary for
`researchers to know the microbiological status
`of each experimental animal available and to
`choose suitable grade of microbiological status
`for their needs.
`
`(2)
`
`[3]
`
`La]
`
`[6]
`
`t7]
`
`[8]
`
`(10)
`
`til]
`
`[12]
`
`(13)
`
`Chay
`
`116)
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Animals
`London,
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`It. A., Stadhouders, A. M., de Boer, H., an
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`‘normal’ mice, Leb. Anim, 18, 188-194.
`Koopman, J. P., Prins,
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`Welling G. W., Kennis, H. M., and Heetors, M. P.
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`Namba, K.
`(1976). Bie Faekalflora bei Menschen,
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`

`

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`"18])
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`
`267
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