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·WORLD HEALTH ORGANIZATION
MONOGRAPH SERIES
No. 23
LABORATORY TECHNIQUES
IN RABIES

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·LABORATORY TECHNIQUES
IN
RABIES
CONTRIBUTORS
D L'ANTONA – P. ATAN ISIU - R. BEQUIGNON - E, FALCHETTI
Karl HABEL - George A. HOTTLE - Harald N JOHNSON - Martin M. KAPLAN
A KOVIAROV - Hilary KOPROWSKI - Pectre LEPINE
Thomas F. SELLERS - Emest S. TIERKEL - C. VIALAT
WORLD HEALTH ORGANIZATION
PALAIS DES NATIONS
GENEVA
1954

·A French edition of this monograph is in preparation.
NOTE
Authors alone are responsible for views
expressed in the lfonograph Series of the
World Ilealth Organization
The mention of manufacturers' products does nol imply that they are endorsed or
recommended by the World Health Organization in preference to others of a similar
nature whkh are not mentioned
FINID N SWIZILAND

·CONTRIBUTORS D d'ANTONA Durector, Istituto Sicroterapico e Vaccinogeno Toscano, Sicna, Italy P ATANASIU Institut Pasteur, Paris, France * R BÉQLIGNON Chief of Laboratory. Virus Section, Institut Pasteur, Pans, France E FALCHETTI Istituto Sueroterapico e Vaccinogeno Toscano, Siena, Italy Karl HABEL, A B, MD Chier, Laboratory of Infectious Diseases, National Microbiological Instituc, Public Health Service, Bethesda, MD, USA George A HOTTLE, Ph D. Chic, Biologics Control Section, Laboratory of Biologics Control. National Microbiological Institute, Public Health Service, Bcthesda, MD, USA Harald N JOHNSON, MD International Health Division, Rockefeller Institute for Medical Research, New York, NY, USA Martin M KAPLAN, VMD, MPH Chief Veterinary Public Health Officer, Dission of Communicable Diseases Services, World Health Organization, Geneva, Switzerland A KOMAROV, MSc. V.MD Director, Government Virus Diseases Laboratory, Haifa, Israel Huary KOPROWSKI, MD Assistant Director, Viral and Rickettsial Research, Lederle Laboratories Division, American Cyanamid Company, Pearl River, NY, USA Pierre LEPINE Chief, Virus Section, Institut Pasteur, Paris, France Thomas F SELLERS, M.Sc, MD Director, Georgia Department of Public Health, Atlanta, Ga, USA Ernest S TIERKEL, VMD, MPH Director, Rabies Control Activities, Communicable Disease Center, Public Health Service, US Department of Health, Education and Welfare, Atlanta, Ga, USA C VIALAT Rabies Section, Institut Pasteur, Paris, France

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·CONTENTS Pare Foreword 9 PART 1. LABORATORY DIAGNOSIS IS Section 1. Shipment of specimens, and techniques for prepara. non of animal tissues for laboratory diagnosis Seeron 2 Rapid microscopxal examination for Negri bodies using simple tissue-apphcation techniques, and preparanon of specimens for biological test Section 3. Histopathological diagnosts Section 4. Mouse inoculation test. Section 5 Serum-virus neutralization test 24 56 69 PART II. METHODS OF VACCINE PRODUCTION 77 Section 6. Phenolized vaccine. Semple type A Sheep-brain vaccine method of Institut Pasteur, Paris & Rabbit-brain vaccine method recommeaded to meet requirements of United States National Institutes of Health Section 7 Ultraviolet-light-irradiated vaccine Section 8 Chicken-embryo vaccine 85 92 99 PART 1 VACCINE POTENCY TESTS . 107 Section 9 General considerations Section 10 Some factors influencing the standardization of sabies- Vaccine potency tests in mice Section 11. Habel test fos potency Section 12 Modified Habe) test for potency 109 112 116

·8
CONTEXTS
PICC
PART III, VACCINE POTENCY TESTS (continued)
117
Section 13. Potency-test requirements of United States National
Institutes of Health (NIH)
Section 14. A potency test in rabbits ...
Section 15. Potency test for chicken-embryo vaccine.
125
128
PART IV. PRODUCTIOY or typTRIMMUNE SERUM
135
Section 16. Method used at Institut Pasteur, Paris
Section 17. Method used at Istituto Sicroterapico e Vaccinogeno
Toscano, Siena. ....
Section 18. Potency test of antirabies serum
137
139
PART V. LABORATORY ANIMALS
Section 19. Breeding and care of laboratory animals,
143
ADDITIONAL RETTRIACE SOURCES

·FOREWORD The World Health Organization has in the past received many requests for information on laboratory techniques connected with various aspects of rabies. As a result of the need for this kind of information in countries of the Eastern Mediterranean, South-East Asia, and Western Pacific Regions of WHO, a meeting was organized for these countries in July 1952 at the Pasteur Institute of Southern India in Coonoor. The meeting provided for lectures, discussions, demonstrations, and laboratory training, and was attended by 55 medical and seterinary officials, including 7 discussion leaders, from 23 different countries WHO consultants on rabies acted as discussion leaders and supervised the laboratory sessions. The working papers, in particular the laboratory instructions prepared by well-known authorities, proved most useful, and it was decided to expand and revise the material for publication The WHO Expert Committee on Rabics, at its second session in Rome in September 1953, discussed the projected manual in detail and made frequent references to it in their report The reports of the committee deal in general with the overall problems encountered in rabies, including prophylaxis in human beings and control in animals, whereas this publication is concerned solely with labo. ratory aspects of the disease The manual is not intended as an exhaustive treatise , its scope has been purposely limited to one or two procedures in each of the major divisions of laboratory techniques in rabies The contnbutors were requested to select and present procedures based on their own esperience which would be dependable and practicable without sacrificing necessary minimal standards, but which at the same time could be adapted to the limited facthties and personnel of many rabses laboratories in different parts of the world The techniques were selected also with a view to encouraging and facilitating unform methods which would permit of a more valid comparison results obtained in different laboratories Certain features of the various sections equire some explanation. • Bid Hith Org techn Rep Ser 1950. 28 1933 82 ·10 FOREWORD Detection of Negri bodies by rapid techniques is the aim of every diagnostic laboratory dealing with rabies, and a choice must be made from a multiplicity of methods described by different authorities. It is believed that the impression method presented here can he mastered relatively easily by most laboratory technicians. Where histopathological sections are concerned, morc-specialized training in pathology is required and this part of the manual has been arranged for such trained individuals, In the laboratory diagnosis of rabies, cramination is frequently limited to the tissues of the central nervous system, and the salivary glands arc entirely overlooked. It has been aptly stated that "animals do not bitc with their brains"-the real risk of contracting rabies is dependent on whether or not virus is present in the salna of a biting animal. However, salivary excretion of virus does not occur in an appreciable but unpredictable number of cases where virus can be recovered or detected in the brain of the animal. Examination of the submaxıllary salivary gland for the presence of virus by mouse inoculation should therefore be carried out wherever possible concurrently with nervous-tissue examination. The necessity for mouse inoculation tests in routinc diagnosis is worthy of special emphasis. Careful studies have shown that up to 20% of animals negative on examination for Negri bodies were positive for rabies by the mouse inoculation test. A description of the preparation of the Semple phenolized type of vaccine, as representative of the most widely used of the "hilled" vaccines, is given. This does not imply that other inactivated or living virus vaccines such as the lempi-, Hoges-, Fermi-, or Harris-type vaccines, to name a few, are not equally effectne, provided they are adequately tested for potency Two methods for producing the Semple-type vaccinc arc gasen: onc according to the procedure used at the Institut Pasteur, Paris, and the other to meet the requirements of the National Institutes of Health of the United States of America These methods were chosen because they cover the largest number of requests for information recived by WHO. It is recognized that modifications of these methods are used successfully in many countries. Descriptions of the production of ultraviolet-light inactivated vaccine and modificd virus vaccine prepared from chicken embryos arc included because of wide interest in these effective and relatively new products. Commercial or large-scale preparation of potent tables vaccines from which the paralysis-producing factor has been remo cd has not, as yet, ·FOREWORD been successfully accomplished, and this procedure is therefore omitted from the manual. The necessity for performing rolency fests on cach batch of vaccine cannot be too strongly emphasized, far ample cenerience has shown that even when routine procedures are closely followed in the preparation of successive batcbes of vaccine there is no automatic assurance of a potent product. Potency tests in laboratory animals provide our only basis, ar presens, for any degree of certainty that a vaccine possesses sufficient immunogenicity to gre dependable results in human beings or animals. Several potency tests of varying complexity are given so that a laboratory may select, or devise, one most suitable to its local conditions and facilities. In the latter instance, strongest consideration should be given to vaccination followed by challenge with street virus, always with an adequate number of control animals. For this purpose dogs are suitable and can be considered as the animal of choice. The important factor to be demonstrated in control tests of rabies vaccines is immunogenicity and not merely innocuity, a mistake which is not uncommon. Hyperimmune serum us a promising addition to rabies prophylaxis in man and animals; Part IV 15 devoted to a description of its preparation, and or potency tests, Laboratory investigations on hyperimmune serum now under way, co-ordinated by WHO, should give us more information within the next year or two on its prophylactic value. The WHO Expert Committee on Rabies dealt with this subject af its second session (sce footnote on page 9) Pari V is devoted to some of the more important problems associated with the use in rabies work of small laboratory animais, a frequent source of difficulty in many countries. The section is necessarily brief and touches on only the chief aspects of the subject-- in particular, diseases encountered in these animals which might affect experimental results. Further information on this topic, and on material dealt with elsewhere in the manual, may be obtained from the additional reſerence sources given on page 147 Work with viruses is a highly developed discipline which permits of inte legitude of reproducible results are to be obtained. However, it is to be expected that the opinion of individual workers on techniques will difter with respect to details The techniques recommended here have been prepared for particular application in rabies work, although it is evident that some of them, such as the serum-virus neutralization test and the mouse inoculation test, are readily appucable, perhaps with slight ·12 FOREWORD modifications, to other virus diseases. It will be noted further that in describing the various techniques a rational and systematic approach has been stressed by the contributors so that errors which might otherwise nullify excellent work may be avoided. An example which may be cited is the advisability of challenging vaccinated animals before, or alternately with, control animals in determining the potency or effectiveness of a Vaccine. Rabies research is far from static, and it is to be crpected that modifi. cations of some of the procedures described will be evolved in the rather near future. It is felt, however, that the techniques given in this manual should be suitable for several years to come, as they are the result of extensive and proven erperience. WHO is grateful to the contributors for the care and trouble they have taken in this work, much of which was hitherto either unpublished. or not readily available in compact form in scientific literature Many of the contributors to this manual are members of the WHO Expert Advisory Panel on Rabies, and their contributions record only a part of their valuable services to the Organization in striving for greater successes in the control of this dreaded disease

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·LABORATORY DIAGNOSIS frozen brain should remain at room temperature until thaned before vection The following information is desirable when animal heads are received examination the species and breed of the animal, and whether it in contact with other animals, whether the animal died or was killed, if the latter, the means used for destroying it, whether the animal was ned and observed for an appropriate time before death, and, if so, ou long, symptoms of rabies, if any, history of vaccination against * 1 5 > Removal of Animal Brain . precautions, including careful operative technique and protection hands with heavy rubber autopsy-gloves, should be taken against ssibility of infection of persons opening animal heads head should be held firmly on a solid table , proper immobilization effected by grasping it with a lion-jau type of bone-holding forceps IG 3 INITIAL SKIN INCISION ALONG MIDLINE OF DOG'S HEAD

·16
E. S. TIERKEL
FIG. 1. DOUBLE CONTAINER WITH ICE FOR PACKING SPECIMENS
By courtesy of U'mirnd Stares Depariment of Healih. Jucatwn anul 1 allure,
Public Ilealth Serrice. Communicante Discuse Center TL'S DIZ 15-PI/S-CDC)
FIG. 2. CONTAINER COVERED AND LABELLED
Caution
In prctau
contains the head
of a dog suspaelad
of having died of
BABIES
RI) of US DIFH-PKS-COC

·17 LABORATORY DIAGNOSIS the frozen brain should remain at room temperature until thawed before dissection The following information is desirable when animal heads are received for examination: the species and breed of the animal, and whether it was in contact with other animals; whether the animal died or was killed, and, if the latter, the means used for destroying it; whether the animal was confined and observed for an appropriate time before death, and, if so, for how long; symptoms of rabies, if any; history of vaccination against rabies. Removal of Animal Brain All precautions, including careful operative technique and protection of the hands with heavy rubber autopsy-gloves, should be taken against the possibility of infection of persons opening animal heads. The head should be held firmly on a solid table , proper immobilization may be effected by grasping it with a lion-jaw type of bone-holding forceps FIG 3 INITIAL SKIN INCISION ALONG MIDLINE OF DOG'S HEAD By courtesy of US DHEW.PHS CDC

·18
E. S. TIERKEL
FIG. 4. USE OF BONE-SAW FOR MAKING TRANSVERSE ANTERIOR INCISION
IN FRONTAL BONES JUST ABOVE THE EYES
.
By courtesy of US DHER-PHS.CDC
FIG. S. CALVARIA REFLECTED
1
re
P
r. of US DIF.PIIS-CDC

·LABORATORY DIAGNOSIS 19 firmly applied to the maxilla. Improvised mechanical devices such as a carpenter's vice have often also been found effective. With section knife or large scalpel a midlene incision is made through the skin, fasciae, and muscles of the cranium, beginning antenotly just above the level of the eyes and extending posteriorly to the base of the skull (see fig 3) The skin, fasciae, and temporal muscles are then dessected away from the cranium and reflected laterally exposing the bone The calvaria is then removed by means of a saw, bone-chisel, or butcher's cleaver. The sawing method is preferred by most laboratories. In this procedure a sterile surgical bone-saw is used to incise the bone of the skull on each side, beginning at the foramen magnum and then sawing anteriorly to the frontal bones The longitudinal cuts are then joined by a transverse incision through the frontal plate just above the eyes (see fig 4) The calvaria is then lifted off with the aid of bone-cutting forceps and/or bone-chisel (see fig 5) With a fresh set of sterile instruments, the brain is now removed from the cranium. The meninges and the tentorium cerebellt which separates the cerebrum and cerebellum are dissected away with tat-tooth ibumb forceps and either scalpel or sharp-pointed scissors. Next, with scalpel or scissors reaching back into the posterior portion of the brain, the brain is severed from its site by cutting into the medulla, cranial nerves, and anterior extension of the thalamus The entire brain is then lifted out of the cranium onto a paper picnic-plate or a large Petri dish (see fig) FIG. 6 REMOVAL OF ENTIRE BRAIN By courtesy of US DHEW-PHS-CDC ·20 E. S. TIERKEL If the body of the animal is available and no typical Negri bodies are found in the rapidly stained films, make a general post-mortem eramination in an attempt to determine the reason for the illness or unusual behaviour of the animal. Wrap the remains in paper and ensure their incineration. Wash the table with 10% cresol solution. Sponge the gloves in the same kind of solution before removing them from the hands, and then sterilize them, with the instruments, by boiling. It is always advisable to record the gross appearance of the brain, indicating especially the state of presersation, the presence or absence of FIG. 7. SKIN REFLECTED AND SUBCUTANEOUS TISSUE REMOVED. EXPOSING SUBMAXILLARY SALIVARY GLAND Submgertiary salivary oland B Submai l'ary lymph sands adhesions and crudate, and whether the blood-sesseis arc injected. There are no gross pathological changes which can be regarded as diagnostic of rabies. Even in the field, precautions should be taken to present bacterisi contamination of the animal brain by the use of sterile instruments in brain removal and dissection ·Removal of Salivary Glands The examination of the salvary glands for the presence of virus is of obvious value in providing definitive evidence of whether or not a bite has entailed a risk. Presence of the infection in the central nerous system does not necessarily induale infective salata. If virus is present in the salivary glands, the submaxillary (mandibular) glands will contain the most virus for removal of the submarillary salivary glands, the head is turned over so that the ventral aspect is facing upward. FIG & LEFT SUSMAXILLARY SALIVARY GLAND COMPLETELY EXCISED A Submarillary salivary gland & Submaxiliary ylands With autopsy kasfe or scalpel, a midine incision is made in the skin covering the area between the ram of the mandible The incision should begin at the mandibular lp and extend posteriorly to the neck The skin is then reflected laterally, exposing the muscles and superficial soft tissues of the lower jaw ·is desired, smears may first be made from the unpresen cd brain and stained, 22 E. S. TIERKEL The submaxillary salivary gland on each side is situated quite superficially and after reflecting the skin back it can be seen in the area of the posterior border of the mandible behind and below the superficial submaullary lymph glands, with which the salivary glands should not be confused (see fig. 7 and 8). The submaxillary salivary gland is elliptical in shape, about 5 cm long and 3 cm wide; it is rounded in outline, greyish-yellow or orange in colour, and covered by a fibrous capsule. With a fresh sterile scalpel and rat-tooth thumb forceps, the gland 01 each side is dissected away from its surrounding tissues and placed in a Petri dish A small portion of each salivary gland is cut with sterile scissors and pooled in a single mortar for preparation of the salivary-gland suspension for the mouse inoculation test (see section 4, page 57) The test preces should be wcighed before emulsification for calculation of the concentration of the tissue suspension (see page 47 for gasserian ganglion) Glycerinated Specimens When facilities for animal inoculation of Negri-negative or doubtful animal brains suspected of rabies infection are not at hand, it is possible to ship portions of brain or salivary gland to a laboratory which performs animal inoculation tests A solution of sterile 50% glycerol-saline is prepared by adding equal parts of chemically pure glycerol to physiological salt solution. This solution may then be placed in small bottles or jars with screw tops, autoclaved, and stored at room temperature. Select fairly large portions of brain or salivary gland, at least about the size of a mouse brain (0.3 g or more) in order to give the laboratory worker sufficient material with which to work. Portions of the brain should include parts of the hippocampus, cerebellum, and cerebral corter from each side. Also take a portion of the medulla or brain stem. Place the tissue pieces in a bottle of prepared 50% glycerol-saline. Rabies wirus: if present, will be preserved during shipment, and no refrigeration is required Glycerinated portions of brain do not usually produce satisfactory smcars on a slide since it is difficult to make the glycerinated brain adhere to a slide. Furthermore, the staining properties of such tissues are changed making recognition of inclusion bodies much more difficult. For this reason, if further examination of brain smears on the questionable specimen and the slides included in shipment along with the glycerinated brain.

·23 LABORATORY DIAGNOSIS Alternatively, brain smears may be made and the unstained slides plunged immediately into chemically pure methyl alcohol (acetone-free) for firation, and then removed and dried at room temperature without blotting These unstained, fixed smears can then be stained at the laboratory to which they are referred. Upon their receipt in the laboratory, glycerinated tissue specimens should be immediately removed from the shipping bottle and placed in a sterile Petri dish. The pieces of tissue are then thoroughly washed in physiological salıne solution by continual addition of the solution, gentle rotary agitation of the Petri dish, and discarding of the liquid. This is repeated several times At this point it is as well to attempt a smear preparation of the washed brain-tissue specimens on a slide for Negri-body staining and microscopic examination. The quality of the smear will depend on the amount of glycerol washed from the brain tissue. The remaining specimens are now ready for emulsification and mouse inoculation For grinding salivary glands in a mortar a small amount of sterile sand or alundum is essential, but these materials are superfluous for making brain suspensions. Note It is advisable to retain in glycerol solution some unused portions of all animal-tissue specimens unts the animal inoculation test either has been reported positive for rabies, or has run its course (at least 21 days), and has been reported negative for rabies.

·Section 2* RAPID MICROSCOPICAL EXAMINATION FOR NEGRI BODIES USING SIMPLE TISSUE-APPLICATION TECHNIQUES, AND PREPARATION OF SPECIMENS FOR BIOLOGICAL TEST The techniques employed in the laboratory diagnosis of rabies should embrace optimum conditions of accuracy, speed, and economy. The method employing the microscopical examination for Negri bodies, using the simple application of brain tissue to a slide and Sellers' technique for staining (see page 32), has been proved to fulfil these requirements. It has been found that Negri bodies, when present, are most readily demonstrated in Ammon's horn (hippocampus major) of the brain and also in the pyramidal cells of the cerebral cortex and Purkinje's cells of the cerebellum; they are found to a much more limited extent in the neurons of the thalamus, sons, medulla, spinal cord, and sensory ganglia. Dissection of the Brain A very simple operation is required to expose Ammon's horn, which is generally the best area for demonstration of Negri bodies in most species of rabid animals With a pair of sterile scissors, a longitudinal incision is made into the dorsal surface of each cerebral hemisphere, about 2 cm lateral to the longitudinal fissure or midline of the brain (sce fig 1). The incision is made from the region of the occipital pole of the hemisphere and is exter.ded forward for 3-5 cm and downward through the grey matter, and then completely through the white matter until a narrow space, the lateral ventricic, is reached the opening is then widened by spreading the incised hemisphere, and Ammon's horn will be rescaled as a semicylindrical, white, glistening body bulging laterally from the ventricie Noor (see fig 2 and 3) It has a spiral contour and, on cross-section, a characteristically rolled surface Disease Center Public Health Services Department or lealth, L. ducation and Heliars • Contributed by Ernest S Tierkel, Director Rabies Control Acuvine Commun.cable Atlanta, Gw, USA ·25 LABORATORY DIAGNOSIS FIG. 1, SITE OF INCISION FOR LOCATING AMMON'S HORN AW AS M By courtesy of United States Department of Health, Education and Welfare Pune Healthy Servire, Contmunicable Disease Center (US DHEW-PHS-COC Preparation of Slide Slides should be made first from Ammon's horn, then from the cerebral cortex, and finally from the cerebellum, Samples (at least sıx) should be taken from these three areas on each side of the brain and examined microscopically before the brain is reported Negri-negative. It is always wise to select another area from each his ampus for good measure, Three recommended methods of applying fresh brain tissue to slides are as follows Impression method With a pair of scissors, $31) transverse sections (2-3 mm in thickness) of brain tissue (Ammon's horn, cerebrum, or cerebellum) are cut and placed on clean blotting-paper or a wooden tongue-depressor, cut surface

·26
E. S. TIERKEL
FIG. 2, INCISION TO LATERAL VENTRICLE SEPARATED, SHOWING AMMONS
HORN
By courtesy of US DHER-PNS-CDC
FIG. 3. CLOSEUP OF AMMON'S HORN. BULGING FROM FLOOR OF LATERAL
VENTRICLE
#oricy of l'S DHE.PHS-CDC
• Indicated by ato

·27 LABORATORY DIAGNOSIS facing upward (see fig. 4 and 5) A clean micro-slide is then touched against the cut surface of the section and pressed gently downwards with just enough pressure exerted to create a slight spread of the exposed surface of the tissue against the glass slide. According to the size of the section, three to four impressions can be made on one slide (see fig. 6 and 7). FIG. 4. TAKING OF SPECIMEN FROM AMMON'S HORN FOR SLIDE PREPARATIONS noin By courtesy of US DHEX-PHS CDC While still motst, the slide is flooded with Sellers' stain (see fig 8), allowed to remain for a few seconds, rigsed under the tap, and dried at room temperature without blotting The preparation is then ready for examination The impression may be examined directly under oil, or covered with a cover-slip mounted in balsam This method is preferred over others because a maximum amount of nerve tissue can be concentrated in a small area with a minimum amount of cellular damage Smear method The spread-smear method consists of placing a very small section of brain tissue on one end of the slide. Another slide is used to crush the

·28
E. S. TIERKEL
FIG. S. TRANSFER OF SPECIMEN TO WOODEN SPATULA BEFORE MAKING
IMPRESSIONS
Br puries of IS DITEM.PH5-CDC

·LABORATORY DIAGNOSIS
29
FIG. 6. IMPRESSION METHOD OF SLIDE PREPARATION
S.
By courtesy of US DHEW.PHS-CDC

·30
E. S. TIERKEL
FIG. 7. CLOSE-UP OF SEVERAL IMPRESSIONS ON A SLIDE
By courtesy of US DIE-PUIS-CDC
FIG..
IMMEDIATE IMMERSION OF SLIPE IN SELLERS' STAIN WHILE
TISSUE FILM IS STILL MOIST
unirsi SUS DNEW.PHS.CDC

·31 LABORATORY DIAGNOSIS section of tissue against the first slide and is then drawn across the lengsh of the slide (sce fig 9). The result is a fairly homogendous spread of a thin film of tissue coicring about three-quarters of the area of the slide. FIG. 9. SMEAR METHOD OF SLIDE PREPARATION By courtesy of US DHEW.PHS.CDC In this spread-smear technique there is a copious concentration of tissue and a rather extensive area for examination. Care should be taken not to use too large a tissue-section, which will result in an etcessitely thick film. making proper staining and microscopic examination impossible. The Impression method, however, gives superior results "Rolling" method The last method, the "rolling" technique, consists of cutting a piece of brain tissue about the size of a fresh garden-pea, and rolling or teasing it gently (cut surfaces downward) over the enure surface of the slide with a toothpick or wooden applicator The staining procedure of Sellers is recommended here because of its accuracy and simplicity In this technique, no preliminary fixation us required, since the tissue film is fixed and stained simultaneously, making tt one of the most rapid and easily handled methods

·32
E. S. TIERKEL
Preparation of Sellers' Stain
Examination of slide
Time may be saved in microscopic cramination by a study of the stained
slide under low power at first, selecting areas containing numerous large
neurons to be examined for Negri bodies under immersion oil (sec fig 10
and 11).
FIG. 10. LOW-POWER VIEW OF IMPRESSION, SHOWING FIELD (UPPER HALF
RICH IN NEURONS FOR EXAMINATION UNDER HIGH POWER
By courtesy of U'S DHEN-PHS CDC
Magnification 1200
Sellers' stain shows the Vegni body well-differentiated in magents or
heliotrope to bright red, with well-demonstrated dark-blue to black haso
philc inner bodies. All parts of the rense cell stain blue, and the interstitial
• This sumettien was very busilly contabuted by Thomas F. Sellers Director, Cross
Department of Puti licalth Attart. 64 YA

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·32
E. S. TIERKEL
Preparation of Sellers' Stain *
Examination of slide
Time may be saved in microscopic examination by a study of the stained
slide under low power at first, selecting areas containing numerous large
neurons to be examined for Negri bodies under immersion oil (sec fig 10
and 11).
FIG. 10. LOW-POWER VIEW OF IMPRESSION, SHOWING FIELD (UPPER HALD
RICH IN NEURONS FOR EXAMINATION UNDER HIGH POWER
BY courtesy of US DHFH.PHS-CDC
Magnification x 200
Sellers' stain shows the Vegri body well-differentiated in magents or
heliotrope to bright red, with well-demonstrated dark-blue to black tava
philic inner bodies. All parts of the rene cell stain blue, and the interstitial
• This subsection » very kindly watsibuted by Thomas Selters Director. Games
Department of Pubic Health Allsnt. Ga USA

·33
LABORATORY DIAGNOSIS
tissue stains pink. Erythrocytes stain copper-colour (orange-tinged red)
and can be easily differentiated from the magenta-tinged red of the Negri
bodies (sce colour plate, fig. 1, facing page 40).
FIG 11. HIGH-POWER VIEW OF NEGRI BODY
By courtesy of US DHEW.PHS CDC
• Indicated by arrow
Magnification x 900
Stock solution
(1) Methylene blue (Colour Index 1, 2 No 922, or Schultza
Inder No. 1038)
10g
Methyl alcohol (absolute acetone-free) to make . 1,000 ml
(2) Basic fuchsın (Colour Index No 677, or Schultz No.780) 5 g
Methyl alcohol (absolute acetone-free) to make
500 mi
Other suitable dyes, such as those from Gruebler & Co., Leipzig,
Gurd & Co, Ltd, London, and National Aniline Co., New York, may
also be used
Society of Dyers and Colourists (1924) F Roue, ed . Colour inder, Bradford
Society of Dyers and Colourists (1929) F Roue, ed. Supplement to colour inder.
Bradford
* Schultz, G (1928-1934) Farbstofftabellen 7 Autt, Leipzig

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·LABORATORY DIAGNOSIS 35 rinsed with tap-rater and others riosed in disolled water containing M[150 phosphate buffer, pH 7.0. The stain must be kept in a tightly-closed container when not in use, 10 prevent csaporation, which tends to make the fuchsin too dominant, The addition of absolute methyl alcohol will restore the proper balance. It is convenient to keep the staining solution in a screu-capped Coplin jar for dasly use If this is not available the stain may be stored in a groundglass-stoppered dropper bottle and the smeas flooded with the stain. Staining in this manner will not be satisfactory unless the entire process can be completed within a few seconds The best results with the stain 3te obtained when the brain tissue is fresh As decomposition sets in, the characteristic colour differentiation Is affected, and although the Negr bodies retain their stamming quality, the smear as a whole becomes too red, or 3t umes too blue, and identification of the bodies becomes more difficult. The Negri Body: Differential Diagnosis Although generally rounded in form, the Negri body may be found to assume any shape. At various times in different laboratories it has been demonstrated to be round, oval, spheroid, amoeboid, clongate, triangular, etc By the same token, there is great analion in size, generally it is found within the limits of 0 24 4 and 270 w. It is characteristically acidophilic in staining reaction, and takes on the pink to purplish-pink colour in fifferential stains which use basic fuchsin or cosin with methylene blue as their base The position of the Negrs body within the neuron is intracytoplasmic Classically, it is found berneen the nucleus and one corner of the neuron, or in the prolongation of the cell body However, it should be stressed here that the intracytoplasmic position of the Negn body can be crpected with reasonable consistency only in histological sections of the brain in the simple tissue-application techniques described above, the histological pattern is disturbed and one may very often see well-formed Negro bodies which appear to be entirely outside the neuron Thus, in methods such as the impression, smear, or rolling techniques, the intracellular position of the Negri body is not required as a diagnostic criterion, and Negri bodies whuck satisfy the requisites of morphological identification, whether inside or outside the neuron, are sufficient to establish a positive diagnosis, The most charactersstic feature of he Negri body is its internal structure It is this feature which serves as the most essentsal criterion for positre identification in the techniques descnbed in this section The matrix of the Negri body has an acidophilic staining reaction, and confained within this magenta-red structure are small inner bodies (Inner.

·36 E. S. TIERKEL körperchen), basophilic granules which stain dark-blue to black. The size of these inner granules generally varies from 0.2 u to 0.5 H. Classically, the well-formed Negri body-the so-called textbook picture--will have its inner granules arranged in rosette fashion, with one large centrally-placed body and a series of smaller granules arranged neatly around the periphery of the Negri body. It should be pointed out, however, that this picture is the exception rather than the rule, and it is very rare indeed that such an orderly arrangement of the inner granules is scen. For purposes of diagnosis it is sufficient to establish the presence of these dark-blue-staining granules, regardless of their numbers or pattern of distribution within the matrix of the Negri body. There is universal agreement that the Negri body is specific for rabies, and its presence always indicates this infection. Furthermore, a fullyformed Negri body cannot be confused with anything else. However, in the diagnostic laboratory other types of inclusion bodies are sometimes cncountered in animal brains and, because of certain similarities, may be mistaken for Negrı bodies. This is particularly true of the dog, for, cal. and laboratory white mouse. In the brains of dogs and fores, the acidophilic inclusion bodies of canine distemper or Rubarth's discasc (canine infectious hepatitis, for encephalitis) are occasionally encountered These scem to occur more often in the thalamus and lentiform nucici than in the hippocampus. By the same token, the brains of non-rabid cats and laboratory white mice occasionally contain non-specific acidophtlic inclu sion bodies when presented for rabies diagnosis. All these non-rabies inclusion bodies have the same staraing characteristics for Sellers' stain, and they cannot be differentiated from each other with the techniques described above. However, the important thing is that these non-rabics inclusions, as a group. can be differentiated from Negri bodies with the use of Scilers' sian. The following outline may be used as a guide in this differentiation Meen buches Presence of busophilic inner granules Heterogeneous matris Loss refractal Magenta (heliotrop) tinge Non-rabies inclusion budid Absence of internal structure Homogeneous malar More highly refractile Colour more acidophilie (pinker) See colour plate fiz .. lacing page 10 Small atypical intracytoplasmic inclusion bodies are sometimes found In animals killed during the early stages of rabies. For that reason. It is imperative to hold buying and suspeci dogs in quarantinc, rather than to Anti them immcdiatch and send the brain to a laboratory for diagnosis (sec page 15) There is touhle reason for this. First, it will allow of ·LABORATORY DIAGNOSIS observation for symptoms of rabies which may make possible a chnical diagnosis of the disease. Secondly, the longer the animal is alloued to live, the better the chance of obtaining a positive microscopic diagnosis. The length of clinical illness in rabies is directly related to the presence, size, abundance, and development of Negr bodies. Thus, if the disease runs its full course, Negr bodies which are larger, more abundant, and fully formed with good internal structure, are more likely to be found. 37 Biological Diagnostic Test: Preparation for Mouse Inoculation Since Negri bodies cannot always be found in the brains of animals dying of rabies, it is important that animal inoculation for demonstration of the virus be done on Negri-negatne specimens Extensive surveys of large numbers of rabies cases have shown that 10%-15% of those cases proved positre by mouse inoculation had been missed by direct-smear microscopic examination for Negri bodies. It is therefore strongly recommended that laboratories which furnish rabies diagnostic services be equipped to carry out animal inoculation tests on Negr-negatxe brain tissues In the past the guinea pig and rabbit have been considered the most suitable test animals for this purpose Since the demonstration that the intracerebral injection of rabies virus into white mice produces typical and constant infection, the white mouse has become the test animal of preference The chief advantages of the mouse are the low cost, making it possible to use several animals for one specimen, the relatively short incubation period for street virus, and the consistency of production of Negn bodies in the brains of mice inoculated intracerebrally with street virus A positive microscopic diagnosis is sufficient proof of the presence of rabies When the microscopical examination proves Negra-negative or questionable, no time should be lost in taking samples of the cerebral cortex, cerebellum, and Ammon's horn on each side of the brain, plus a ample from the medulla-brain-stem These should be pooled in the emulfier in preparation for the mouse inoculation test described in section 4, tge 56. It is quite important to achieve complete representative sampling all those parts mentioned on each side for pooling, since there is often great variability in the virus distribution through the brain. Antibacterial Agents for Contaminated and Decomposed Specimens It is often difficult to obtain animal brains from the field which are bactenologically sterile The head may have been in transit for a long ·38 E. S. TIERKEL time, or picked up long after death, or shot, or clubbed in the head. Also, the cause of death may have been a bacterial encephalitis. Intracerebral injection of bacteria may cause the death of inoculated mice in one, two, three, or more days, before the rabies virus, which may be present in the inoculum, has had its full incubation period. On the other hand, injected mice may live long enough for rabies incubation to be complete, may pass through the typical rabies symptoms of tremors, paralysis, and prostration, followed by death, and may show typical Neem bodies and many bacteria in their brain smears. When bacterial contamination is suspected, for example, if the animal brain is decomposed or when many bacteria are demonstrated on the original brain smears, it is best to treat the brain suspension with an antibacterial agent before inoculating it into mice. Or the following agents, penicillin and streptomycin 4 will give the best results. If these antibiotics are not available, any of the other agents may be used-e...: (1) Glycerol. Place the brain specimen in pure glycerol for 48 hours. (2) 65% phcnol. Make up 0.5% phenol in physiological salt solution, This is used as the diluent for making up the 10% tissue inoculum, Hold for 6 hours or overnight. If held for 6 hours, keep the suspension at room temperature. If held for over 6 hours, keep the suspension Overnight in the refrigerator. (3) 7:5,000 thiomersal (merthiolate). Make up a 1:5.000 solution of thiomersal in physiological salt solution. This is used as the diluens for making up the 10% tissue inoculum. Hold for 6 hours or overnight If held for 6 hours, keep the suspension at room temperature. If held for oler 6 hours, keep the suspension overnight in the refrigerator. (4) 10% ether. Place one part ether in ten parts tissue suspension : shake in a stoppered bottle and leave at room temperature for two hours: use a sterile conton plug to allow for evaporation of ether. Caution if all the cther has not evaporated, its intracerebral injection will cause the almost instant death of the mice. (5) Penicillin and streptomicin. Add 500 units of soluble sodium penicillin G and 2 mg streptomycin per ml of tissue suspension. Allow stand for 30 minutes at room temperature before injection. This amount is usually enough, but for very heavily contaminated brains or safnary glands, as many as 1,000 units of penicillin and 3 mg of streptomycin may be used In order to help detect it. tuossible presence of contaminating bacteria, a portion of all time Chi sists should be cultured in dertrose infusion • Johnson, 13 I 181, 18 ·LABORATORY DIAGNOSIS 39 broth or similar media, and streaked on a blood-agar plate. The recommended amount is about 01 ml of emulsion in 3 ml of broth. Incubate overnight at 37 5°C. Early deaths (1-3 days) among the infected mice may be attributed to the presence of contaminating bacteria if the cultures show moderate to heavy growth, and if many bacteria are found in the brain smears of the dead mice.

·40 DESCRIPTION OF PLATE Fig. 1 is reproduced from Risers, T. M. cd. Viral and rickettssal infections of man, 2nd ed. Philadelphia, London, Monireal, 1952. by courtesy of J. B. Lippincott Company: fig. 2 is reproduced by courtesy of Lederle Laboratories Division, American Cyanumid Company, Pearl River, N.Y. USA, fig 3-8 were kindly supplied by Professor P Lépinc, Institut Pascur, Paris. Fig. 1 Sellers' stain showing three large Negri bodies with Inrer. körperchen"; the small round cells stained red are crythrocytes (Magnification x900) Fig 2. Fox: non-specific inclusion body in cytoplasm of degenerated neuron (anterior hom cell). Note homogeneity and lack of inner structure of Inclusion body. (Hann stain: magnification 1.440) Fig. 3. Street rabies. The cell right of centre shows the difference between the nucleolus (stained violet-red inside the nuckus) and the Negri body (in the same cell just below the nucleus) Other Negri bodies can be seen in the cytoplasm of the cell at the 10 o'clock position (Mann stain magnification Y680) Fug 4 Street rabies-11. Note Megri body in cytoplasm of neurons below centre of figure. (Cremsa stain: magnification Y 680) nigs Street rabies typical een bodies, several showing internal structure, many micro-bodies present : ses cral, stuated at the prestongations of the neurons, appear to be etira-cellular. (Lépine fuchsin-safranine-blue stain magnification x 680) Fug 6 Fixed-urus rabies--1. Typical or philic nuclear degeneraison chara, 'crizing fited-trus lesions. (Mann stain. magnification Fig 11 virus rabies-11 Similar type of orphilic nuckat Thon in a neuron in the outer layer of Amnon's horn. (Gemsa 1.ation - 690) FIE nukar degeneration resulting in Lied-irus rabies polyurome bodies fill the nucleus of the "ected stan maga calon - 600) nej ·8 لوگ

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·HISTOPATHOLOGICAL DIAGNOSIS nciple, the histopathological diagnosis of rabies consists in recoge the presence in the animal of acute encephalomyelitis which scribed to a specific agent, namely the rabies virus e animal has died from rabies, it is normally easy to detect the lesions , if the animal has been killed, death may have occurred the appearance of specific lesions (Negri bodies). Thus, all animals -h the cerebrospinal axis (brain, medulla, ganglia) shows the slightest of lesions, particularly infiltrations, should be regarded as suspect. utter how small the lesions may be. fter correct removal, the brain should be carefully dissected. Smears mpressions should first be examined for Negr bodies (sce section 2. ..-" -hoz horor erog Bedding by a rapid method. At least six samples should be examined, rresponding respectively to the cornu ammonis (both sides), the cerebral irtex (motor area), the cerebellum, the medulla, and a ganglion (gasserian r upper cervical). The sections should be examined for (a) Signs of meningo-encephalomyelitis, 1.c, meningitis, meningeal Infiltration, perivascular cuffing, parenchymatous infiltration, formation or encephalitic nodules (Babès' nodules), and ganglionar infiltration with satellitosis and neuronophagy (lesions of van Gehuchten and Nélis (scc fig 1 and 2) These lesions may be detected by any staining method (hacmatein-cosin, polychrome blue) They show the existence of encephalomyelitis and enable a probable positive diagnosis to be reached (b) Specific lesions The different types of neurons should be examined for Negri bodies and lesions of fixed-virus rabies • Contributed by Pierse Lepsne, Virus Section Insutut Pasteur Pati, France - 41 -

·42
P. LÉPINE
FIG. 1. NORMAL
GASSERIAN GANGLION
FIG. 2. RABIES : INFILTRATED
GASSERIAN GANGLION
Gangtoneurons with a single Layer
of salelite cells
Jnftration, sa'entosio and neuronoply
(lesion of van Gehuchten & N+1)
FIG. 3
RABBIT
STREET RABIES
van on
POR - 7.000
Nort
el

·LABORATORY DIAGNOSIS The Negri bodies are found especially in the central pyramidal layer of Ammon's horn (see fig. 3) and the hippocampus, in the lower loop and the middle layer of the ganglioneurons of Ammon's horn and, less frequently, in the neurons of the cerebellum (motor areas), and of the medullary nuclei. They may be present in very large numbers in the ganglia but are generally small in size. + -- FIG 4 RABBIT: FIXED-VIRUS RABIES Lepina fuchxin-satranine-blue) slain magnification x 770 Polychromatic degeneration of neurons in outer layer of Ammon's horn The lesions of fixed-virus rabies are found exclusively in the middle zone of the external layer of the cells of Ammon's horn (see fig 4 and 5). They always co-exist in a varying proportion with the lesions of street-virus rables. These lesions can be detected only by special staining methods (Mann, Giemsa (sec colour plate, fig 4, facing page 40), Sellers, etc ). Their presence enables a definitely positive diagnosis to be made.

·42
P. LÉPINE
FIG. 1. NORMAL
GASSERIAN GANGLION
FIG. 2. RABIES : INFILTRATED
GASSERIAN GANGLION
Ganglioneurons with a single layer
ol satellite cells
Infiltration, satellitosis, and neuronophagis
(lesion of van Gehuchten & Nelis)
FIG. 3
RABBIT : STREET RABIES
:
Vann A
magnification 2000
*** bodies in revions of Ammon's horn (inner layer)

·LABORATORY DIAGNOSIS 45 occipital bone, followed by two more cuts, one on each side, along the temporal bonc in prolongation of the cuts already made. Join up the saw. cuts by means of bone-cutting forceps, and lift up ard push backwards the top of the skull In the case of very large animals (large dogs, cows, etc.) a different method is preferable: make a longitudinal saw-cut on each side of the midline at about 15 cm from it, and join up these cuts by one or two transverse saw-cuts above the orbits and at the occiput so that the calvaria can be removed in two symmetrical pieces Once the calvaria has been removed, use fresh instruments to open the meninges, making use of serrated dissection forceps and a pair of fine. sterile scissors The operation is performed by making an incision in the meninges, starting from the median region, along and on each side of the longitudinal sinus, next, a second incision is made perpendicular to the first and the meningeal flaps pushed upward and backwards Again change instruments; cut through the medulla with a scalpel as low as possible and lift up the brain proceeding from back to front, and successively severing the pairs of cramal nerves At the erd of the operation the brain is toppled over forwards into a large sterile Petri dish where it rests on its upper surface In hot weather, or if the brain is soft (cadaveric brain), it should be cooled to +5°C (refrigerator) to give it a firmer consistency before dissection. Eramination of the brain Note whether or not there is congestion of the cerebral vessels or exudate in the meninges, etc. Dissect the brain as follows: (a) With a brain knife separate the two hemispheres longitudinally, after having detached the cerebellum and the medulla. (6) Look for the hippocampus and the cornu ammonis. This may be done in two ways (1) Cut across the brain transversely, starting from the base behind can easily be removed (2) Alternatively, a longitudinal incision may be made externally in the posterios thusd of each cerebral hemsphere about 1.5 cm from the midline, the incision is continued through the grey matter and the white matter until the third ventricle is reached in the form of a narrow fistica tricle later Than

·P. LLPINE
FIG. 5. RABBIT : FIXED-VIRUS RABIES
Lépine (fuchsin-safranine-blue) staln
magnification x 1.450
Detail of affected nourons pyknosis of cellular bodies, with multiplicity of
polychromatic nuclear debris
BRAIN REMOVAL, AND PREPARATION OF MATERIAL
Large Animals (Dogs, Cattle)
The animal is firmly secured to the autopsy table or, better, the head
is separated from the body and strapped to a wooden block hollowed out
for the purpose The operator and his assistants should wear thick rub
ber gloves to protect their hands. Make an incision on the midline of the
skull through the skin, push aside the flaps of skin, and reflect the muscles
and fascia as far as the base of the skull, proceeding from the crown
to a horizontal line passing through the ches
Saw transuersely through
the skuli st eye leset and make a symmetrically placed saw-cut through the

·LABORATORY DIAGNOSIS 47 of eversion which completes the clearance of the field of operation. The Farabeuf and bone forceps are then laid aside. With the fine forceps and the scissors the meninges are cleared aside and the anterior part of the brain sectioned at the olfactory lobe, the medulla y severed behind the cerebellum, and after the brain has been raised in order to cut the optic chiasm, it is placed in a Petri dish where it is dissected With the brain lying on its dorsal surface in the Petri dish, the ventral surface facing upwards, cut the cerebral trunk at the peduncle; then cut through the encephalic mass obliquely along a transverse slanting plane, starting from the optic chiasm and going towards the convexity, parallel to the posterior surface of the hemispheres and to the cut surface of the cerebral trunk. A second cut made in the same way, parallel to the first and from 5 mm to 8 mm behind it, gives a transverse section of the brain which includes the gyrus hippocamp and the cornu ammonts as well as the basal optic ganglion-areas of choice for the detection of Negri bodies, in addition to the cortical motor area (see fig 6 and 7, and colour plate, fig 5, facing page 40) A transverse section of the cerebellum makes ut possible to examine Purkinje's cells and the peduncular region. Finally, a slice cut from the end of the cerebral trunk gives a section of the medulla To reach the gasserian ganglion, cut through the petrosal bone with the bone forceps at its insertion into the sella turcica; make the cut surface gape open by everting the temporal bone downwards and outwards The gasserian ganglion is easily recognized from its whitish and nodular aspect and its almost fibrous consistency. The ganglion is carefully removed, freed from fragments of bone, placed on a piece of filter paper, and immersed in the fixing agent with the other samples Gumea-pig, hamster The animal is secured to the autopsy tray and the head thoroughly freed from skin The skull is rapidly flamed With the second pair of large sterile scissors, open the skull by means of four incisions encircling the skull cavity, the first one joining the two orbits and the other three made successively at the sides and the occipital bone Remove the brain with the fine forceps and scissors and dissect it as described above for the rabbit It is more difficult to locate the gasserian ganglion than with the rabbit, but, with care, this can be done successfully Mouse The mouse is pinned, ventral surface downwards, on a sheet of cork, the limbs being spread out and fixed First of all the four paws are fixed, the base of the tail is secured with a fifth fin, ard a sixth is passed through ·46 P. LEPINE Cut transverse sections 1-2 mm in thickness from each hippocampus Take similar samples from the cerebral cortex (motor area), the cerebellum, and the medulla, and immediately submerge them in the fixing agent selected. When impressions are to be examined at least sir slides (two for each hippocampus, one for the cerebral cortex, and one for the cerebellum) should be carefully inspected before deciding that the results are necalne, If the results are negatne, however, histological examination and animal inoculations are carried out. Preparation of the tissue samples for histological eramination Ir the tissue is soft and difficult to section, prepare pieces of filter poser slightly larger than the tissue sample to be collected. Apply the piece of filter raser to the cut brain surface, grasp the edge of the filter saper with fine forceps held in the left hand, and with the right malc a cut with a scalpel parallel to the filter paper ard 2 mm or 3 mm from 11. so as to semove the piece of brain, which is immediately submerged in the living agent together with the fragment of filter paper to which it is adhente Removal of matcrial,or iroculanon In operating, care should be taken to put on one side fragments asepti cally removed from the same areas (cortex, hippocampus, cerebellum, medulla) for use in animal inoculation (see section 6 (A), page 77) the brain is received in good cordition and can be assumed to be sterile. the fragments are remoied before any examination is made. When the brain is infected, the fragments may be removed at any time and antibiotics added, as described in section 2. page 38. Small Animals (Rabbit, Guinca-pig. Jamster, Mouse) Rahhur The animal, resting on its sentral surface, is attached 10 the autopsy tray, with the head at the edge of the tray. Using a scrrated dissection forcers, the scale and the scissors, completely scalp the hood from the nape to the muzzle, removing the cars and the upper cyclus. Moisten the csposed surface of the head with lodved alcohol and rapidly flame it with ubunsen hurner Holding the muzzle of the animal with a Farabeuf forcess in the lelt hard. open the brain pan with three cuts of the bone forcers Make the first two in the front part of the head, from cach orbit to the midic s sn upward and outward movement of csersion, opening the brain rin netai ard temporal bores) in two flaps, to the right and the left th: ad cut is made at the occiput, with a backuard musemeri ·LABORATORY DIAGNOSIS 47 of eversion which completes the clearance of the field of operation. The Farabeuf and bone forceps are then laid aside. With the fine forceps and the scissors the meninges are cleared aside and the anterior part of the brain sectioned at the olfactory lobe; the medulla is sciered behind the cerebellum, and after the brain has been raised in order to cut the optic chiasm, it is placed in a Petri dish where it is dissected With the brain lying on its dorsal surface in the Petri dish, the ventral surface facing upwards, cut the cerebral trunk at the peduncle; then cut through the encephalic mass obliquely along a transverse slanting plane, starting from the optic chiasm and going towards the convexity, parallel to the posterior surface of the hemispheres and to the cut surface of the cerebral trunk. A second cut made in the same way, parallel to the first ard from 5 mm to 8 mm behind it, gnes a transverse section of the brain which includes the gyrus hippocampi ard the cornu ammonis as well as the basal optic ganglion-areas of choice for the detection of Negri bodies, In addition to the cortical motor area (see fig 6 and 7, and colour plate, fig 5, facing page 40) A transverse section of the cerebellum makes it possible to examine Purkinje's cells and the peduncular region. Finally, a slice cut from the end of the cerebral trunk gives a section of the medulla To reach the gasserian ganghon, cut through the petrosal bone with the bone forceps at its insertion into the sella turcica , make the cut surface gape open by everting the temporal bone downwards and outwards The gasserian ganglion is easily recognized from its whitish and nodular aspect and its almost fibrous consistency The ganghon is carefully removed, freed from fragments of bone, placed n a piece of filter paper, and immersed in the fiung agent with the other mples Guinea-pig, hamster The animal is secured to the autopsy tray and the head thoroughly freed from skin The skull is rapidly flamed With the second pair of large sterile scissors, open the skull by means of four incisions encircling the skull cavity, the first one joining the two orbits and the other three made successively at the sides and the occipital bone Remove the brain with the fine forceps and scissors and dissect it as described above for the rabbit It is more difficult to locate the gasserian ganglion than with the rabbit, but, with care, this can be done successfully Mouse The mouse is pinned, ventral surface downwards, on a sheet of cork, the limbs being spread out and fixed First of all the four pays are fixed, the base of the tail is secured with a fifth sin, ard a sixth is passed through

·FIG. 6. SECTIONS OF RABBIT AND MOUSE BRAIN
A
B
E
5
b
8
9
10
12
For esplanation of this figure, so page 11

·Rabbit brain and Section on't horn (uddle 1 corpus ca inver). & Ammon's horn finner layer). LABORATORY DIAGNOSIS 49 the anterior extremity of the muzzle which is firmly stretched forwards. Remove the skin from the head. Treat the skull with nodized alcohol and flame it very gently with the pilot flame of the bunsen burner. Open the skull with the fine Scissors, commencing by joining the orbils, then cutting laterally through the skull fairly low, and finally pushing back the flap thus obtained Sever the medulla and the chrasm. Remove the whole of the brain mass and place it in a Petri dish. Separate the cerebellum. Cut through the brain transversely at the optic chiasm and along a plane parallel to the posterior surface of the brain, so as to obtain a cross section which is placed in the fixing agent, the anterior portion being reserved for sinears and inoculations Fux also a section of the cerebellum. If it is wished to examine a ganglion, the upper part of the spinal cord should be detached and removed together with the upper cervical ganghon. EMBEDDING, STAINING, AND EXAMINATION FOR NEGRI BODIES Rapid Mercury Sublimate Fixing Agent We recommend the following mixture for rapid fixation of the nervous system.equal volumes of glacial acetic acid, acetone, and saturated solution of sublimate (HgCl) in absolute alcohol. The mixture is made in advance and keeps perfectly well. Prepare the saturated solution of sublimate in absolute alcohol in hermetically sealed bottles, hasten saturation by keeping in the incubator at 37°C The sections should be treated with Lugol's solution or nodized alcohol before staining, in order to remove the sublimate Mixtures containing other components, particularly formol, give distinctly inferior results, as do mixtures into which water or hydrated alcohol has been introduced in order to slow down Key to fig 6 (papr 18) Brain (mouse, rabbit) 1 cerebrum, 2 cerebellum. 3 medulla oblongala B Section of normal mouse brain cerebral cortex, 2 lateral ventricie (choroid plexus). 3 Ammon's horn. 4 mesencephalon, inner nuclel of grey matter, 6 median ventricle, 1 corpus callosum C Section of normal rabbit brain 1 tko ventricle, 2 Ammon's horn (middle layer). 3 thinal fistute.choroid plexus, infundibulum, 6 gyrus of hippocampus, 7 lateral genleviate ganphon.& ventral nucleus of thalamus Rabbit brain atter removal from cranial cavity Inverted, lateral aspect Left-anterior por. tron, centre-temparat and parietal lobet, right-cerebelturn Section is performed anterior to a through optic chiasm and posterior to the portion a-d is immersed in fizative E 2 third ventricle, 3 lateral rentricle, 4 choroid 7 Ammon shorn (outer layer), 8 lateral geniculate ganglige, ventral nucleus of thalamus, 10 lateral chorord plexus, 11 runas fissure, 12 infundibulum ·SO P. LÉPINE fixation. Tissues other than those of the nervous system are hardered by this fixing agent. Rapid Method for Histological Embedding For slices of brain tissue I mm in thickness, fixation is complete in 15.50 minutes. The tissue is transferred directly to absolute alcohol, with which it is treated for 20-30 minutes in two baths, followed by two charges of foluer.e ard tuo of paraffin, each lasting 15 minutes. Including the time necessary for cutting ard stairing the sectiors, the preparation is ready for microscopical examination from three and a hair to four hours after autopsy of the animal. Embedding Using Dioran It is sometimes advantagcous to use a solvent for paraffin which is also miscible with water. The number of manipulations is reduced ard for fairly thin tissue fragments there is a pain of time. The dioun technique is suitabic for use in this connexion. For the tissue in thin slices, not crceeding 5 mm in thickness. In principle, ary fiung agent can be used It is preferable, however, to employ Bouin's dioran mixture as given by Lison : dioun saturated with pictic acid 8.5 volumes commercial formol 1 volume glacial acetic acid 0.5 volume Neut embed without washing, following the procedure grven below: dioran 1 1 hour dioran 11 I hour dioun 111 2 hours followed by paraffin 1 15 minutes parafin 11 15 minutes parallin III 2 hours Use fresh paraffin for embedding Tale care to Icep the dioun in well-stoppered bottles (nisk of chroric poisoning accompanied by anaemia caused by the vapour of the coun is lept in open holtles), and ensure that it remains anhydrous by adung pieces of Cao.

·51 LABORATORY DIAGNOSES .. 23 mi Staining of the Negri bodies by Mann's Method This classic method gives sections which are permanently stained, with very fine differentiation of the Negri bodies. It is an excellent demonstration method, but it requires time and a certain knack for full success. The procedure is as follows: Prepare the mixture at the time of use: methyl blue (not methylene blue), 1% aqueous solution 18 ml 1% aqueous eosin solution distilled water 49 mi Stain for 24 hours at laboratory temperature, or for 6-14 hours at 38°C in this case first treat the section with alcohol-formol to render the gelatine insoluble since otherwise the sections come loose). Was, first wah tap-water, nert with absolute alcohol (rapidly) Differentiate 10 alcoholic caustic sodi solution 1.5% solution of caustic soda in alcohol. 1 m? absolute alcohol. 30 ml un the section is stained pink (about 10 minutes) As soon as this stage 15 reached, wash the preparation well with tap-water. The section should take on a sky-blue colour, if not, treat it with water containing acetic acid (2 drops of acetic acid in 40 ml of distilled water) for 1 minute Dehydrate rapidly (absolute alcohol); treat with xylol, mount in balsam. Result. Negri bodies, vermilion red , nucleol of the neurons, violet red, chromatin, blue; cells, dark blue; stroma, pale blue, erythrocytes, pink (see colour plate, fig 3, 6, and 8, facing page 40). On substituting phlorin B for cosin in the same proportion, the preparahons obtained are less attractive (purplish-blue or mauve background instead of sky-blue) but the inclusions (Negtı bodies) are more sumerous and more striking Staining of the Negri Bodies by the Fuchsin-Safranine-Blue Method After being well fixed, the tissue is embedded in paraffin, cut into thin sections, and freed from the paraffin, Stain for 10 minutes with the following mixture (1) basic fuchsin 50% alcohol 200 ml (2) 02% aqueous solution of safranine ·52 P. LÉPINE Mix equal parts of (1) and (2) (drop-bottle, the mixture is fairly stable and keeps for some time). Discard the stain, cover the section with alcohol-acetone (equal partsi to remove ercess stain, wash rapidly: the section is coloured red Stiri for from 15 seconds to one minute with Unna's polychrome blue (109 dilution) or with permanganate blue prepared by Stevenel's method 11 and used undiluted. Discard the stain: the section is deep violet in colour Differentiate in alcohol-acetone for a few seconds; the section becomes blue; immediately wash the preparation in running water (tap-water) 10 remove the excess of blue stain, and again treat with alcohol-acetone: next, without washing, commence dehydration by shaking in a Borrel tube filled with absolute alcohol. The remaining stain is removed from the section which becomes differentiated, taking on a pink-lilac lint varying in paleness according to the thickness of the section. Rapidly complete dehydration in absolute alcohol, carefully remove the alcohol in severa! changes of xylol, and mount in balsam. Result stroma, very pale pink with nerve fibres a decper pink ; neuroglia and leukocytes, purplish-blue : neurons, light blue: chromatin, deer purple with the nucleolus a vivid red; pathological formations particularly well shown nuclear inclusions and oryphilic substances, vivid pink; Negri bodies, poppy-red to mau e pink, with the internal structure lilac (scc colour plate, fig 5 and 7, facing page 40) Detection of Vegri Bodies with the Fluorescence Microscope This method makes use of the fluorescence of the Negri hodies when they have been impregnated with a fluorochrome and subjected to light with a wavelength of 4.000 Is When equipment for (luorescence microscopy is available this is a rapid and sure method of rabies diagnosis. (1) fit the brain tissue and cmbed in paraffin as usual finc sections are made which arc mounted on Wides and treated successively with xylol. absolute alcohol, and water to remove the paraffin (3) Net, stain by immersion for 20 minutes in an aqueous 0 2. solu tion of thioflavine S Without washing it, treat the preparation for a few seconds with absolute alcohol and then with toluene Mount in balsam between the slide and the cover-slip, upplying as thin a layer of balsam as possible It should first be ascertained that the balsam docs not contain substances which become fluorescent in ultraviolet light (3) Vest cumine with the fluorescence microscope, using a filter 10 block uiclengths longer than 1 - 5.1501 Locate the cornu ammons ·LABORATORY DIAGNOSIS S3 under low magnification and cumine orth high magnification, using glycerol or liquid parafin as immersion liquid The Negri bodies immediately stand out because of their vivid fluorescence (see fig. 7 and 8) and brilliant azure-blue colour. The background of the preparation is pale-yellow, cellular protoplasm brilliant yellow, nucleolt and erythrocytes pale-blue. FIG. 1 STREET RABIES : AMMON'S HORN (MIDDLE LAYER) Mann starn magnification * BOO Appearance under white light, many Negrl bodies Method of Stoiall & Black A popular staining method used in the USA is that described by Stovall & lack 12 using acetone fixation and a sequence stain. The following description is taken from Lillie. A variant of this method is also given by Lillie "1 Stain 2 minutes in a 1% alcoholic solution of ethyl eosin ( CI No 770, sodium cthyl cosinate) adjusted to pH 30 with N/10 hydrochloric acid 2 Rinse in water 3 Stan 30 seconds in 23% methylene blue in 22% alcohol buffered to pH 55 with the acetic acid-sodium acetate buffer

·54
P. LLPINE
4. Then differentiate in 0.38acetic acid in water (13 drops IR 60 cc) uail sectes
are brownish red.
5 Wash, dehydrate, and clear.
6. Mount in balsam
Results: Negri bodies are brownish to pure red, nucleoli pale bluc, other structuring
pink.
FIG. &
STREET RABIES : AREA OF BRAIN, TISSUE BLOCK, AND CELLS
IDENTICAL WITH THOSE OF FIG. 7
magnification 20
Mann utan
Ted by thionavine and landing out clearly, because of their muorescence, Point
Appearance, under same magnification but with fluorescent light of Negri bodies
the dark background of the preparation
• turunt of the method which we have used successfully,
In 90 * 10), wohet or 94 95 alcohol, 3.25 cc 1;, acetic acid, ang akit
le muke 111) ... dissode 950 mg chyl cosin Stain in this for 2 minutes
? Then with ir skohol and in water
kountetyt on turmalin material in 0s, methylens Hue tn 25 dicohol, ahorel
to materiali ulum hematorylin
["Plein 2: acetic acid for 2 to S minutes
$ 1124 1..Ji!, and clear
Viruntim
+
6

·LABORATORY DIAGNOSIS Other Staining Methods SS Among the innumerable methods which have been recommended for staining Negri bodies the following also give good results: (1) Lentz's method .? this is a variation of Mann's method. (2) Gallego's method, using acid ferric chloride as mordant, followed by staining by Ziehl's method and with picro-carmine. (3) Romanowsky-Gremsa method: a buffered Glemsa following the technique of Lillie & Pasternack.? (4) Schleifstein's method : 10 a combination of Sellers' method with the rapid dioran-embedding method REFERENCES Gallego, A (1925) Z. Infeht Kr Haustiere, 28, 95 Lentz (1907) Zbl Baht, 1 Abt Ong 41, 374 Lépine, P (1935) C R Soc Biol (Paris), 119, 804 Lépine, P & Sautier, V. (1936) Bull Histol Tech micr 13, 287 5 Levaditi, J. Lépine, P & Augier, J (1948) CR Acad Sa (Paris), 227, 1061 6 Lille, R D (1948) Histopatholugic technic, Philadelphia, p 225 7 Lille, RD & Pasternack, JG (1936) 1 rechn Meth 15, 65 8 Lison, L. (1936) Histochimie ammale Methodes et problemes, Paris 9 Mossman, HW (1937) Stain Technol 12, 147 10 Schleifstein (1937) Amer I publ Hilth, 27, 1283 11 Stevenel (1918) Bull Soc Path crot 11, 870 12 Stovall, W D & Black, C E (1940) Amer chn Path 10,1 ·Section 4 * MOUSE INOCULATION TEST The mouse inoculation test, in spite of its simplicity, deperds grezy on the accuracy of its performance for dependable results. Choice of Mice Sirun Usually white mice of any breeding strain may be considered suzlat Preference should be given to the Swiss albino strain since it is very suset? cible to rabies virus and it is easy to maintain the breeding stock on its laboratory. If the Swiss albino stock is not available, however, almace any breed of mice, except grey wild mice, can be used, because a genencally resistant strzen kas not yet been found. Grey wild mice should be exclude, not because of insusceptibility but because of the difficulty in restraisin? these animals in cages during the observation period Agc Nfice of all ages are susceptible to intracerebrally introduced rabies virus. However, it is easiest to inoculate, maintain, and obscnc minut which are 21-35 days old (8-12 g weight) at the time of inoculation. Ser posable to keep the older mice of the same set in one care since the elder Alice of both sexes are equally suscepuble to rabies virus It is inged animals, particularly the males, are apt to kill each other in fighis before the observation period is completed General health Isis imperatis, that the animals chosen 10 be inoculated he in goud health It is important to know the history of the breeding colony, and i IS Chle to ir ut the animals closely before inoculanon. 1 Talerle ir te 110in Amenaan Cinamid Company, Pean Aitct. T.654 in Korrons, Atwstant Director, Varal and Rulelwal Rewah, ·LABORATORY DIAGNOSIS Ectoparasites on the animal, ruffled fur, and unmistakable signs of diarrhoea, should disqualify the animals immediately. If the mice are sent to the laboratory from a considerable distance, it is advisable to postpone inoculation for a minimum period of three days in order to let them rest and become adjusted to changed conditions. In such cases it may be equally important to leave a few animals uninoculated in order to observe the death-sate among "normal" mice as compared with inoculated animals, 57 Preparation of Supposedly Infectious Material to be Inoculated Choice of tissue Either the brain or the salivary-gland tissue of a suspectedly rabid animal may be used for virus isolation Detection of the virus is more frequently possible in the brain than in the salvary gland However, from the epidemiological and epizootiological points of view, Jt is important to examine the salivary glands for the presence of virus Although it is relatively immatenal which part of the brain tissue is chosen for the preparation of the suspension, preference may be gnen to Ammon's horn, the cerebellum, and parts of the corter. When salvary. gland tissue is chosen, the submaullary glands should be considered as those most likely to show the presence of rabies virus Also, when salivarygland tissue is used, it is always advisable to mince it before grinding, Grinder The choice of grinder depends to a certain degree on the amount of tissue available If more than 3-4 g of material are available, a small Waring blendor should be preferred over any other type of grinder (see fig 1) However, if the amount of tissue is less than 3g (which 's more usually the case), or if a Waring blendor is not avaslabic, the following grinders or grinding devices may be suitable in the order of preference listed (a) Ten Broeck grinder (see fig 1) This grinder is a handy tool, easy to assemble, manipulate, clean, and sterilize, but only brain tissue may be used in it since salivary-gland tissue is too tough for it to grind properly There is a slight disadvantage with respect to its fragility If improperly used, it may break while being manipulated, and the gainder say be easily damaged during cleaning, sterilizing, etc From the point of view of personal safety, the Waring blendor and the Ten Broeck grinder, if properly used, have some advantage over the other grinding devices (b) Mortar and pestle (see fig 2) This is a time-honoured method of grinding which has one advantage, namely, that with the help of an abrasive (eg, stenle sand) even the toughest tissue can be properly ground up ·58 H. KOPROWSKI FIG. 1. GRINDERS FOR PREPARATION OF TISSUE 1998 A-Mortar and pes. B - Ten Broech grinder C - Waring blendor (amall vite The mortar and pesile cannot be operated under as sterile conditions as cat the Waring blendor or the TenBreech grinder , it can, however, be easil cleaned and sterilized, und sinds wear ard tcar for a long period of time (c) Maheshift deres 11 Waring blendors. Ten Brocck grinders, o mortars are not available, the following makeshift tools may be considered (1) Concare dish and paint brush Any concave dish and medium-s1/04 paint brush which can be sier led muy be used for grinding brain tissu alone Thc sussension can be mide quite simply and us as homogeneou as one prepared in a mortur and pesttc, although more time is required The brush should be scrupulously cleaned and properly sterılızed anel USC (11) Mincing with scissors. If no grinders at all arc available, the possibility of mincing the tissue curied scissors may be considered. be itch-glassicpt inside a Petri dish. JI is the since some picces of tissue will always 1. etc This may ting. i ·59 LABORATORY DIAGNOSIS FIG 2. PREPARATION OF TISSUE SUSPENSION BY EMULSIFICATION WITH MORTAR AND PESTLE By courtesy of United States Department of Health, Education and Welfare Public Health Service Communicable Disease Center (US DHEW-PHS-CDC Diluent The choice of diluent may be left to the user but it is preferable to use an isotonic salt-concentration. However, mice have been known to withstand casily an intracerebral inoculation of sterile distilled water The following diluents may be considered in order of their availability. (a) A physiological salt solution containing varying amounts of animal serum (10%-50% concentration) This is by far the most common diluent used It should be carefully ascertained, however, that the donor animal has never been vaccinated against rabies It is therefore usually advisable to avoid the use of dog, cat, or cattle serum Normal sheep serum seems to possess some " antiviral " properties which are absent in rabbit serum. Thus, if it is available, the rabbit should be considered as the first source of blood It is preferable to inactivate the serum for 30 minutes at 56°C before using it as a component of the diluent Diluent containing serum can be sterilized only by filtration through bacteria-retaining filters ·60 I, KOPROWSKI (b) Other diluents : (i) skimmed milk (11) albumin solution in a balanced salt concentration (iii) physiological salt solution with distilled water These are not particularly recommended, although radies virus seems to be relatively non-susceptible to the inactivating action of salt solution, in contrast to such viruses as castern or western equine encephalomychtis Note: If a tissue suspension has been frozen and stored, a 50% scrum: water solution should be the preferred diluent. preseri. Bacterial stenhly There is no need to add antibiotics to suspensions of brain tissue if the material has been handled with reasonable precautions at the autopsy. and dispatched in a sterile container. A 50% glycerol solution is highly recommended for preserving the material since, in addition to its ing qualities, it exerts a strong bacteriostatic action (sec section 2. page 38) If there is any doubt, however, it may be safer to add enough streptonijcis and penicillin to the suspension to obtain a concentration of 2 mg of streptomycin and 50 units of penicillin per 1 ml of the final suspension, If antibiotics are added, it is best to let the suspension stand for at least 30 minutes before inoculating the animals. It is always advisable to add antibiotics to suspensions of sahrary glands Regardless of the addition of antibiotics, the salivary gland Uissue suspension should always be cultured for possible bacterial contamination, Becf-infusion broth, thioglycollate medium, and blood-agar are considered to be good culture media for this purpose. If bacterial growth is observed. an attempt should be made to identify the bacterial agent. If the results of the mouse inoculation rest arc equivocal (scc page 67) it may be advis: able to test the pathogenicity of the bacterial contaminant for micc injected intracerebrally Concentration of infected issue in the suspension This is optional. If the suspension is for storage, it is advisable 10 prepare a 20º, suspension by ucight. The weight of the tissue in grams multiplied by + gises the correct amount of diluent in mullitres to be added Houeser, if the suspension is to be used for inoculating mice intracerchrally, a 10% suspension by weight should be gren preference. Tither dilute the 20%, suspension by adding an equal volume of diluent. or prepare the suspension by multiplying the weight of tissue in frams hy 9 in order to obtain the required amount of diluent in millilitres.

·LABORATORY DIAGNOSIS
61
FIG. 3. CENTRIFUGATION OF TISSUE SUSPENSION
Ry courtesy of US DIFW.PUS-CDC
1.000 rpm for 5 minutes
FIG 4 APPEARANCE OF CENTRIFUGED TISSUE SUSPENSION
B, courtesy of US DHEW-PS-CDC

·62 H, KOPROWSKI In attempting to isolate certain strains of rabies street virus, the "selfsterilizing neuro-infection " phenomenon may be very occasionally suspected, and in such rare cases it is advisable to dilute the tissue suspension more than 10% (see page 67-68). Centrifugalion and filtration If the equipment is available, it is advisable to centrifuge the tissue suspension for 5 minutes at 1,000 revolutions per minute (r.p m.) in order to remove the gross particles (see fig. 3 and 4). However, if ro ceninluge is available, it is perfectly feasible to inject mice intracerebrally with a 10% uncentrifuged brain suspension. Salivary-glard suspensions, if uncentrifuged, must be filtered through one or two layers of storite gauze in order to prevent the death of the animals from trauma. Inoculation of Mice Choice of springe Syringes should be chosen which can measure accurately 003 ml (single mouse dose). Thus '/-ml tuberculin syringes should be considered first, followed by !:-mi or i-ml tuberculin syringes for intracerebral inoculation, 27- or 26-gauge (0.40-0.45 mm) needles 1-1.5 cm long should be selected. Larger-gauge needles cause trauma to the brain substance. Anaesthesia It is strongly recommended that mice be anaesthetized before they are injected. inhalation of ether is the best form. A battery jar with a specs cially fitted wire bottom may be used for the purpose (fig. 5). If no such device is available, pentobarbital sodium injection should be considered. It is a good procedure to have the work-labíc used for mouse inoculation drawn well away from the wall, so that an assistant can etherize the mice from a position opposite the operator. Inoculation reclinique (see fig. 5) There are as many methods of inoculation as there are insestigators. The present author is right-handed and prefers the following technique. Anacsthetized micc are laid on the Icft side, the legs pointing towards the inoculator With the thumb of his left hand, the inoculator supports the lower jaw of the animal, with his inder finger behind the shull of the animal Pressure should be very slight in order to asoid the asphyxiation ard death of the animal In his right hard, the iroculator holds the syrirge ·63 LABORATORY DIAGNOSIS FIG 5. INTRACEREBRAL INOCULATION OF MICE in a horizontal position parallel to the table surface and perpendicular to the head of the mouse, with the needle pointing towards the inoculator. With a quick thrust, the needle is pushed through the skull of the animal at the place which can best be described as the apex of an imaginary angle of which the arms point to the animal's right eye and right ear. The needle easily penetrates through the bone and should then be further inserted for about 01-02 cm into the brain tissue When the inoculator uses a 1 5.cm needle, he should be careful not to penetrate too far since he may then end by giving the injection into the base of the skull The plunger is pushed to the next O 03-ml mark and the needle is then gently withdrawn. For intracerebral inoculation, move the inoculated mice away from the the inoculated mice to the h may result in catching a Ver ti.. 1.1 The inoculated mice are immediately placed in a can or box previously prepared and identified by a tag bearing the mouse-group number or any other particular identification mark. If a large series of inoculations is made, it is worthwhile checking the number of living mice in each series ·11. KOPROWSKI after the entire experiment is finished. If any animals are found dead, an equal number of living animals should be inoculated and added. Under no conditions should the same syringe be used for inoculation of two different suspensions. If an adequate number of sterile syringes is not available, each syringe should be boiled between inoculations, ard care should be taken to let it cool before filling it with inoculum. It is well always to use the same rigid precautions to establish goou habits in virus work. For example, the rapid emptying of a stinge into a pan of water will produce an acrosol which can cause inhalation inlection in the operator, or the animals with which he is working, if the disease can be transmitted by such exposure. Virus may be spread from table to hands, and if these are not washed properly it is possible to contaminate subsequent specimens during grinding of tissue in a mortar--for example, virus may fall from the hands or sleeves into the mortar. Mice laid on the table after etherization may awake and have ether jar, thus contaminating it. Should other studies be carried out using the same ether jar, virus may be deposited on the heads of normal mice and be carried into the brain by the inoculation procedure. The worktable must be considered contaminated until washed with soap and water. Dilute bichloride of mercury is a good disinfectant for table tops. Cresol and phenol solutions are no better than water for the most common Sirus contaminant of mouse tissue, namely, the mouse encephalomyelitis virus, and several other viruses; phenol has no appreciable vincidal action on rabics Wrus When inoculation of all the mice is finished, the syringe and needle may be rinsed in water, provided this is done gently with the point of the needle well below the surface of the water in the sterilization tray. Sterilize all syringes and needles by boiling for five minutes. be put back in the Observation of Inoculated Mice Although rabies virus will only rarely cause signs of illness in mice before the fifth day after intracerebral inoculation, it is advisable to check mice daily. beginning with the first post-inoculation day. The number of mice found normal, sick, or dead is recorded on a mouse-history card (sec fig 6) which remains on filc as a permanent record of the crperiment. The observation period should estend for a minimum of 21 days after inoculation. Only rarely will rabies virus be detected in the inoculum later than 21 days after the inoculation, The following signs may be noted (a) Roughing fur. (h) tremors when held in the air by the tail with a pair of forceps. (c) lack of co-ordination of hind legs-note galt when ·LABORATORY DIAGNOSIS placed on table and made to move; (d) paralysis, (e) prostration (ntar death). Letter symbols denoting the different signs are recorded each day on the mouse-bistory card. FIG. S. MOUSE HISTORY CARD Na Data Vyrun Stroin tanog Preporalion Volum Dision CATE UI 7 3 2 $ 5 10 IN » IS119? 3 3 3 w G Checked or Aqua Mice Route Lee Deaths ia mice occurring 24-48 hours after intracerebral inoculation are attributable to causes other than rabies virus (trauma, bacterial contamination, other viruses). For diagnostic purposes, one or two mice may be sacrificed each day, beginning on the fifth day, and a search made for Negri bodies, Frequently, an carly diagnosis is thus obtained, particularly in instances where certain strains of street virus might take between 1 and 3 weeks to kill the mice Nate Cincal signs of stiness in inoculated mice cannot be considered characteristic of rabies Although signs of paralysis 5 days or more after troculation may give grounds for suspecting the presence of rabies vines, the same signs may be observed in numerous other viral, bacterial, and protozoal infections which involve the central nervous system of the mouse. Defimte evidence of the identity of the virus is obtained with the serumvirus neutralization test (see section 3, page 69) Further Passages of Infected Material If it is desired, btain tissue from mice which have succumbed to infectron after moculation with the original surus may be made into a suspension as described ahore and stored, or used in neutralization tests, or inoculated into another group of Bice

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·LABORATORY DIAGNOSIS 67 thereby exposing the brain. The brain is removed with curved scissors (see fig. 7) and is transferred to a sterile Petn dish. A thin section of the brain is cut out, just anterior to the cerebellum, and is transferred to a wooden tongue-depressor or a paper towel. A clean microslide is then pressed lightly against the cut surface of the section; the pressure should be sufficient to create a slight spread of the exposed surface against the glass slide. Negri-body stain should then be applied to the slide. Complications of the Mouse Inoculation Test Bacterial contamination of inoculum If bacterial contaminants caused the death of the mice in spite of the addition of antibiotics, and if the original suspension was preserved, the following methods may be tried in an effort to overcome the interfering action of the bacteria. (a) Filtration through bacteria-retaining filters: The supernatant liquid of a suspension centnfuged at 1,500 rpm. for 15 minutes should be used for this purpose Since rabies virus is a fairly large particle and since, in general, the concentration of virus in specimens submitted from the field is not very great, the virus may be lost in the process of filtration (6) Dilution method. Sometimes the suspension can be diluted beyond the end-point of bacterial contamination with retention of viral activity, but this happens very rarely (c) Prolonged storage In some instances, it is easier to combat acterial contamination after the tissue suspension has been stored for a period of time either at freezing temperatures or in glycerol (see pages 38 and 60). (d) Parenteral inoculation The Syrian hamster, which is the animal most suscepuble to parenteral infection, may be chosen for this purpose. Mice are relatively insuscepuble to parenteral infection with rabies. Presence of fuo uruses This is particularly confusing of the second virus has pathogenic properties similar to those of rabies Again, intracerebral or parenteral noculation of animal species other than the mouse may be attempted, articularly in view of the extremely wide host-range of rabies virus Self-sterilizing neuro-infections" or interference phenomenon In certain instances, either because of the properties of a particular ain of rabies virus or because large amounts of inactive virus particles ·68 H, KOPROWSKI may interfere with the living virus, it may be necessary to dilute the ina culum ten- or a hundredfold, or even more. There is no rule to determine when this should be done. However, if failure to isolate rabies virus is consistently encountered in the same species of animal in a particular geographical area, the possibility of an interference phenomenon should bc seriously considered, and tissue suspensions should be diluted beyond 10% concentrations, ·Section S SERUNI-VIRUS NEUTRALIZATION TEST If properly executed, the serum-sirus neutralization test constitutes one of the best proofs of identny of rabies vitus. Although the intracerebral type of neutralization test is less sensitive than a test in which injection is carried out in mice parenterally, it is impossible to apply the latter test in identifying rabies virus, since the vifus has very low invasive power when injected parenterally in mice This test will be described as though attempting to identify a virus recently isolated from the field. However, the technique may be applied to any neutralization test soutinely performed in the laboratory. Choice of Mice The same criteria should be used as those described for the mouse inoculation test (see section 4, page 56) Preparation of Virus Suspension Usually the first mouse passage of a virus in the laboratory can be used as the source of infected malerial However, in some instances the concentration of virus is too low and the test will fail, because too small a number of nuce injected with a mixture of virus and normal serum will dle of the infection. In such cases, the second or third laboratory passage in mouse brain should be used the preparation of the mouse-brain suspension should follow the technique described in section 4, page 57, except that it may be more convenient to prepare a 20% suspension by weight of infected tissue. Also, the use of 10% normal-serum: physiological-salt-solution or 10% normal-serum distilled-water-solution as diluent may give more uniform results than other diluents. Storage of Virus For certain technical reasons it may be advisable to store the virus before its use in the neutralization test Severa) methods may be recom. mended for the preservation of the rabies virus The best is to keep the Lederle Laboratories Division. Amencan Lyanamid Company, Peat! Biver NY, and Harald N Johnson, International Health Diveston, Rockefeller Institute for Medical Research New Work, NY USA ~ 69 -

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·LABORATORY DIAGNOSIS 71 morning and have food removed from the cages the night before so that clear serum will be obtained. Immunization of guinea-pigs should be carried out with guinea-pig brain virus The use of mouse-brain virus may result in the production of antibodies to mouse encephalomyelitis virus of this is present as a contaminant in the mouse-passage strain. This may lead to a false interpretation of the results of the specificity test. Immune serum from guirea-pigs, prepared as described above, may also be used for the complement fixation test. Rabbits are also suitable animals for preparing immune serum (see section 6 (B), page 85). Performance of the Test Serum Prepare two rows of sterile 12 x 75-mm tubes, each row containing five tubes. With a wax pencil mark each set of tubes lo', 10", 10, 104, 104, Mark one set with letter N (normal serum) and the other with letter I (immune serum) In cach tube of the set marked N, place 02 ml of normal serum, measured from a l-ml pipette calibrated in 0 01 ml. Do the same with the set marked I, using immune serum. (Use separate pipettes for each serum ) Virus Prepare a set of 12 x 110-mm tubes, also marked 10 to 10. Leave the first tube empty.into each of the remaining tubes Pipette 2.7 ml of diluent (preferably 10% normal serum physiological-salt-solution. Into the first, empty, tube marked 10-, pipette the previously prepared 20% suspension of infected mouse brain Transfer 0 3 ml of this suspension into the tube marked 10-2 and mix it well with the diluent, using another pipette (1-ml), by blowing the fluid back and forth about 12 times. Following that, transfer 0 3 ml into the tube marked 10-, mix well, etc, as before Measure 0.2 ml of the contents of the tube marked 10-1 into the tube containing normal serum (N-10-1) and then 02 ml into the tube contain ing immune serum (1-10-"). Follow the same procedure with the remaining ditutions of virus, using a fresh pipette for each dilution Shake the rack well and place it in either a water-bath or an incubator set at 37°C for one and a half hours. Note In preparing serial dilutions it is essential to use a fresh clean pipette for each transfer After transferring the desired quantity to a subsequent tube, the fresh pipette should be used for miung, and then for transfer to the next tube A routine should be establtshed whereby the tube to which the virus has been added is set apart from the other ·72 II, KOPROWSKI & H. N. JOHNSON tubes in the rack before mixing. Mixing is accomplished by filling and emptying a l-ml pipette about 10 to 12 times; avoid frothing. A noe uncommon error with inexperienced technicians is to transfer without mixing, or to skip a tube and so fail to carry on any virus. Under no circumstances should titration be attempted iſ sufficiert pipettes are not available for changing with each dilution, because the titration will be meaningless. By titration with a single pipette it is possible to obtain infection in mice with 10-12 dilutions, etc., because a little tissue virus keeps coming off the inside of the pipette. This may be illustrated by diluting methylene blue in tenfold steps using a single pipette. Inoculation of Mice Remove the tube-rack from the incubator or water-bath and place it in a tray containing water and ice-cubes. Inject the contents of cach cube intracerebrally into six mice (0.03 ml per mouse) following the technique described in section 4, page 62. The series of mice receiving the immune serum : virus mixtures should be inoculated first, One tuberculin syringe may be used for the immune-serum series and one for the normal serum. However, in that case start the inoculations with 10 dilution and proceed backwards to 101 dilution. While changing dilutions, always rinse the syringe with the "new" dilution before filling it up. Be certain 10 have six lning mice injected with each dilution at the end of the test. Observation of Inoculated Mice Follow the procedure described in section 4, page 67. Terminate the test on the 21st day after inoculation unless the presence of contaminating mouse encephalomychtis virus is suspected (see page 73). If untform mortality ratios arc obsersed, the test may be terminaied on the 14th day. Calculation of Rosults Hypothetical results of a typical test are summarized below: Vartoliaty rolu elmice mixulured LD Mouse LT. Surom #ith seram and dilullens of us nire el pirkt 10-1 10 of Trut 10-3 10 10-5 Normal 6 6 06 10-123 Immune 66 06 06 06 10-13 316) nuruhi • Sec kction 11, page 111 for logarithms cakulations + Antiluanim of 25 the difference tween 425 and 1 76 ·LABORATORY DIAGNOSIS 73 Interpretation of Results If 100 LDS or more of virus is neutralızed by the serum, the identity of the virus is established. In case of equivocal results it is desirable to repeat the test. Sometimes neutralization of 75 LD of virus may be considered to be a sufficient criterion of identification. If the rabies virus has been lost and replaced by another virus the titre will be the same in the control and rabies-immune serum groups. Viruses isolated or maintained in mice are hable to be contaminated by mouse encephalomyelitis virus, and this virus may be passed in scries with rabies virus. Most strains of mouse encephalomyelitis virus are apt to produce symptoms somewhat carlier than the rabies fired virus, that is, on the second or third day Mice showing symptoms at this time should be suspected of harbouring mouse encephalomyelitis virus, and brain tissue should be taken for microscopical study and for subpassage. So-called "highly invasive" strains of mouse passage rabies virus which overwhelm immunity produced by vaccination with rabies vaccine must be checked for the presence of mouse encephalomyehtis virus. A single subpassage through rabbits will remove the mouse disease. The inoculated mice should be held for at least 30 days, as mouse encephalomyelitis virus, if present, may produce paralysis three to four Weeks after inoculation It is difficult to isolate virus from mice that develop paralysis at this time if they are infected with the mouse encephalomyelitis virus. The mouse encephalomyelitis virus is maintained best by subpassage at three-day intervals The mouse disease virus is not apt to be present in high titre Testing of Half-Log Dilutions of Virus For certain studies at may be advisable to include half-log dilutions of virus. When preparing the usual tenfold dilutions of virus (1 ml plus 9 ml dilment set in alternator 1 part in 3 16 or half-log) Do not mix the half-log dilutions until the tenfold dilutions have been prepared Then take a clean pipette and, beginning with the highest dilution of half logs, transfer and mur successively throughout the series to the most concentrated tube, using the same pipette It is also possible to my the half-log dilutions in a flask with rotation agitation without using a pipette ·74 1. KOPROWSKI & ft. N. JOINSON Modification of the Test : Dilutions of Serum with Same Virus Concentration If the titre of the virus is known beforehand, the test may be modified by the use of a constant dose of virus representing approumately 100 LD: per mouse dose when mired with an equal amount of scrum The procedure is similar to that described above, ercept that the serum is to be diluled instead of the virus. This test saves mice, since it is not necessary to use more than one dilution of normal serum. The immune serum may be diluted tenfold or fivefold, depending on its previously determined neutralizing potency. This is a more sensitive test than the virus dilution test but it has more application as a standard laboratory procedure than as a method of identification of an agent of unknown titre. It is particularly useful in determining relative titres or potencies of scrum samples.

·Part II METHODS OF VACCINE PRODUCTION

·No text on this page.

·Section 6 PHENOLIZED VACCINE: SEMIPLE TYPE A. SHEEP-BRAIN VACCINE: METHOD OF INSTITUT PASTEUR, PARIS General formula for vaccine 101 1.-.-.-L... Rabies strain employed The strain employed is the Institut Pasteur fixed virus (Louis Pasteur strain) isolated on 19 November 1882, which has been maintained solely in the rabbit since that date On its 90th passage this strain was used for the first human immunization (6 July 1885) and since then it has been constantly employed at the Institut Pasteur in Paris for immunization purposes. The behaviour and characteristics of the strain are periodically examined and have formed the subject of much research. At the present time, titration of the strain in the rabbit shows that a 1 15,000 dilution of the fixed virus kills rabbits six times out of seven, whereas a 1. 16,000 dilution kills them five times out of ten, a 1 : 17,000 dilution rarely kills them, and a 1, 18,000 dilution, never. Since 1 April 1952 the Pasteur strain, which formerly was continually passed in the rabbit, has been regularly passed once a month. For passages of the strain, at least two rabbits with a minimum weight of 2 kg are inoculated intracerebrally with 0 25 ml of a 1 10 suspension of brain tissue prepared from the preceding passage (the brain is kept in glycerol at +500). The rabbits, which are paralysed on the sixth day, are sacrificed by bleeding on the seventh day. The brains, which are extracted under sterile conditions, are subjected to bacteriological sterility tests, histological examination for the detection of typical fixed virus lesions (see colour plate, fig. 6, 7, • Contnbuted by Pierre Lépine (Chich and R Bequenon Virus Section Institut Pasteur, Paris, France (A) and Martin M Karlan Chief Veterinary Public Health Officer, Division of Communicable Diseases Services World Health Organization, Geneva, Switzerland (B) - 77 - ·78 P. LÉPIXE & R. BÉQUIGXOY and 8, facing page 40) and titration of the sinus by inoculation of a dilu:e suspension of fresh virus into rabbits. Immediately after removal, the brain is placed in purc, sterile glycero! and dispensed into wide-necked bottles. Labeis indicating the number of the rabbit and of the passage are placed on all bottles, which are kept in the refrigerator at between +2°C and +5°C. By 1 June 1953, the Pasteur strain had undergone 1,908 passages since its isolation. Preparation of inoculation virus For inoculating sheep the last passage of the strain is used, i.e, a brain from the regular series of passages, kept in glycerol in the refrigerator for more than a week but less than a month. A fragment of brain is removed. rinsed under sterile conditions with sterile distilled water, and ucighed It is then ground up in a chilled mortar so as to obtain finally a 1:10 cmulsion of brain (by weight) in physiological salt solution. This constitutes the inoculum. Each sheep is injected intraccrebrally with 0.5 ml of the inoculum at one point of the brain, using the technique described below. Preparation of sheep For vaccinc purposes only young sheep in good health are used. The average weight of the animals is 42 kg, and the brain of such animals weighs about 80 g. The animals are shorn and kept under observation for FIG. 1. TABLE FOR RESTRAINING SHEEP FOR INTRACEREBRAL INOCULATION ·79 METHODS OF VACCINE PRODUCTION FIG. 2 SITE AND TECHNIQUE FOR TREPHINING OF SHEEP three weeks and all animals which do not appear to be completely normal are eliminated On the day before inoculation the head of the sheep is shaved and the skin then disinfected by vigorous brushing with a brush steeped in cresyl solution The skin of the skull is then painted with tincture of iodine This last procedure, which is carried out in the morning and evening, is repeated on the morning of the next day At the time of inoculation the shaven skull of the animal has thus been desinfected with cresyl and with three applications of tincture of iodine. The animal is taken to the inoculation laboratory and immobilized on a special table (see fig 1) The skin is held taut by an assistant, and trephıning performed, without a previous incision, using a trephining drill with a point 13 mm long and 2 mm in diameter (see fig 2) The point of the instrument is directed towards the lateral-external region of the left hemisphere of the brain (lateral face of the frontal lobe) As mentioned above, cach animal receives 0 5 ml of inoculum in a single injection Several sheep are always inoculated during the same session, the usual number being eight sheep per session The sheep show the first symptoms of rabies seven to eight days later, and all are sacrificed on the ninth day when they are already lying on their side but still alive The animals are ·82 P. LÉPIVE & R. BÉQUIGAON FIG. 5 EXTRACTION OF SHEEP BRAIN Lett-itenilo Pyrex-glass jar In which brain to kept at low temperature cooled by a jucket filled with crushed ice. Two brains at a time (about 160 g of brain) are placed in the apparatus. Grinding is performed without diluent, with the machine running at full speed for a period of four minutes. A small amount of sterile physiological salt solution is then added. Alles having allowed the blendor to run for three minutes to ensure that the mixture is homogencous, samples are taken for the control culture and the final dilution is made in large Pyret containers holding five litres. Thc diluent consists of sterile physiological salt solution which is phenofized at the time of use. To do this, a s solution of purc phenol is made with physi ingical salt solution and is diluted immediately before use in the propor'. necessary to obtain the final concentration of vaccine The propor': . 1. arc calculated according to the scight of the brains as follows 15 16 ml Sheep nein Sterile pulogical salt solution Spher a physiological salt solution ·METHODS OF VACCINE PRODUCTIOV 83 The vaccine mixture is agitated and then left for 24 hours in an ovca it 22°C. On removal from the oven, the vaccine is immediately dispensed into 5-ml ampoules. It is essential for the mixture to be absolutely homogeneous so as to ensure that the quantity dispensed into cach ampoule is Fig. 5. DISTRIAUTION INTO AMPOULES OF ANTIRABIES VACCINE Note turbo-strrer immersed in vaccine suspension wuch ensures dispersion of us during distribution exactly the same For this purpose it is necessary to keep the vaccine stirred during the process of filling into ampoules In order to do this, special adapters and shaſts of an electric turbo-stirrer are used. The stainless-steel shaft (about 35 cm long), turbine, and adapter can be detached ·84 P. LÉPINE & R. BÉQUIGNOY from the driving motor and sterilized in advance. The turbo-stifter has the advantage over ordinary stirrers in that it cnsures a much more homo geneous mixture without frothing. In this way unlimited quantities of vaccire can be filled into ampoules with the certainty of rigorous homogeneity and, consequently, of correct dosage (sce fig. 6). The vaccine ampoules are kept for at least a weck at between +2"C and +5°C before being sent on to the packing service. During this minimum period of storage, which completes the inactivation of the virus, the final control of the vaccine is carried out. Final control of vaccine Before being distributed for use, the vaccinc undergoes the following tests (1) Sterility test, as described above. (2) Inactivation test, by intracerebral inoculation of the rabbit. (3) Potency test. For this test cither Habel's method (scc sections 11 and 12, pages 112 and 116) is used or the rabbit control test described by Béquignon & Vialat (sce section 14, page 125). A rabbit scighing 2 kg receives the vaccinc subcutaneously for ten consecutive days at the rate or 2 ml per day. On the 30th day it should be protected against the intracerebral inoculation of a fixed-virus dilution of 1: 10,000. Furthermore, from time to time vaccine samples are subjected to an additional control consisting of the assay of the dried extract and of the phenol, as well as determination of the pl. Period of use The vaccinc can be used during a period of five months dating from its distribution, prouded that it is kept at a temperature of between 2 and SC throughout this time If low-temperature storage cannot be guaranteed, the period of use of the vaccine is decreased to two or three months, according to the climate of the region where it is employed Phonetized vaccine should not be frozen at any time during its propter ration or storage Method of use The vaccine is supplied in bores cach containing 20 S.ml ampoules. representing a complete treatment in the case of sesers bites and his on the face, 30 injections are recommended instead of the 20 which con. stitute the standard treatment The volumes injected are reduced by half in the case of children aged less than 10 years ·85 BIETHODS OF VACCINE PRODUCTION B. RABBIT-BRAIN VACCINE : METHOD RECOMMENDED TO MEET REQUIREMENTS OF UNITED STATES NATIONAL INSTITUTES OF HEALTH The following procedure is recommended so meet the stringent requirements of the National Institutes of Health (NIH) in the United States of America? Horses, sheep, and goats can be and are used instead of rabbits for the preparation of vaccines, particularly for veterinary purposes (sce section 6(A), page 77). Formula of vaccine The vaccine comprises 20% inactivated rabies fixed virus in phosphatebuffered saline (pH 7), 0 25% phenol, 1:10,000 thiomersal (merthiolate). The NIH requirement for vaccine to be used in human beings stipulates that each dose of the final vaccine should contain not less than 20 ml of 1 5% brain-tissue suspension, or its equivalent in another concentration. The dose of a 20% suspension would therefore be o 5 ml. With respect to preservative, in addition to the final concentration of 0.25% phenol, phenylmercuric borate 1 • 12,500 or thromersal 1. 10,000 may be used. Phenol concentration in the finished vaccine should not exceed 0 25% for vaccines containing 10% or less of brain tissue, and should not exceed 04% for vaccines contaroing more than 10% brain tissue, Virus strain The Pitman-Moore strain, or other suitable fixed virus strain which through experience has gnen satisfactory results, should be used Maintenance of virus strain Virus passage for vaccine production is always through rabbit brain, A seed virus is used for any convenient period (usually not more than three months) as long as full potency is maintained as shown by mouse tistration, A 25% virus suspension in buffered distilled water (pH 7) is quick-frozen and stored, preferably in a CO, chest (-70°C) but at least at below--15°C. * The procedure described is het on aurrectfit. - ·86 M. M. KAPLAN Seed virus must be of sufficient strength to kill all mice injected intracerebrally with 0 03 ml of the rabbit-brain suspension in a dilution of not less than 10, The purity of the virus strain is tested periodically by titration in mice against specific antırabies hyperimmune serum which has proved protective against street virus (see section 18, page 139). Production of seed-sirus 25% suspension Rabies-infected rabbit brains are weighed and then emulsified in 3 parts of buffered distilled water (pH 7). Emulsification is cffected in a small (200-ml capacity) Waring blendor for 3 minutes ; l-ml amounts are placed in 5-ml ampoules, sealed, quick-frozen in dry-ice and alcohol, and stored, preferably at dry-ice temperature (-70°C), but at least at below --IS C. Sterility 0.5 ml of 25% virus suspension is inoculated into cach of tuo tubes containing 30 ml of sodium thioglycollate medium or other suitable mel. for a 14-day sterility test, including a subculture incubated for seven days (sec page 90). Timur titration At least two intracerebral mouse titrations of 10°. 10. and 10 dilutions are performed. The test period is 14 days, and all mice receiving the 10- dilution should die. Inoculation ichnique Crom the 25% suspension of sccd vicus a final 1:1,000 dilution of virus is made in 2 normal serum in distilled water. The diluted srus suspension is centrifuged in an angle centrifuge (approximately 1,000 ) for 15 minutes, and the supernatant fluid is used as the inoculum. A fresh dilution is made for cach day's inoculation, and is tested for sterility in ihroglycollate medium Healthy rabbits weighing 3-4 kg arc firmly secured and 0 2 ml of the inux ulum is introduced by å sterile 2-mi syringe and 23-gauge 3/8-inch (0 65 mm 1 cm) reedle through a puncture made in the săull 1.3 m from the outer canil us of the eye This is usually into the right herrisphere only although quv inocula of 005 ml cach can be used for the tuo hemispheres À Stainless-steel axl or a punch can be used to make the puncture (see fiz ! ard 2)

·METHODS OF VACCINE PRODUCTION
FIG. 1. USE OF PUNCH FOR INTRACEREBRAL INOCULATION
By courtesy of Dr F Pere Gallardo Madrid
FIG
2 INTRACEREBRAL INOCULATION TECHNIQUE
By courtesy of Dr F Perez-Gallardo, Modrid

·88 MM, KAPLAN Progress of discase in rabbits On the 4th day rabbits show marked signs of paralysis, which inctease rapidly to complete paralysis on the 5th (or occasionally as late as the 6th) day. Killing of rabbus Moribund rabbits which have shown rabits symptoms for at least 24 hours, and are completely paralysed except for the chest muscles, are killed by intravenous injection of 10 ml of air into the marginal vein of the ear. Death occurs in from one to two minutes. The brains from freshly killed rabbits only are used for vaccine production; animals already dead from rabies or other causes are not used. Autopsy To guard against the use of unsuitable rabbits, autopsies are performed after brain removal. The brains of rabbits showing the presence of coccdiosis elsewhere in the body are not used because of the chance that coccidiosis rather than rabics may have caused paralysis. A record is made of all autopsy firdings. Brain harias! The rabbit is washed with crcolin or another suitable antiseptic solution Decapitation is performed as close to the shoulders as possible; the severed head is fastened securely into a special holder, swabbed with tincture of rodire, and taken into a " sterile room " where actual brain removal is performed The sien is sitt down the middle of the head and folded back and 11c crposed top of the shull us washed with iodine from a small dropper bottle. The top of the skull is then removed with sterile bonc-shears, and the bruin is transferred with sterile scissors and forceps into a suitable sterile container A small piece of the brain tissue is removed with a sterile saad trd tremulsiod in u tube of 15 mi or thoglycollate medium for a sterilay Bruins are stored at 150 or loucri preferably much louer, until needed for ucine Any brain showing bacterial contamination is dis carded Iest Manufacture of Vaccine : 40Suspension Imulsification Frozen bruins are weighed and placed in a 2-litre Erlenmeyer fast as # 4*, suspension in serie phosphate-buffered sodium chloride solution ·METHODS OF VACCINE PRODUCTION 89 L.1.-1.a. containing sufficient phenol to give a final concentration of 0.5% (see under Diluent below) After the brains are thawed, the flask is shaken by hand until they are well broken up. This partial emulsion is then trans- "'ication other inning the emulsifier long enough to generate appreciable heat. From two to three minutes are usually sufficient for a Waring blendor to emulsify the brains adequately. The container can be surrounded with a jacket of chipped ice and salt to prevent overheating of contents Diluent (a) Phosphate-buffered sodium-chloride solution : prepare a solution containing 2 parts M/10 Na,HPO, and 1 part M/10 KH,PO, To one part of this solution add four parts o 85% NaCl of pH 70. (b) To make a 40% tissue-concentration containing 0.5% phenol, add to phosphate-buffered saline solution 68% of 90% fine reagent phenol (this allows for volume of emulsified brain tissue), make a 40% weight) volume with the diluent according to weight of the brains. Note. After phenol has been added, great care should be taken not to freeze brain-tissue suspensions, either in the concentrated 40% form or in the final vaccine dilution. Freezing of phenolsed suspensions destroy's antigenicity Virus titre of brain-1155ue pool before incubation A sample of 40% tissue suspension is weighed (because of air content from action of the mill), diluted immediately, and titrated in mice weighing 16-20 g Dilutions of 104, 10-, and 104, representing dilutions of brain-tissue content, are injected intracerebrally and the titre (L.Dso) should be at least 10% (see section 11, page 113 for calculation) incubation The emulsified vaccine containing o 5% phenol is brought to 37°C in a water-bath for 1 hour and then placed in an incubator at 37°C for about 72 to 76 hours (the longer period is used if the preliminary safety test fails) The vaccine is agitated mechanically during the entire period in the incu. bator Note The period in the incubator required for inactivation will vary with different virus strains and Metbods of emulsification of brain issue (fineness of particles) The time required should be established in each ·90 M. M. KAPLAN laboratory through experimental trials, c.g., intraccrebral inoculation of mice with suspension at 6-hour, or less, incubation intervals. Dilution to 20% vaccine The 40% vaccine is diluted with an equal volume of the sterile phosphate-buffered salt-solution containing 1:5,000 thiomersal, to make the finished product 20% tissue, 0.25% phenol, and 1:10,000 thiomersal Diluted vaccinc is passed through seseral layers of gauze or silk filter for the purpose of removing any unground particles of brain tissue. A convenient batch of vaccine is about 2 litres in volume. It is subjected to the safety and sterility tests described below, and is stored at 12-67. Sterilication A sterility test of the finished vaccine is made in sodium thioglycollate medium or other standard media. Ten mi from cach bulk container of vaccine is divided into 3 portions and inoculated into 3 bottles cach containing 225 ml of medium. The test is mixed and placed in an incubator at 310-32°C. The medium is mixed carefully after a 48-hour incubation period. Tests are observed cach day for 7 days for bacterial growth. At the end or 7 days the bottles are mixed carefully, and I ml from cach bottle is inoculated into tubes containing 40 ml of fresh sodium thioglycolla18 medium. Original bottles and subculture tubes are incubated for another 7 days Any Vaccinc showing contamination is reiested, using a double volume for the test. Safeu lest Immediately after dilution and at the time of sterility testing, a 2-ml sample is taken for the intracerebral inoculation of 5 mice with a 0 03-mi dosc At least 3 of the 5 mice must survive without symptoms of rabies Final raichung The final mixture is made after the end of the preliminary safely test, or after re-incubation of the vaccine at 264C if at least 3 of the mice tested have not survised the preliminary safety test Batches are combined in : boule of adequate size for filling ampoules. Samples are taken for the following tests Aralı sical disas A sample of finished vaccine is re-incubated for 24 hours at 37°C 10 ensure the killing of the sus, and the following determinations are made: total solids, phenol, pil, and thiomersal.

·METHODS OF VACCINE PRODUCTION 91 Sterility Test performed as described under Sterilization, page 90. Innocuity Not less than 2 rabbits and 2 mice are used Each rabbit is injected intracerebrally with not less than 0 25 ml and each mouse (weighing 18-20 g) with 0 03 ml of the vaccine containing not less than 5% of brain tissue. All test animals are observed for not less than 14 days, during which time each animal must remain free from symptoms of fixed rabies or of other central-nervous-system disease. Final standardization to meet NIH Requirements Potency: (see section 13, page 117). Safety: A safety test is made on the contents of a final container selected at random from each filling of each lot, or portion of a lot The . AA death At least 2 animals of cach species are used, and the observation period is not less than 7 days. Dating The expiration date is not more than six months after the date of manufacture or date of 15$ue. For dating purposes, the date of manufacture may be considered as the last date of passing a satisfactory potency test The date of issue may be three months after the date of manufacture. The product is stored at 20-5°C at all times. Freezing of phenolized suspensions should be carefully avoided ·Section 7 ULTRAVIOLET-LIGHT-IRRADIATED VACCINE Principles and Methods The ability of irradiation by ultraviolet (UV) energy to cause bacterial sterilization has been known for several years, but the effectiveness of UV-inactivated microbiological antigens as vaccines is a more recent application of that knowledge. UV energy has little ability to penetrate biological substances since it is absorbed rapidly. Therefore, for electic exposure, the material being irradiated musi be presented to the incident UV rays in a very thin film The present practical use of UV-irradiated rabies vaccinc became possible only after the development of apparatus which would accomplish this thin-film type of exposure. It is also well known that as the amount of UV energy absorbed by biological material increases, the degree of chemical change in that material increases, so the length of ciposure must be as short as possible while accomplishing the purpose of inactivation, other: Misc breakdown of the antigen will occur. The biological effect of UV Irradiation on viruses is instantaneous, for all practical purposes, and once this immediate effect has taken place, no further action ensues. There is no cudence that any secondary by-products are formed in the irradiation process which would subsequently be deletcrious to the antigen, Besides the necessity for a controlled length of exposure and for the material to be irraduated in a thin film, the practical production of large solumes of vaccine requires that the apparatus used should allow for a continuous ſon of the antigen suspension through the equipment. Although quarts is a good conductor of UV energy and has been used extensively for making various types of container in which an antigen could becisosed, in general an apparatus designed to eliminate the passage of the UV rays through quartz gives more eficient radiation. Case of operation and the use or un casily swallha incurense source of UV arc, of course, other desirable features of any practical arradiation apparatus. • Cerimot hy hat Hentatzef 1shorziory of Infectious Distant s'nal Mar wolnllue Punte Heal-XII Hatha MUSA -- 92 - ·93 METHODS OF VACCINE PRODUCTIOV Apparatus Several types of apparatus have been designed which in some way incorporate the principles listed above. Two types are available commercially in the United States of America and both have been proved to be FIG. 1. CONTINUOUS-FLOW UV-EXPOSURE APPARATUS A dye solution is being used for illustrative purposes satisfactory in the production of potent rabies vaccines The “ Dull" apparatus employs a standard high-energy germicidal lamp that has a long effective life, and is easily available and relatively inexpensive. The Oppenheimer-Kettering" apparatus uses a special highly efficient lamp which is more expensive I Dill Irradiator. I J Dill Co. 1302 Bixby Road Kalamazoo, Mich USA Oppenheimer Kettends UV radiator Michael Reese Research Foundation, 29th Street and Ellis Ave, Chicago, USA ·94 X. HABEL However, the construction of the type of apparatus of thich the Dill machine is a modification can be accomplished by any modern machine-shop from basic materials that are readily available and not crcessively expensive. Thc shop-made model used in our laboratory consists of a long hollow metal or glass tube, preferably of stainless-steel, so that the inner surface over which the virus suspension flous is very smooth and casily cleaned FIG. 2. INTAKE END OF UV-EXPOSURE APPARATUS This tube is of such dimensions that a germicidal lamp suspended through the centre of it will have one centimetre of clearance from the inner wall. This tube is fitted 10 slide smoothly into an outer, more sturdy, tube made of brass, which is mounted in bearings at cach end. These tubes are lo he rotated at a specd hetueen 500 and 1,000 resolutions per minute ( p m), so a driving force must be applicd. This may be accomplished by a pale or helt-drive, but in our own apparatus is achieved rather simply by making the outcr brass tube the armature of a small AC motor of the tyre used in electric fans for long runs in large-scale production thus muy result in greater heating of the tube ·95 METHODS OF VACCINE PRODUCTION A suitably designed collecting-cup fits tightly to the discharge end of Lahannarntne FIG. 1 COLLECTING END OF UV-EXPOSURE APPARATUS inner side of the rotating inside tube, where it is immediately spread in a film approximately 1 mm in thickness which progresses the length of the tube The film is thus continuously erposed to UV light and is collected through a closed connexion into a flask at the opposite end. A burette or inverted fiask may be used as the original reservoir of virus suspension, and is connected to the intake-needle by rubber tubing with a valve for adjusting rate of flow inserted in the line (see fig 1-3) Blueprints of this apparatus are available from the author at National Insututes of Health, Bethesda, 14 Md, USA ·94 K. HABEL However, the construction of the type of apparatus of which the D.!! machine is a modification can be accomplished by any modern machine-shery from basic materials that are readily available and not excessively copersise The shop-made model used in our laboratory consists of a long hollow metal or glass tube, preferably of stainless-steel, so that the inner surface over which the virus suspension flows is very smooth and easily clancy FIG. 2. INTAKE END OF UV.EXPOSURE APPARATUS This tube is of such dimensions that a germicidal lamp susper the centre of it will huse one centimetre of clearance from ih This tube is fitted to shde smoothly into an outer, more sturdy of br.18, which is rounded in bearings at cach end. These he rulated atosphetween 500 and 1,000 resolutions per min 50 mng for ust be applied. This may be accomplishe or helt af... hu in our own apparatus it is achieved rathe making the Artuss tuh. armature of a small AC moto used in clair1 ans une runs in large-scale products result in " tube ·METHODS OF VACCINE PRODUCTION 97 times the minimum inactivating exposure is necessary before any marked drop in antigenic potency occurs. Virus Emulsion All the factors of importance in rabies-vaccine production by any other method of inactivation are of equal importance for material to be irradiated. The virus titre of the original emulsion should be as high as possible, but variations of as much as one log in the titre will not noticeably change the necessary exposure time. Since UV light will not penetrate biological materials to any appreciable depth, it is quite important that the emulsion be a uniformly fine one. To ensure this it is desirable to screen the emulsion through several layers of sterile surgical gauze or a wire screen. In general, a $% whole emulsion of infected nervous tissue is used with the irradiation equipment, but no difficulty is encountered in inactivating suspensions up to 10% in concentration. Presen anon and storage of irradiated vaccine If irradiated vaccine is to be kept in the liquid state before use, a chemical preservative should be added Thiomersal (merthiolate) at a l. 8,000 dilution, or 0.25% phenol, appears to be superior to formalin. irradiated vaccine, because it contains no substance in itself deleterious to viral antigen, may be kept frozen or may be dned from the frozen state. The latter process of freeze-drying will cause an immediate drop in immunizing potency, but the vaccine will then hold that potency indefinitely of sufficiently dry. Testing of irradiated vaccine Starta en more consistently by the irradiation method than by any other method of Wactivation There is no evdence to date that the incidence of post-vaccinal complications is any greater after the use o uradiated vaccine than with other types Steps in an irradiation run (1) The inner tube is cleaned with soap or detergent solution, thoroughly nised, and then cleaned with ether, ·98 K. HABEL (2) The apparatus is assembled, and the UV lamp inserted through the tube ard clamped into its sockets. (3) The collecting end-piece, which has been autoclased, us conrada! to the apparatus and, through sterilized rubber tubing, to the collect": flask. (4) Two reservoirs, one with sterile distilled water and one wh rs emulsion, are connected by a Y-tube to the rubber tubing going to the intake-needle through a flow-valve, (5) The intake-needle is adjusted to direct flow against the inrer sc's of the rotating tube, and is fixed in that position. (6) The UV lamp is turned on for 5 minutes to sterilize the inside of the rotating tube, the motor of which has been started. (7) Distilled water is run through the apparatus to wet-down the ruhe. and the flow is adjusted to the desired rate. (8) The flow of the distilled water is stopped, and that of the virus emulsion started. The rate of low is rechecked. As soon as the emula aprears at the collecting end, a fresh collecting flask is attached, ard tim of the run started. The time is again taken at the end of the run, ard tº: volume of collected emulsion is measured.

·Section 8 * CHICKEN-EMBRYO VACCINE At present the Flurya and the Kelevi strains of rabies virus adapted to and modified in the devcloping chicken embryo are used for the produc. tion of chicken-embryo rabies vaccines employed in the prophylactic immunization of dogs. Vaccire made with the Flury strain has been used on a worldwide basis and therefore the description of the production procedure will be mainly confined to the Flury-strain vaccine. The same prin. ciples, however, are followed in the preparation of chicken-embryo rabies Vaccine with the Keley strain The Flury-strain vaccine for dogs represents the 40th-50th egg-passage level of the virus. At this ego-passage level the Flury virus is fully virulent by the intracerebral route for mice, cotton-rats, hamsters, and guinea-pigs, while rabbits are more resistant. Hamsters, mice, and cottonrats are susceptible to extraneural injection, but guinea-pigs are more Tesistant On the other hand, rabbits and dogs fail to show signs of infec tion when inoculated intramuscularly with concentrated suspensions of the virus. Further modification of the Flury strain has been achieved by continued egg passage (180th passage), and this has resulted in a lessened pathogenicity for experimental animals while its antigenicity has apparently been retained. For the present the high egg-passage level vaccine has been recommended for use only in cattle and the low egg-passage level vaccine, described here, for use only in dogs and cats 3 The Kelev chicken-embryo vaccine represents the 60th-70th egg-passage level of the virus At this egg-passage level the virus does not produce any signs of infection in hamsters, guinea-pigs, or rabbits inoculated either extraneurally or istracerebrally Suckling mice are, however, susceptible when inoculated intracerebrally Dogs inoculated intramuscularly with concentrated suspensions of virus fail to show signs of infection, Preparation of seed material At the proper egg-passage level of the virus (Flury, 40th-50th, and Kelev, 60th-70th egg-passage level), a sufficient quantity of 60% embryo Contbuted by A Komarov, Director, Government Virus Diseases Laboratory, Haifa, Israel . These strains are available 10 national laboratories on request to the World Health Organization, Palais des Nations, Geneva, Switzerland - 99 - ·98 K. HABEL (2) The apparatus is assembled, and the UV lamp inserted through the tube ard clamped into its sockets. (3) The collecting end-piece, which has been autoclaved, is conrected to the apparatus and, through sterilized rubber tubing, to the collecting flask. (4) Two reservoirs, one with sterile distilled water and one with virus emulsion, are connected by a Y-tube to the rubber tubing going to the intalc-needle through a flow-valve. (5) The intake-needle is adjusted to direct flow against the inner sal of the rotating tube, and is fixed in that position. (6) The UV lamp is turned on for 5 minutes to sterilize the inside of the rotating tube, the motor of which has been started. (7) Distilled water is run through the apparatus to wet-down the tube. and the flow is adjusted to the desired rate. (8) The flow of the distilled water is stopped, and that of the virus emulsion started. The rate of flow is rechecked. As soon as the cmul on aprears at the collecting end, a fresh collecting Nask is attached, ard timing of the run started. The time is again taken at the end of the run, ardere olume of collected emulsion is measured.

·Section 8* CHICKEN-EMBRYO VACCINE At present the Flury and the Kelev strains of rabies virus adapted to and modified in the developing chicken embryo are used for the production of chicken-embryo rabies vaccines employed in the prophylactic Immunization of dogs." Vaccine made with the Flury strain has been used on a worldwide basis and therefore the description of the production procedure will be mainly confined to the Flury-strain vaccine. The same principles, however, are followed in the preparation of chicken-embryo rabies vaccine with the Kelev strain. The Flury-strain vaccine for dogs represents the 40th-50th egs-passage level of the virus At this ego-passage level the Flury virus is fully virulent by the intracerebral route for mice, cotton-rats, hamsters, and guinea-pigs, while rabbits are more resistant. Hamsters, mice, and cottonrats are susceptible to extraneural injection, but guinea-pigs are more resistant On the other hand, rabbits and dogs fail to show signs of inſecuon when inoculated intramuscularly with concentrated suspensions of the virus Further modification of the Flury strain has been achieved by continued ees passage (180th passage), and this has resulted in a lessened pathogenicity for experimental animals while its antigenicity has apparently been retained. For the present the high egg-passage level vaccine has been recommended for use only in cattle and the low egs-passage level vaccine, described here, for use only in dogs and cats 3 The Kelev chicken-embryo vaccine represents the 60th-70th egg-passage level of the virus. At this egg-passage level the virus does not produce any signs of infection in hamsters, guinea-pigs, or rabbits inoculated either extraneurally or intracerebrally Suckling mice are, however, susceptible when inoculated intracerebrally Dogs inoculated intramuscularly with concentrated suspensions of virus fail to show signs of infection Preparation of seed material At the proper egg-passage level of the virus (Flury, 40th-50th, and Kelev, 60th-70th egg-passage level), a sufficient quantity of 60% embryo Israc1 • Contributed by A Komarov Director Government Virus Discascs Laboratory. Haifa, These strains are available to national laboratories on request to the World Health Organization, Palais des Nations, Geneva, Switzerland - 99 — ·100 A. KOVAROV suspension in distilled water is prepared, tested for polency (see section 15. page 128) and preserved either frozen or in its dehydrated form. This constitutes the seed material. Just before use a 20% suspension of seed material in distilled water is prepared and tested for viability as follows. Inoculate a group of six mice intracerebrally with 0.03 ml of a 10-9 ditu. tion of the seed material (use a 0.25-ml tuberculin syringe with a "cinch 27.gauge (0.40 x 6 mm) needle). If the secd matenal contains lise sinus the mice will sicken on the 6th-8th day. Should inoculsted mice remain unafected by the 9th day, discard the inoculated eggs. In the case of the Kelev strain the viability test must be carried out in suckling mice. Inoculonon of eggs The number of eggs to be inoculated will depend on the amount of vaccine required and storage facilities. On a large production scale the average yield is 4-6 dog-doses per haricsted embryo. Embryo mortality due to non-specific causes docs not usually exceed 15%. Fertile hen eggs incubated for 7 days are used. It is best to obtain ferile incubated eggs for saccine production from a commercial hatchery and transfer the cges to the laboratory one day before inoculation. On the 7th day of incubation the eggs are candled : unfertilized eggs and those with dead or weak embryos (poor vascularization, sluggishness), and eggs showing displaced air sacs are discarded. While candling, the boundaries of the air sacs are marked on the shell with a pencil and the eggs are arranged air sac uppermost in a tray (a commercial cardboard ces-tray serves the purpose well) The shell is painted at the top of the air sac with 70" akcohol. and flamed briefly. By means of a carborundum disc attached to a rotary motor tool, or a dental drill, make fuo crosswise cuts, or penetrate the shell directly at the top and centre of the air sac. If this type of cutting machine is not available, piered the shell at the indicated point hy means of any sharp instrument (see fig. 1) The eggs are inoculated in the poll sac with 0 3 ml of the 20* suspen. sion of seed material, using a Syringe with a ',-ach to l-inch 19.1020-Faure (6-25 mm · 090-1.10 mm) needle till the syringe with the inoculum .....he hand nastal with the rable with the left hand uit 1 the egg and deposit 0.25 ml of the inoculum (ke tig ! The inccultat est are replaced in the tray Oncompleti 77 of th: 1706 station that material

·METHODS OF VACCINE PRODUCTION
101
FIG. 1. PIERCING AIR SAC
r.
By courtesy of Dr F Perez-Gallardo Afadrid
FIG 2. YOLK-SAC INOCULATION OF EGG IN HORIZONTAL POSITION
a
By courtesy of DI F Perez-Gallardo. Madrid

·102 A. KOVAROV is tested for bacterial sterility, and the cut portion of the shell is sealed by means of a melted mixture of paraffin-baseline (two-thirds paraffin, ose third vaseline), or collodion, After inoculation the eggs are incubated at 36 SC for a period of 9-10 days Hariesi At the end of the incubation period, only living embryos are hanesied Yolk, yolk-sac, extra-embryonic tissue, and fluid are discarded. The eggs to be harvested are arranged in a tray, and the shells are sprayed or painted with 70% alcohol and flamed. The live embryos are harvested by either of the following metheus. (a) Cut off shell over the air sac by means of sterile sharp pointed scissors (see fig. 3) Tear away the membrane, thus exposing the embro Pass a wire hook attached to a handle under the nech of the embro, and pull the embryo slowly upward. The embryo will be pulled out of the cge, frec from the yolk and extra-embryonic material. FIG. 3. CUTTING OFF SHELL COVERING AIR SAC O! Di. f Perr: Gulur, ·METHODS OF VACCINE PRODUCTION 103 FIG. 4. SEPARATING EMBRYO FROM MEMBRANES ch By courtesy of Dr F. Perez-Gallardo, Madre Alternatively, lift the embryo out with one pair of forceps while separating the membranes with another (see fig. 4) (6) Put on sterile rubber gloves. Crack the shell of the eggs at the centre by means of a sharp sterile instrument. Open the shell sith gloved hands and pour the contents over a sterile wire gauze. The fluid and yolk should drop through the meshes into a sterile container placed under the wire gauze layer. Collect the embryos from the layer of gauze into a proper container. Preparation of embryonic suspension The harvested embryos are placed in a chilled weighed container. The total weight is noted and then the embryos are transferred into a chilled Waring blendor. Sufficient chilled sterile distilled water (containing 500 units of penicillo and 1 mg of streptomycin per millistre where laboratories find that contamination occurs) is added to the blendar to prepare a 33% embryo suspension. The embryos are ground three or four times, each time for about 3 minutes, chilling the blendor between each grinding. The resulting suspension is filtered by squeezing it through a layer of gauze into a chilled container The filtered embryo suspension is placed in 5-ml ampoules so that each ampoule contains 3 ml of 33% embryo suspen- ·104 A KOVAROV sion-this equals one dog-dose. Multiple doses can be used without increasing the volume of liquid, by preparing a 66% embryo suspension. Desiccated raccine The embryo emulsion is desiccated from the frozen state. After desicca. tion, the ampoules are sealed either in vacuum or in an atmospherc of ritrogen, and stored at 4°C. In our hands, after desiccation with an Edwards apparatus, the virus titre is approximately 1055 to 104. Bacterial safery test Desiccated material is rehydrated and 1 ml is injected subcutaneously into 8 mice. At least 7 out of the 8 mice must survive an observation food of 14 days. Potency test Virus titre is distinct from the potency of the vaccinc. Titres may be as low as 10-1 and yet the vaccine can be potent. Vaccine should be be released for use unless tested for potency. A potency test for chick-cmbryo rabies vaccine is described by Ko roushi (see section 15, page 128). The test consists of the intramuscular inoculation of guinea pigs with vaccine, followed 21 days later by intra muscular challenge of the vaccinated animals with street virus. The challenge virus consists of an emulsion of rabies-infected dog salivary giards Preliminary intramuscular titration of the virus in guinca-pigs is made and the highest dilution of the virus which kills 70%-100% of the control animals is used for challenge. In the actual test, 10 guinca-pigs each receive 0.25 ml of 5%-suspension chick-embryo rabies vaccire. ard. 10 animals are left as controls. Twenty-one days after the vaccination, animals are inoculated intramuscularly with 0.1 ml of dog saluaro-glar.d challenge virus A vaccine of good potency will protect 70% of the 130€** nated guinea pigs REFERENCES 1 Lomaros A Herenstein, K (1993) Cornell t'a 43, JH : koprowskı, II & Cus H R (1949) Immunol. 60, 533 World Ilealth Organization. Expert Commille on Rabics (1994) HW W O'R trcan Rep Sm *!

·Part III VACCINE POTENCY TESTS

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·Section 9 GENERAL CONSIDERATIONS Although the need for evaluating the immunizing potencies of rabies vaccines has been recognized since the early Pasteur days, and a large volume of experimental work aimed at this goal has been carried out over the years, most laboratories producing rabies vaccine have not made a routine practice of testing their products until secently. Thus, when prac. lical potency tests became available many laboratories were surprised to find that they had been labouring under a sense of false security since the vaccines they had been issuing for years, prepared according to sellestablished production procedures, were actually of very low or negligible immunizing ability The now well-known characteristic property of all viruses, including rabies fixed strains, to mutate and change in their properties with animal passage, makes 31 obvious thas the known requirements for the use of any one virus strain in preparing a potent vaccine 10 years ago may not be valid today It has always been difficult to evaluate vaccines on the basis of results obtained in human beings because of the lack of controls, the relatively small number of human cases of rabies, and the impossibility of taking into consideration all the many factors which tend to make human exposures uncomparable Furthermore, the need is for an evaluation before a vaccine is released for human or veterinary use There would appear to be three important considerations, among others, in assessing any potency test of rabies vaccines First, the lest procedure should actually evaluate the property of the vaccine which determines its effectiveness in the prophylaxis of rabies in man or animals, Using a naturally susceptible host, the ideal test would simulate conditions of natural esposure and usual prophylactic treatment. This would mean the use of street virus introduced those wh. doses of vacci has, of course where adminis, AUVIL UI vaccine is started after etperimental exposure of the rest animal Most tests, therefore, involve multiple doses of vaccine (as administered in man) followed by subsequent challenge with fixed virus Contributed by Karl Habel Chef Laboratory of Infectious Diseases, National MITO biological Institute Bethesda, MD, USA

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·Section 9 GENERAL CONSIDERATIONS Although the need for evaluating the immunizing potencies of rabies Vaccines has been recognized since the carly Pasteur days, and a large volume of experimental work aimed at this goal has been carried out over the years, most laboratories producing rabies vaccine have not made a routine practice of testing their products until recently. Thus, when practical potency tests became available many laboratories were surprised to find that they had been labouring under a sense of false security since the vaccines they had been issuing for years, prepared according to well. established production procedures, Mere actually of very low or negligible immunizing ability. The now well-known characteristic property of all Viruses, including rabies fixed strains, to mutate and change in their properties with animal passage, makes it obvious that the known requirements for the use of any one virus strain in preparing a potent vaccine 10 years ago may not be vald today. It has always been difficult to evaluate vaccines on the basis of results obtained in human beings because of the lack of controls, the relatively small number of human cases of rabies, and the impossibility of taking into consideration all the many factors which tend to make human exposures uncomparable. Furthermore, the need is for an evaluation before a vaccine is released for human or veterinary use. There would appear to be three important considerations, among others, in assessing any potency test of rabies Vaccines. First, the test procedure should actually evaluate the property of the vaccine which determines its effectiveness in the prophylaxis of rabies in man or animals Using a naturally susceptible host, the ideal test would simulate condztions of natural exposure and usual prophylactic treatment. This would mean the use of street virus introduced through a bite-wound, followed by daily doses of vaccine in the case of those vaccines intended for human use This has, of course, been found to be impracticable, as have most types of test where administration of vaccine is started after experimental exposure of the test animal Most tests, therefore, involve multiple doses of vaccine (35 administered in man) followed by subsequent challenge with fixed virus Contnbuted by Karl Habel Chef Laboratory of Infectious Diseases, National Microbiological Institute, Bethesda, MD USA -107 ·108 K. HABEL given intracerebrally, as being a more easily standardized type of challenge While far from being a duplication of the situation with natural exposure and the usual use of vaccine, this type of test has been shown to react the relative ability of a vaccine to protect under more natural cordille"... The second important factor in a potency test is its practicability to case with which it is carried out, the availability of the material used, and the cost and time involved. Not all laboratories can obtain large cumbers of experimental animals casily, nor can they, perhaps, support the cirease of repeated tests when animal costs are high. The time factor is important. since newly prepared vaccine must be held until potency tests are comples. and this is time deducted from its period of effectiveness. The third important factor is the standardization of the test prazdort so that there may be comparability results from one laboraton 1 another In the following sections of Part III five types of rabies potency lest are described in detail. All these may be used for evaluating vaccines for both human and veterinary use, although the street virus guinea.fi test (see section 15, page 128) is specifically designed for testing the live vires chicken-embryo type of vaccine for use in animals. The decision as to which test to use in any particular laboratory w:] depend on the type of information required and the facilities available. !! a laboratory has limited facilities for animal procurement, and wiss mercly to screen its vaccines for potency or lack of potency, then the riche simple test, such as the modified Habel test in mice, should be used the section 12, page 116). If facilities are available, and a quantitative cvaldstion of potency is desired as a continuing check on efficiency of production then one of the more standardized tests is indicated. The original Nake! tutes of Health test (section 13, page 117) are both quantitatne testes, et test in mice (section 11, page 112) and the newly adapted US National Irso second being a more standardized procedure. The disadvantage of this en animals. The Pasteur Institute test in rabbits is also a quantitate tes? lies in the necessity for using a standardized vaccine and a large number of similar to the Habe! tost in mice.

·Section 10 SOME FACTORS INFLUENCING THE STANDARDIZATION OF RABIES-VACCINE POTENCY TESTS IN MICE For approximately the past decade the potency of rabies vaccines distributed under licence in the United States of America has been controlled by the use of an immunity-breakdown type of test in which all mice are vaccinated with the same dose of vaccine given in sit injections. At the time of challenge the mice are divided into five groups, each group receiving a different tenfold dilution of virus. The potency of the vaccine is determined by calculating the number of LD sy of virus which will overwhelm the immune mechanism in half the vaccinated mice An acceptable vaccine must protect mice against at least 1,000 LD50 of virus When the potency test described above is used for evaluating the same vaccines in more than one laboratory, certain variables have been recog. nized which could lead to vastly different potency-test results, both in the different laboratories, and in the same laboratory from one test to another. Some of these vanables are the strain of challenge virus, the technique of handling the challenge virus, and the strain of mice. The first variable, that of the strain of challenge virus, has been studied carefully by Habel and Wright and has been eliminated by the establishment of the standard challenge virus (CVS) strain of rabies virus which is supplied to all laboratories interested in the potency evaluation of rabies vaccine. The technique for handling the challenge virus was also standardized by Habel & Wright and a complete description has been published ? It has since been found that less variation is encountered in the titre of the challenge virus from test to test if, in preparing the challenge virus for each test, the virus suspension is diluted to 10% brain tissue with 2% serum in distilled water, and clarificd by centrifuging for 15 minutes at a relative centrifugal force of approximately 1,000 times E. In this way all larger particles of brain tissue are removed, and a slightly opalescent supernatant fluid is obtained which contains the virus in undiminished titre • Contnbuted by George A Hottle Chiet, Biologics Control Section, Laboratory of Biologics Control, National Microbiological Institute, Public Health Service, Bethesda, Md. USA 1 Habel, K & Wneht, J T (1948) Publ Hith Rep (Wash ) 63 44 - 109 - ·110 G, A. HOTTLE The problem introduced by the use of different strains of mice is ! serious one. Instances have been reported in which potency values for the same lot of vaccinc have varied from 100 LD,, to 100,000 LDA tion when diferent strains of mice were used. The first step which mit! be taken to control this variation would be the use of a reference 1321" which is put on lest each time a lot of vaccine is to be evaluated While I is desirable that the reference vaccine show the minimum acceptable poters, i.e., 1,000 LD, protection, this is not essential. The potency of the refereret vaccinc must be determined in mice of average immunizing capacity, 2-4 the potency value obtained must be accepted by all participating laborat ries. After the potency value of the reference vaccine has been established the results of each potency test can be evaluated on the basis of the accepty potency value of the reference vaccine. Although such an evaluation will not rescal a quantitative relationship between an unknown vaccine ard the reference vaccine, it is possible to get a qualitate comparison between tno vaccines by means of the immunity-breakdown type of potency test Because of the instability of rabies vaccines, it is suggested that a dritet reference vaccine be made available. This can be accomplished by susperu. ing the infected brain tissue in distilled water, inactivating the surus ar ultraviolet irradiation, and drying the vaccine from the frozen state A dried reference vaccine prepared in this way will have retaired a large part of its original antigenicity and will remain fully active for at least ore year Suggestions for a morc quantitative evaluation of ino saccines have been made on the basis of an antigen-extinction test in which groups of mice recere graded doses of vaccine. All mice are then challenged with the same dose of virus A 50% effective dose (ED) for each vaccine calculated as the amount of vaccine which will protect half the mice sairst the challenge dose of virus. Because of the variation in challenge dose of virus from one test to another, a reference vaccine is essential in this rest, and potency results are always gren in terms of the reſercnce vaccine. With a test of this type the number of injections of vaccine gren to mice may be sex, luur, ihrce, or csen iso doses. When fewer dous ! Saccine are injected into the mice, the number of LD, of virus in te challenge dose must likewise be reduced. With three or four doses or accore, the chef'enge dose of virus is usually 50-S00 LD, With Iwodows of vaccine the challenge dose of virus is 5-50 LDA. The third revison of the US National Institutes of Health in respinge requirih PTS sw'... jaccine, dated 3 february 1953. fines the daisi's of the sre: en-extli un type of test which will be used for evaluation all lots of rahisi diving distributed under US licence (see section 11. page 112. In thos.cat many of the details with regard to the challenge virus, ·VACCINE POTENCY TESTS used with the immunity-break down type of test, have been left unchanged However, in the vaccination of the mice, O 5-ml amounts of fivefold dilutions of vaccine are injected intraperitoneally, 16 mice being used for each dilution The mice receive two injections of vaccine one week apart. One week after the second injection all vaccinated mice are challenged by the intracerebral injection of 5-50 LDs of the CVS virus. The mice are observed for 14 days All mice dying after the fifth day following challenge, and all mice showing paralysts, are regarded as deaths An ED30 for each lot of vaccine is calculated as the amount of brain tissue in milligrammes needed to protect half the mice against the challenge virus The potency of the vaccine is expressed as a factor relating to the potency of the reference vaccine, which is arbitrarily assigned unit potency. With the present reference vaccine, lot 150 C, a factor of 0 67 has been set as the minimum acceptable potency for any lot of rabies vaccine ·Section 11 HABEL TEST FOR POTENCY Nice White Swiss mice, 4-6 weeks of age, and uniform in weight, are used: their sex may be selected at random, or mice of only onc ses may be used if preferred. Immunization of mice Sixty mice receive 0.25 ml of a vaccine diluted to give a 0 5% emulso of original wet-weight of brain. Inoculations are given intraperitoneally on Monday, Wednesday, and Friday during two successive wechs (a total of sıx doses). Thirty mice should be kept apart from the rest at the beginning of the immunization for use as controls at the time of challenge. Challenge A challenge test is performed on the 14th day from the first dose of vaccine. At this time iso ampoules of the standard challenge fixed wines (scc section 10, page 109, for its preparation) should be thawed out and diluted to a 104 suspension Serial tenfold dilutions from 10' to 10 are then made, using 2% horse or rabbit serum in distilled water as diluen. It is recommended that the dilutions of challenge virus be held in an into water bath during the performance of the tesi to prevent loss of war Groups of 10 vaccinated mice are then challenged intracerebrally with 003 ml of the 10%, 104, 107, 10", and 10h dilutions of virus in tha? A single syringe and needle may be used for all inoculsters, provided the cmply syringe is rinsed ses cral times in the next dilution before being refilled. With a new syringe the control mice are then inoculated with the it! 104, and 19 dilutions of virus in order to determine whal dilswt represents one ID. If the challenge virus is fully active, these teste dilutions will usually give a range of from 100% 10 less than 50surname Scattered deaths si Jl three dilutions should be viewed with suymacian titre order Euskal)"stute kein Ma, USA is consecue Flahe! Clicf, Laboratory of Infecturus Diseases. Ng'wval Mate - 112 ·VACCINE POTENCY TESTS 113 For the potency test to be valid, the 50% end-point of rabies deaths should be beyond the 10 dilution in the control mice. Al mice are observed for ---ar the fifth day should be considered daily and symptoms recorded. Mice sultanga. showing definite symptoms of rabies should be considered as rabies deaths in potency calculations - Determination of degree of protection 50% end-points of rabies mortality are determined by the method of Reed & Muench, as illustrated in Annex I. This gives the dilution in w where theoretically, 50% of the mice пр~- the vaccinated, the log of the number of Luzo buru... To meet minimum standards, this should be log 3 or 1,000 LDS Modification of technique for kve vrus or arenuated accines The 6 intraperitoneal doses of O 25 ml of equivalent 0.5% emulsion should be made starting with the least virulent of the vaccine doses and grading to the most virulent, if human doses vary as in the original Pasteur type of vaccine If all human doses ate the same, then no change from the original procedure is necessary Annex 1 PROCEDURE FOR DETERMINING 50% END-POINTS OF ITABEL-TYPE POTENCY TEST IN MICE Results of typical protocol Vaccinated mice Difunton Survived rabies 10-1 4 6 10- 5 5 10-3 7 104 8 2 10- 10 0 Accumulated totals survived rabres 20 9 14 12 9 20 2 30 0 Percentage mortality 83 6) 43 9 قا 0 Actual figures are accumulated from 101 to 10-for survivors and from 10' to 10- for rabies.

·or Percenisse Menuirr 100 Dilution 10-3 10-6 10-7 Lascinated mice survnied rabics 0 10 4 6 9 1 | 7 7.31 calculations by making use of a sheet of some K. HABEL Now make a caculation thus: (50% minus the % next under 50% 50-43 7 0 39 % next above 50% minus % next under 50%) 61 - 43 18 Now subtract this figure (0.39) from the log of the dilution which had a mortality next below 50%: 3.000 0.390 2.610 This is the log of the 50% end-point for this protocol, namely, 10*** Next calculate the control 50% end-point in the same way. Accumulated totals Surved rabies 0 17 4 7 13 50 - 7 43 = 0 75 64 - 7 57 7 000 0.750 6.250 The 50% end-point of controls is 10%. To find the LDO of protection afforded by the vaccine, subtract the le? or 50% end-point in the vaccinated mice from the log of 50% end-point in the controls 6.25 2 61 3.64 Thus this vaccine protected against 10" LD., or 4,366 LD... The figure should be sounded out to the ncarest hundred, ie , 4,400 LD, Graphical methods calculating LD:0 Approximately ili ume result, or one of sufficient accuracy.cara derised without fr.: logarithr is parer tecimul disisions. It is sufficient to know **:

·VACCINE POTENCY TESTS 115 cumulative totals of the mice which have survived and those which have died at the doses on either side of the 50% end-point, as calculated above. The number of mice is indicated on the arithmetic scale of the sheet as an ordinate, the virus dilution being entered on the logarithmic scale as an absçissa The four points corresponding to the mice surviving and mice dying above and below the 50% end-point are ploited in this way FIG 1 GRAPHICAL METHOD FOR CALCULATING LD 18. PROTOCOL ENDPOINT CONTROL ENDPOINT Cumulative Potel of mic. 3 locraying dilutions of chollongo virus NO )322 D-Survivals (cumulative) *Deaths (cumulative) against the corresponding concentrations. The concentrations of challenge virus are entered on the logarithmic scale (abscissa), the left-hand extremity of the sheet corresponding to the higher concentration, and the right-hand extremity to the higher dilution, of virus. The figures corresponding respectively to the number of mice which have died and the num. ber which have survived are joined by straight hnes The point of intersection of the two lines gives the 50% end.point, the value of which is read off on the logarithmic scale Fig 1 uses the same figures of the preceding example of a test The LD50 of protection afforded by the vaccine is arrived at in the same manner, 1 e., subtraction of the two logarithms ard derivation of the antilog of the result, ·It is advised that at periodic intervals a complete standard potency Section 12* MODIFIED HABEL TEST FOR POTENCY For many laboratories producing antırabies vaccinc, the lack of rela: tively large numbers of experimental animals makes the routine use of the standard mouse test for potency impractical. Especially is this true in those laboratories where multiple small volumes of vaccine are prepared at frequent intervals. In these situations a simplified potency test whxh would screen out vaccines of below-standard antigenic potencies, and which at the same time would be comparable in its minimum requirements to the results obtained by the more complete standard test, would be of value. In checking results of many potency tests with vaccines of high, ww, and intermediate levels of antigenicity, it has been apparent that affrou. mately 500 LD so as an intracerebral challenge dose brings down less than half the immunized mice if the vaccine by the complete iest has a potency of over 1,000 LDs protection, which is the minimum requirement. The technique of the test is the same as the standard procedure isce section 11, page 112) using sit intraperitoneal doses of a 03. suspension? of nervous tissue (0.25 mi), and intracerebral challenge on the 13th clue followed by 14 days of observation. Only 20 mice arc immunics, art 15 additional mice are held as controls. On the 14th day the standard potency-test challenge virus, diluted to contain 500 LDA per 003 ml. is gren intracerebrally to the vaccinated mice, and at the same time groups of control mice receive the challenge virus diluted 10 104, 104, and 10? (sce section 11, page 112, for analogous procedure). To be a valid tcst, the control titre should indicate that the challenge given 10 the succinated mice was between 100 and 1,000 LD... and Sp of the vaccinated mice should surve in order that the vaccine pass the screening potency tese (for calculation, sce section 11, page 112). test (sce sections 11 and 13, pages 112 and 117) be performed to obtain a mure 46.initiate evaluation of the vaccinc over long periods of produces tion in euh lihoratory (net, CJ HiChief Laboratory of Infectious Dixesxt News! Iluta cv_ 111 USA — 116 - ·Section 13 POTENCY-TEST REQUIREMENTS OF UNITED STATES NATIONAL INSTITUTES OF HEALTH (NII The standard challenge virus (CVS) The CVS is supplied periodically, usually once a year, to laboratories in the United States of America This provides a nearly uniform challenge virus and makes possible the evaluation of different lots of vaccines as well as of vaccines from different laboratories. Laboratories are urged to follow closely the procedures outlined CVS is available to matonal laboratories upon request to the World Health Organization, Palais des Nations, Geneva, Switzerland. The working CVS In the USA, CVS is supplied as a 20% mouse-brain suspension in a *% horse-serum distilled water druent. This has been stored under dryice before shipment, it should be used only if received in the frozen state, and should be retained in this condition until used The contents of the ampoule should be thawed rapidly under cold running water and then dduted 1.4 with the 2% horse-serum diluent. This gives a 5% suspen- $ion, which is centrifuged for 15 minutes at a relative (entrfugal force of approximately 1.000 & The supernatant fluid is diluted to 107 and, using this dilution as the inoculating dose, a sufficient number of normal nice are injected with 0 03 ml intracerebrally to produce the amount of working CVS needed for approximately one year (One mouse brain will yield approximately 15 ml of a 20% suspension ) When an inoculated mouse has shown signs of rabies for a period of 24 hours, the brain is harvested and mediately frozen wità dry-ice 3 he harvested brains are placed in a common container and when the collection is complete they ·I'SA on toyu. When the contents of each ampoule are dissolicit 8 ml of distant water containing 0.25% phenol and o 01 thiomerul 118 M. M, KAPLAN are thawed, weighed, and ground to pulp, and enough of the 2 hors serum diluent is added slowly, while grinding, to yield a 20", final sus pension. The suspension is given a lot number and without straining or centrifuging it is distributed into ampoules, 20-2.5 ml to each ampak The ampoules are flame-sealed, and the contents are quich-frozen :-) stored at dry-ice temperature (approximately -70*C). Each step in preparing the working CVS must be carried out prompty so as to ensure the survival of the maximum possible amount of virus Before use as challenge virus, the LD:n value of the lot should be deter mined in six-week-old mice. The lot is satisfactory provided the ID. value occurs between the 10-3" and 10-* ' dilutions, inclusive. The mat mum variation in the titre obtained from test to test should not citu one tenfold dilution when the same lot of challenge virus is used when all the ampoules of a lot have been used, or at the end of a one-year fond. a new reference CVS is obtained from the NIH for preparing a rewly of working Cys. This is essential in order to assure uniformity of the working CVS among all laboratories producing rabies vaccine. Note The difficulties in shipping frozen material necessitate that WHO send the CVS strain in dried form to laboratorics requesting i A central laboratory can prepare its own primary pool of CV'S by ons passage of the strain in a suficient number of mice calculated to meet !! needs for several years. If dry-ice facilities are available, the priman pool in flame-scaled ampoules should be kept frozen ar -- 70°C, ard tit procedure followed as described above. If dry-ice facilities are lucken the material can be kept frozen at -20°C or lower, the lower the better, but periodic litrations in mice are required to determine possible Salsa titre It is not advisable to work directly from the dried form of cus because the titre may be too low. Tope of test mouse The test is based on the use of white Swiss mice approximately for Mechs old, un form in weight (11-15 g), and of one set. Reference de cine An NII Rabies Reference Vaccine (ultraviolet-light inactmated and dricus is supplied by the NIII to saccine-producing laboratories in the reference and is served at 29-10°C at all times, whether in the **** or the dried itt !!! reconstitution, the vaccine is used not later this? Iwo week, st. and in is added.

·VACCINE POTENCY TESTS 119 At the request of WHO a hmited quantity of NIH Rabies Reference Vaccine 150 C has been made available, and will be supplied by WHO to national laboratories upon demand Since only a very limited quantity of the dried reference vaccine will be available, it is suggested that national laboratories prepare a sufficient quantity of a dried vaccine of their own to meet their needs for at least one year, and then test their own dried Vaccine with the NIH Reference Vaccine. The advantage of using a dried reference vaccine is its stability, and thereby its usefulness for future comparison of vaccines Immunization of mine Three or more dilutions of each vaccine under test are prepared in - 1 1 h Vaccine to protect 50% of the mice The range will depend upon the strength of the challenge dose, the kind of mice used, and the potency of the vaccine With a challenge dose of 20 LD, (see page 120) a dlution range of 10%02%-0 01% of brain tissue has worked out well for both the NIH Reference Vaccine 150 C, and commercial Vaccines produced in the USA. At least 16 mice are injected intraperitoneally with 0.5 ml of each dilution of vaccine. Two doses of vaccine are given to each mouse one week apart Enough control mice are set aside at the time the test mice receive the first dose of vaccine for an adequate utration of the challenge virus to be made with at least 10 mice for each dilution of virus (a total of 30-40 control mice). For vaccination of mice use a 1-mi or 0 5-ml tuberculin syringe and give each mouse 0.50 ml intraperitoneally. For rapid work it is necessary to etberize the mice Grasp the mouse by the skin of the neck between the thumb and forefinger and clasp the tail between the little finger and palm The mouse is then held firmly on its back and cannot get away even if not etherized The needle best suited for vaccination is the 25-gauge 19-or-inch (0 50-mm x 12- or 18-mm) needle Inoculate in the midline Immediately above the umbilicus Thrs avoids the stomach, liver, and bladder Insert the needle, with the bevel up, at a 45° angle from the head end of the mouse, and inject the vaccine suspension rapidly (see fig !) If there is any doubt as to whether the needle point is in the peritoneal cavity, move the point from side to side and observe the skin. * 15 ml MA15 KH,PO., 85 ml M/15 NazHPO, 12H30 900 mt distulled water ·120 M. M. KAPLAN FIG. 1. INTRAPERITONEAL INOCULATION OF MOUSE Br courtesy of Dr F Pere Gallurds, Mudru Vaccine The order of vaccination is r.ot important, so that all the mice to be Vaccinated with one lot of vaccinc may be kept in one or two bores Challenge of control and test mice All mice arc challenged intracerebrally 1 days after the first dose a One ampoule of the pooled first-passage working CVS is thawed rapidly urder cold running water and diluted 1 * with 2%, horse-serum : destilledwater diluent This mixture is then centrifuged for 15 minutes at 3 relative Centrifugal force of approumately 1.000 g. The supernatant fluid is. 10 dilution of the challenge material, and is used to make further 1!! dilutions are made in the same diluent as originally used It is recommended that the dilutions of the challenge situs be held in a aquivalent. during the performance of the test 19 order to pre The im it challenged with a dilution of virus utih has been four loss to contain approumately 230 1. DANS virus. S dilutions ICC-ujter thach UL ·121 VACCINE POTENCY TESTS Each laboratory, therefore, must determine for itself the dilution of working CVS which will contain the test dose of 20 LDso virus; for example, if the working CVS has been found to have a titre of 1045 or 1 3,160,000, then the 20 LD so would be contained in a dilution of l: 158,000 or 10 This dilution is prepared from the 5 x 10 supernatant described above, as follows: I ml(of 1 20) + 4 ml diluent = 1:100 I ml (of 1: 100) + 9 ml diluent = 1:1,000 I ml (of 1 : 1,000) + 9 ml diluent = 1:10,000 I ml (of 1. 10,000) + 14.8 ml diluent = 1:158,000 After all immunized mice have been inoculated with the test dose of virus (003 ml of the 1.158.000 dilution in the crample given above), one group of control mice is inoculated with the same test dose of virus. Two tenfold dilutions of the test dose are then prepared for injection of the remaining control mice. It is desirable to inject the 1.100 dilution and the 1 10 dilution of the test dose of virus in that order. In this way onc syringe may be used to challenge all the immunized mice and one group of control mice, another syringe must be used to challenge the remaining control mice. All mice are observed for 14 days from the time of the challenge injection Only those deaths occurring after the fifth day and those preceded by signs of fixed-virus rabies (paralysis, convulsions) are considered deaths from rabies Any mice becoming paralysed but surviving the 14-day observation period are considered as equivalent to deaths from rabies The definitions of paralysis " and "convulsions ", as applied to mice following the injection of the challenge virus, are as follows Paralysis is the partial or complete loss of motor power of one or more legs Convulsions are indicated by violent and abnormal muscular contractions of the body-often termed spasms These are brought about by external stimulation, such as a disturbing sound, or handling For the test to be considered valid, the results obtained after challenge of the immunized mice must show that the dilutions of the reference vaccine encompass the 50% end-point--that is, the majority of the mice receiving the highest dose of vaccine survive, and the majority of the mice receiving the smallest dose of vaccine die For the test vaccine, unless an exact end-point is especially desired, it is only necessary that the majority of the mice receiving the highest dose of vaccine survive The virus titre of the test dose of challenge virus is not particularly important, provided that it falls between 5 and 50 LD: It is necessary, however, that all the control mice receiving the test dose of virus die. Because of the nature of the virus ·122 V. M. KAPLAN ard the test insoled, it is difficult to obtain cucuy the same vil from one test to another. Poienai rest on lire or altenuated situs roccire Vaccines containing lise or attenuated virus, cicept for chiciero Vaccines (see section 15, page 125), can be tested in the summer m2*** 1:lled-virus vaccines. Calculation of potenci (pritigenic ralue) 50cnd-points are determined for both the refererce vaccire 3rd the test vaccirc by the method of Reed & Yuench. The end-point is caku 25 2 50% elfective dose (ED.) of vaccine in milligrams of ongiral brzina tissue which will protect 50% of the mice. An abstract of this retouis given in Anner i below. By dividing the ED., of the refererce 1200 by the ED:n of the test vaccine, a value is obtained which is the 3700 Salue of the rest vaccine in terms of the reference vaccine. The LD challenge virus received by the immunized mice is calculated by curs the dilution of virus used as the test dose by the 50 crd-point c*** of virus in control mice, as calculated by the method of Reed & Match Anugenic value requirement (see page 133 for compulation) The antigenic value of a vaccine urder test should be 0 6 or highet 13 relation to NIH Reference Vaccine 150 C. For cach new lot of referent vaccine, an antigenic value will be determined in terms of the previous seference vaccine. It is anticipated that there will be miror differences !? poiency of future batches of reference vaccires in spite of erorts to lepo these variations to a minimum. Anner 1 VIETHOD OF DETERMINING 50%. END-POINTS BY THE VIETJIOD OF REED & MUENCH: AS APPLIED TO THE POTENCY TEST OF RABIES VACCINE IN MICE 1954. uh with r. ve Sp'ED) is calculated as the amount of train ",", of the mice against a subsequent cha! 's of the challenge are arranged B7 Columns . • Pet !

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·VACCINE POTENCY TESTS indicating the number of mice which survived and shich died on each dose of vaccine. The value for survied and dead in the columns under the heading Reconstructed totals are then obtained by adding the survivals from the high to the low dilutions, and the deaths in the opposite direction. 12 Dilutuins of S. brain hssue raccine EXAMPLE Brain Aumber 11330 of mg) mice Surnal 10 Deud 125 1 135 Reconstructed totals Percentare Sarssved dead murtalary 16 16 16 04 10 8 A19 1 6 8 15 6 From this tabulation, the 50% end-point dilution lies between 2 mg and of brain tissue, and is obtained by the following calculation: 10 6/15 - 24 14,23 - 61 mg 29 А 61 (mortality nert over 50%) - 50 61 (mortality next over 50%) - 24 (mortality next under 50% x the logarithm of the dilution factor (in this example dilution factor is 5) = logarithm of dilution factor at 50% end-point or 37 * 0699 = 0 208 (log of dilution factor at 50% end-point) B Antilog of dilution factor at 50% end-point X amount of brain tissue injected into mice at dilution at which more than 50% of mice died + EDS since antilog 0 208 = 1 61 then 1 620 mg = 3 22 mg = EDS The antigenic value of a test vaccine is obtained by dividing the ED of the test vaccine into the ED50 of the reference vaccine, eg, reference vaccine ED50 3 22 mg BT (brain tissue) test vaccine No 1, ED:0 107 mg BT 3 22 antigenic value of vaccine No 1 = 30 1 07 The challenge dose of virus is calculated from the results of challenge of the control mice The results of the challenge are arranged in columns and a Reconstructed totals column is obtained in a manner similar to that described above ·124 M. BI. KAPLAN EXAMPLE PAUN mvla.lt Diuron Number of of Trus MICS 10 10 10 10-3: 10- : Surrned 0 4 8 Dead 10 6 2 Reconstructed totals surrned deal 18 8 81-6 . Since the 50% end-point dilution or LD:n lies between 10-** and 10"? the logarithm of the LD dose is found as follows: 67 - 50 Log of LD so dose = 6.2+ 67 -14 17 = 6.2 + 53 = 6.2 + 0.32 6.52 The LDsn dose, therefore, is 104S, or the undiluted sirus suspension contained 3,310,000 LD3n. Since a 10-: dilution of virus was used as the challenge dose, that dose contained : 3,310.000 or 21 LD: 158,000 ·VACCINE POTENCY TESTS 127 challenged similarly with a 1:5,000 dilution (3 LD.). All vaccinated animals should survive and the controls should die. Note: The test technique described above was devised for use with the Louis Pasteur strain of fired-sirus rabies at its present stage of adaptation to the rabbit at the Institut Pasteur, Paris (1,901 st passage 1 January 1953) When another strain of fired-virus rabies adapted to the rabbit is used, It is as well to perform a preliminary titration of the virus in the rabbit by the intracerebral route, determining the dilutions of virus on either side of the LDSO For intracerebral test on the 30th day in rabbits which have been Vaccinated for 10 days, 1.5 LD,, should be used, in rabbits which hase been vaccinated for 20 days, the concentration should be 3 LDS ·(10) dilution (ISI!!! Pasteur-strain fred virus; Group 15 a 2- g rabbit intracercbrally inoculated on the 30th day against a 1:10.my 20 consecutive days, it does not protect the rabbit against a 1:16,071 wiednirol Thirty is after commencing the administration of 1,61 Groups and in challenged intracerebrally with 0 3 mois 126 R. BÉQUIGNON & C. VALAT It would appear unnecessary to interpret these results mathematicali by the cumulative method of Reed & Muench, the minimum lethal dose being casy to determine and not varying under the experimental con ditions. However, it is clear that this virulence titre varies not only accord.rs to the strain of the fixed virus selected for the test, but also according to the number of passages undergone by the strain, the wcight and charien teristics of the domestic rabbits used, and the climate where the test 15 carried out. Nevertheless, there is no reason why these factors should alter the minimum lethal dose provided this has been determined under the same experimental conditions (see Note on page 127). The results of the test are available in 8 days, but it is advisable to keep the animals for a month after the challenge inoculation to male certain of the results, since in exceptional cases (2% of cases) rabies can develop with some delay. Vaccination Once the vrulence titre is known, it is possible to evaluate the pro tective power of the rabies vaccines by testing batches of five rabbits en the 30th day of vaccination with incrcasing dilutions of fixed virus Thus the Paris Institut Pasteur phenolized vaccine (sce section 6 (A); page 77), on being injected subcutaneously into the flank of the anima! at the rate of 2 ml per day during 20 consecutive days, protects a 2; rabbit inoculated intracerebrally on the 30th day of vaccination against al 5.000 virus dilution (3 LD,.). If the volume of the vaccinal injections (s.c., the quantity of acting inoculated and the duration of vaccination) is varied, the results oht.sired show that massive injections are more effective than repeated ones when 2 ml arc injected daily for 10 consecutive days, phenolized vaccine protectes virus dilution (IS LDO). However, when 1 ml is injected daily for dilution, thus tending to confirm the importance of carly presente treatment and massive and carly injections in man after a suspected the for an actual test a minimum of 6 rabbits is used, although it." preferable to use 12 anımals, divided into groups of equal number. Gro? recenes 2 ml of sacure is tissue suspension) for 10 days. Group, recenes 2 mt of vaccine and issue suspension) for 20 days Group in 1 ·129 VACCINE POTENCY TESTS FIG. 1. INOCULATION INTO MASSETER MUSCLE, USING DOG By courtesy of D F Perla Gallardo Aladrid 28-35 days old These mice are observed for 21 days and the number of dead mice is recorded daily. After the period of observation, the LDS titre of each preparation is calculated (for calculation see section 13. Page 122) In order to make a large visus pool, only those glands of which the Este exceeds 1045 LD so are pooled. Preparation of the pool The glands are removed from the freezer, thawed, cut into small pieces with scissors, and put into a Waring blendor with enough sterile 10% nortnal rabbit serum physiological-salt-solution to make a 20% suspension by werght The blendor is set in motion for 2 or 3 minutes only, in order ·Adult dogs are injected sath 0.1 ml of a suspension of infected c3^**** submarillary-gland tissue kept frozen at -70°C. The injection is bilder into the masseter muscle, using a l-ml syringe and a 20-gauge, Ir of rabies, or when they are sacrificed when moribund, the submit gland tissue is removed, a small piece is separated by cutting with sisin and the remainder is frozen in a large Petri dish and lent in a rel' SOC to - 70C The selected portion is ground in a morty crouch 10% normal rabbit serum physiological-salt-solution to malcal suspension by weight Thuis suspension is then centrifuged in 37 head centrifuge for one minute at 1,000 crolulions per minute ( and the supernatant liquid is separated. Tenfold dilutions of the same solution and o03 ml is injected intracerebrally into Swiss Bird 1 Isderle Laboratorio Dixon American (yanamid Company, Pearl Rise, TU Contributed by Hilary Kortouski Asystant Director. Viral and Rultetas Section 15* POTENCY TEST FOR CHICKEN-EMBRYO VACCINE The test consists of the intramuscular inoculation of guinca-pigs :: nichcn-embryo vaccine, followed three weeks later by challenge of 1*: inimals with street virus. Ullmnea-pigs Guinea-pigs weighing not less than 400 g should be chosen. The bike of lighter animals is inadvisable. Inimization procedure If the presently available chicken-embryo vaccine (33% tissue suspa sion) is used, o 25 ml of 5% tissue suspension per guinea-pig show's a injected intramuscularly into the gastrocnemius muscle of the right to: leg of each guinea-pig. The vaccine should be rehydrated with ster; distilled water shortly before inoculation and the required dilution scene be made in sterile, distilled water, Preparation of challenge matcrial at nalunt liquid are made in 10° normal rabbit serum: shysioloomakes -- 128 ·TABLE I. RESULTS OF POTENCY TEST FOR CHICKEN EMBRYO VACCINE Results of Intramuscular challenge with street virus Batch of vaccine LD titre in mice gulnea-pigs dogo mortality ratio of challenge animals mortafity ratio Immunized with dilutions of vaccine 15 1 20 1 80 1 320 non- Vaccinated controls 1 1.280 A. 10-275 1 5,120 vaccinated 08 nonvaccinated controls 09 05 0/10 08 B 10-25 10,10 05 00 0/10 3,6 5/10 5/10 9 10 с 10-03 10/10 08 061 two testo performed 08 277 3 $ 10 10 68 D 10-31 10/10 VACCINE POTENCY TESTS 08 two tests performed 0,6 3/10 & 9 0/10 0/9 E 1090 88 0 10 10,10 16 4'5 04 F 10-30) 89 0,6 18 25 05 03 1/4 G 15 10-390 8 9 08 3 15 18'25 13'16 017 35 • 1 ml of vaccino containing 5% tissue lui pension was injected into each Quinea-pigi in all remaining Instances O 5 ml was used. (Noto 025 ml of 5% tissue Suspension is now used for routine testing) 5 ml of 20% tissue suspension were used for vaccination 131 ·130 I: 10 to present heating well chilled, ard procedure is res. through one li The filterer quantities. Ti an alcohol-C ranging from Ease bersedia Na obsend:012:19 cznine sainen : Tout and prepared as c - LOCulum tool animals shoulders enies should sunuchare: rabies This test is dix}"! mi can be performe !!! sith vaccice 2'c: cena mixally isolated I strict *ed to evaluathi phical and contact --- 3. 125 a disadvantage virus House. :: Challenge ? Twenty. S non-acc animals sh of a diluti known to The c! and dead i with street The te 1 sd, a relatnosti: phuch can be ir: Alternate A $31 A group of intramuscl 05 ml o At the er should be observatio should sut Diccion Asum pussed pole has proses street vi protcuted. immunued m.ntioned tuule the the past t'i. 1 ·Part IV PRODUCTION OF HYPERIMMUNE SERUM ·132 H. KOPROWSKI animals. So far, only two street-virus pools have been used for chal... purposes in this laboratory, and it has been observed that 0.1 ml of 1:8 dilution or 02 ml of 1:80 dilution of canine salivary-gland tissues, 1? fected with the New York City strain and prepared as outlined on me 129, constituted an excellent challenge inoculum. In the actual test, 80% of the control animals should die of rabies ar 70% of the vaccinated guinea-pigs should survive challenge inoculos without showing any symptoms of rabies. This test Sery closely paralles events occurring in nature and can be performed with relatnely liike labour. Only one inoculation with vaccine and one challenge inoculitics are required. By employing locally isolated street-virus strain for challenge purposes, the test can be used to evaluate the potency of chicken-embro vaccines in different geographical areas of the world against different strains of rabies. It has a disadvantage in that it requires dogs for the preparation of challenge virus. However, iſ a potent preparation of salivan. gland tissue is employed, a relatively small number of dogs will suflice to yield a preparation which can be used for a long period of time for challers purposes.

·Part IV PRODUCTION OF HYPERIMMUNE SERUM

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·Section 16 METHOD USED AT INSTITUT PASTEUR, PARIS The Institut Pasteur antirables serum is prepared from horses hyperimmunized with fixed wrus. This serum can be produced either in the crude state, or purified and concentrated Rabies strain The rabies strain used is the Institut Pasteur fixed-virus strain which has always been employed there for immunization against rabies. The characteristics of the strain and the method of maintaining it in the rabbit are dealt with in section 6 (A), page 77 Method of immwisation The horses are immunized by subcutaneous inoculation. They are fisst given an initial series of injections of phenolized rabies virus, 1 e., antirabies vaccine prepared in the same way as that for human use, as described in section 6 (A), page 81, but employing rabbit brain instead of sheep brain The vaccine is used for the first injections after incubation for eight days at +4°C Thereafter, more freshly prepared vaccines are used, After this initial immunization with phenolized vaccine, the horses next recenve the non-attenuated fixed virus Finally, according to the titre reached during immunization, the injections are continued with the alternative addition of adjuvants Once a suitable tstre has been reached, the horses are re-inoculated every month and are then bled again Immunization schedule and doses Young horses in a good state of health are used. Immunization is carried out according to the following plan First series phenolized fired us First injection 40 ml One week afterwards 80 ml 5% rabbit brain, phenolized One week afterwards 120 ml Coninbuted by Pierre Lépine (Chief, Virus Section) and P Atanasiu, Institut Pasteur, Paris, France 135 -

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·Section 16 METHOD USED AT INSTITUT PASTEUR, PARIS The Institut Pasteur antirabies serum is prepared from horses hypero immunized with fixed virus This serum can be poluced either in the crude state, os pursfied and concentrated. Rabies strair The rabies strain used is the Institut Pasteus fired-sirus strain which has always been comployed there for immunization against rabies. The characteristics of the strain and the method of maintaining it in the rabbit are dealt with in section 6 (A), page 77. Method of immunication The horses are immunized by subcutaneous inoculation. They are first given an initial series of injections of phenoluzed rabies sirus, i e.. antirabies vaccine prepared in the same way as that for human us, as described in section 6 (A), page 81, but employing sabhit brain instead of sheep brain The vaccirc is used for the first injections after incubation for eight days at +4°C Thereafter, more freshly prepared 13ccines are used, After this initial immunization with phenolized accire, the horses next receive the non-attenuated fixed virus Finally, according to the titre reached during immunization, the injections are continued with the alterna. tive addition of adjuvan's Once a suitable titre has been reached, the horses are re-inoculated every month and are then bled again Immunication schedule and doses Young horses in a good state of health are used. Immunitation ss carried Out according to the following plan First series First injection One week afterwards Ons weck afterwards phenolized fred sus 40 ml 80 ml 5.rabbit brain, phenolized 120 ml Coninbuted by Pierre Lépine (Chief. Virus Section) and P. Atanasu, Institut Pasteus. Paris France -- 135 ·136 P. LÉPVE & P. ATANASIV For this series, as for those following, the injections are made at several soints so as to distribute the injection mass and to ensure rapid absorption, Little if any reaction is observed at the site of the injections. Second series : fresh fired trus The second series commences immediately after an internal of one xech. rirst injection hall a rabbit brain (fresh fired virus) suspended in 50 ml of distilled Witcr Second injection: one whole rabbit brain in 100 ml of distilled water Third injection: the wholc rabbit brains in 150 ml of disailled water A control bleeding is carried out a wech after the third injection of the second series. According to the results and the titre obtained, immunization is stopped, or the second series is repeated, adding suppicmenuary artigen injected intramuscularly with adjusants. Various adjuvants may be employed, but the best results are obtained with sodium alum in a concentration of 4:1,000 in the suspension injected. Mineral-oil denatnes with an emulsifier totalling one-third the quantity of injected material can also be used, but they are less advisable than sodium alum. The final titre of the antirabics scrum is normally reached befuccn the second and third month (sec section 18, page 139, for potency test). Momnienance of immunication Blood is hari ested at this stage and the horses are allowed to rest for a month Subsequently, immunized horses reccisc cicry month to rabbit brains (fresh fixed rabies virus), injected as with the third injection of the second series. The horses are bled a neck after this reinforcing Injection. They are allowed to rest completely for ore month during the year ·Section 17. METHOD USED AT ISTITUTO SIEROTERAPICO E VACCINOGENO TOSCANO, SIENA of the various animal species, whether small or large in size, the equine in particular the horse--should be considered the animal of choice for the preparation of antirabics serum, The production of hyperimmune serum in the horse, started by Fermi in 1909, is related to the quality of the antigen used, the doses injected, and the rhythm of the injections. The antigen used by the authors con sists of Fermi's phenolized antırabies vaccine ("Sassarı" virus), freshiy prepared, and injected subcutaneously in a dose of 10-20 ml every day for a month A bleeding 7-8 days after the fast injection of the first series shows, however, that the presence of virucidal antibodies in the animals' sera is still not high at this stage, although they are clearly present in all the sub jects treated, On giving the animals a rest period of a month and carrying out a second series of injections, a serum whose virucidal value is double, treble, or even higher is obtained After a further rest period of from two to three months, and following another series of vaccine injections, a very powerful serum 15 obtained, of which a l-ml dose is capable of neutralizing up to one million or more doses lethal for the guinea-pig The production of a good antirabies serum in a fresh horse appears, however, to be very slow, calling for several rest persods alternating with an immunization series On the other hard, antibodies are rapidly and Intensively produced in horses which have undergone previous immunzation Animals which have received a basic immunization with phenolized vaccine are also not harmfully affected by the injection of high doses of living and virulent virus, thus making it possible to bring about immunization more readily As in the production of antitoxic sera (diphthena and fetanus), animals which are good producers of serum are encountered side by side with mediocre or even refractory subjects. On a reduced scale, the course of antirabies serum production is very similar to that of antitetanus serum, the rapidity and intensity of production of this is well • Contributed by D d Antona & E Falchetti Istituto Sieroterapico e Vaccinogeno Toscano, Siena, Italy -- 137 - ·138 D. D'ANTONA & F. FILCHETTI known in the case of horses subjected to repeated cycles of immunization, or to previous vaccination. It is thus possible to prepare a powerful mixed antitetanus and antirabics serum at the same time by simultancous injec. tion into the animal of the corresponding antigens (tetanus toroid or toun, and antirabies vaccine). As with the tetanus antitoxin, the virucidal antibodies seem to be distributed in the pseudoglobulin fractions of the scrum, and to a reduced extent in the cuglobulin fraction. The antırabics serum may be concentrated and purified, however, by the same techniques as apply to the antitoxic sera, including reptic digestion. The antirabies serum is remarkably stable and retains its properties in a refrigerator for several years, as well as resisting escn prolonged heating at 58-60C. Titration of the virucidal antibodies is carried out by the authors using cs of different dilutions of the scrum and an emulsion of Virus (brain tissue) with a definite, Inoun virulence. After standing for one hour at 37°C, the mixtures are injected intracerebrally into guincipios in a dose of 0.2 ml, and the titre is calculated on the basis of the dilutions able to protect 80% of the animals. in ·Section 18+ POTENCY TEST OF ANTIRABIES SERUM Potency test This test is essentially the same as the serum-virus neutralization test in mice described in section 5, page 69 The test must be performed with serum before the addition of any chemical preservatives Test animal: Normal mice of either set weighing 10-14 g cach are used in any one test, mice of only one sex are used. Test urus: Any standard strain of rabies visus of noun potency may be used Reference antirabres serunt A reference serum distributed by WHO will be available for use in the potency test for comparative purposes when standardizing all antırabies sera This serum will be supplied in the dry form, and instructions for redissolving will be sent with each shipment, Its potency is adjusted so that when mixed in final concentration with not less than 31 6 LD, and not more than 316 LD., of one part of virus sus. pension, the minimum protective titre (50% end-point) will be not less than 1 300 dilution, Procedure Serial twofold dilutions of both the serum under test and the reference serum are prepared in 2% normal serum in distilled water or physiological salt solution Sıx serial twofold dilutions, starting at 1.50 and continuing through to 1:1,600, are usually sufficient for the reference serum, and 1 125 through to 1 4,000 for the serum under test These dilutions will reveal a 25 potency factor required of the serum urder test Equal quantities of a suspension of test virus are added to the dilutions of serum. The mixtures are incubated in a 37°C water-bath for one hour, and 0 03-ml quantities are injected intracerebrally into the mice. At least 10 mice are injected for each mixture The amount of virus used is such that each mouse receives not less than 31 6 LD., and not more than 316 LD:0. The mice are observed for two weeks for a serum under test, its minimum acceptable potency should be 2.5 times that of the reference serum Contributed by Hilary Koprowski Director Viral and Rickettsial Research Lederle Laboralones Dosscon American Cyanamd Company Pearl River NY, USA 139 ·140 I, KOPROWSKI Sterkty iest The contents of the final container should be sterile, as indicated by culturing the entire recommended dose, except that the amount cultured need not cicced 5.0 ml. The culture should be made in one or more tubes of fluid thioglycollate medium or other stand3rd media with suflicient dilution for the preservatric no longer to crert a bacteriostatic effect. Incubation is at 32°C, with observation for at least 7 days.

·Part 7 LABORATORY ANIMALS

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·Section 19 BREEDING AND CARE OF LABORATORY ANIMALS Breeding stock This is perhaps the most important matter in the establishment of a breeding colony for mice or other laboratory animals Every variety of animal has certain natural diseases, many of which can be eliminated by proper segregation of mature animals and testing for the presence of disease in isolated families. The inbred white mouse is the most widely-used experimental anumal in Virus laboratories. Certain genclic lines of white mice have been found to be highly susceptible to a wide variety of virus diseases Other genetic Hices or varieties of white mice are known to be resistant to certain virus diseases; for instance, genetic variety of white mice is resistant to 17D yellow-fever-vaccine virus, ard when inoculated intracerebrally with this urus the mice do not sichen ard die, while other genetic types uniformly die of this infection Breeding stock can be obtained from mouse colonies that have been tested and found free of paratyphoid and salmonella intestinal bacterial infections, bartonella ard toroplasma parasites, and the more common Viruses such as mouse encephalomyelitis, lymphocytic choriomeningius, and ectromelia It is possible that vaccinja virus used for immunization of man may be introduced into a mouse colony and may produce a disease similar to, and antigenically identical with, ectromelia disease There is a mite-borne rickettsial infection of wild mice caused by Richettsia aharı, which could be introduced into breeding colonies of mice Faecal droppings of wild mice can introduce mouse encephalomyelitis virus into breeding boxes and so infect breeding stock previously known to be free of this disease All lines of white mice appear to be uniformly susceptible to rabies street virus inoculated intracerebrally Housing The colony of normal mice should be in a room by itself and inoculated mice must not be allowed in the same unit It is preferable to have * Contributed by Harald N Johnson Virus Research Centre Poona, India 143 -- ·11. N. Jou SOS the breeding colony on another floor or in a different building from the room used for inſected animals, The windows of the breeding room must be screened 10 leep our Nies and mosquitos, as these insects may introduce bacterial, parassic, and virus infections. The entry to the breeding room should have a sestibule with a screened door. The floor should be washable and of a matcrial which would present entry of wild mice or other rodents. The roof should be constructed or reconditioned to present the entry of rodents and birds. Mice cannot stand high temperatures, and the temperature of the breeding room must be kept at under 100°C. Caretakers for animals Persons who work in the rooms containing infected mice should not be allowed to do any of the work in the breeding quarters for normal mice. Caretakers should be instructed to scrub the hands with a brush in soup and water, and to clean the finger-nails with a stick before beginning work and whencier there is a likelihood of contamination. Intestinal in ſections of mice, such as paratyphoid and salmonella, may be introduced by carctakers' handling food or water-bottles with contaminated hunds Cages anel bedding Cages for mice are ordinanly constructed from galvanized iron with a heavy wire-mesh section in the lid for ventilation. Water-bottles are necessary for breeding horns of the ishing Wood The ܝܕܐciܠܐܪ play ve used for bedding. Food An food must be protected from rodents and insects by storage in metal containers with suitable lids. Whole grain such as unmilled wheat is a complete food for mice. For breeding stock it is advisable to include poudred milk in the deck An ersclient method of feeding mice is to prepare a whole-heat ccreal containing reconstituted powdered milk and salt, allox it to sct, and then cut it inio blocks for feeding This furnishes hoth foodid water Method of Home up breeding colony For rapid sreding it is best to lccp fuo females and one male together all the time the breeders are discarded. This ensures a new tuttet ·LABORATORY ANIMALS 145 every three weeks and simplifies the handling of the animals. For some purposes it may be preferable to have fise females and one male in a stock breeding box and to remove the females when they become obviously pregnant : these are then put into a bor by themselves and returned to the same breeding box at the end of the weaning period This method Elles a check of the number of mice per litter and a more cract record of age, but it is not justified in most diagnostic faboratories as it takes too long to produce a litter from each mouse. Breeder females which do not become pregnant in three wecks are removed from the breeding colony and used for routine purposes. Testing for presence of disease in breeding colony It is possible to test for the presence of mouse encephalomyelitis virus by collecting a stool-specimen pool from a box of 50 mice of weaning age. This material should be ground in a mortar with salıne solu make a concentration of about 10% and centrifuged at top speed in an ordinary horizontal or angle-head centrifuge for one hour to sediment as much of the bactenal content as possible. Then take off the supernatant fluid, add to it penicillin to a concentration of 500 units per ml and streptomycin to a concentration of 1 mg per ml, and test this material by inoculation of 003-ml amounts intracerebrally into cach of 10 mice Úse six-week-old mice for the test. Jf paralysis develops in the inoculated mice at from 4 10 30 days aſier inoculation, check brain impressions for bacteria by staining and culture methods; if no bacteria are found in the stained preparations, subpass to other mice and, if these are consistently infected, check specificity of the virus by serum neutralization test if encephalomyelitis virus is present in the colony there should be so difficulty in obtaining the virus by the method outlined above Most laboratory colonies of white mice harbour this virus in the intestinal tract and only rarely do mice develop paralysis The virus may be isolated in subpassage studies by chance selection of a mouse having a systemic infection with the natural virus There are instances where subpassage in mice of a neurotropic virus, such as rabies fixed virus, may result in loss of the fixed virus and the maintenance of a fixed strain of mouse encephalomyelitis virus of about the same incubation period as that produced by fixed rabies virus. Subpassage of the virus through rabbits wul eliminate the mouse encephalomyelitis virus infection, Hamsters are susceptible to infection with the mouse encephalomyelitis virus when inoculated intracerebrally For lymphocytic choriomeningitis virus and several other chronic infections of mice, it is best to test individual spleen specimens from at least 10. mice selected at random from among two to three-week-old mice obtained from the breeding colony Prepare suspensions of spleen tissue ·146 11. N. JOM\SOY by grinding in a mortar in saline solution to make a 10% tissue suspension, Centrifuge at low speed for 10 minutes, or preferably allow to settle by standing in the refrigerator for 30 minutes; then take off the supernatanı fluid and test by intracerebral inoculation of 0.03 ml into cach of 10 mice. Toroplasma infection may also be picked up by this method, although if this is suspected it is best to test the whole tissue suspension without centrifugation. The presence of toroplasma in brain impressions can be determined by firation of the impressions in methyl alcohol and staining with Giemsa stain. The course of the discasc and incubation period of toxoplasma infection and radies fixed-virus infection is similar in mict infected by intracerebral inoculation. Information on other discases will be found in the UFAW handbook edited by Worden, for which the reference is given on page 149, Ectoparasites of mice Mice should be examined for the presence of ectoparasites such as mites and mange parasites. If found, such conditions may be handled by dipping the mice or treating them with insecticides. Natural infections of mite origin must be presented by not allowing wild mice access to the mouse breeding quarters ·ADDITIONAL REFERENCE SOURCES ·볼 ·ADDITIONAL REFERENCE SOURCES English American Public Health Association, Diagnostic procedures for virus and richertsial diseases, New York, 1943 Banks, H S, ed Modern practice in infectious feners, London, 1951 Beveridge, WIB & Burnet, F M The cult anon of viruses and richestside in the chick embryo, London, 1946 (Med res Coun Spec. Rep Ser No 256) Bozeman, V. Torp, IT & Berry. B (1950) A modified Habel-Sockrider ultraviolet Irradiation apparatus for use in serum and vaccine production, J. Immunol 64. 65 Conn, H J Biological stains, 5th ed. Geneva, NY, 1946 Cunningham, C | A luboratory guide in strology, Minncapolis, 1948 Farris, E J. ed The care and breeding of luboratory animals, New York, London, 1950 Habel, K (1947) Ultraviolet irradiation in the production of potent antirabres vaccines, Puhl With Replash 62, 791 Habel, K & Socknder, B T (1947) A continuous flow method of exposing antigens to ultraviolet radiation Immunol 56, 273 Horsfall, F Lijr, ed Diagnosis of viral and richerisial infechons Symposium held at the New York Academy of Medicine, Junuary 29 and 30, 1948. New York, 1949 Hyde, R R & Gardner, R E Lahoratory outhne in filterable viruses, New York, 1937 Kolmer, J A, Spaulding, EH & Robinson, HW Approved laboratory technic, Sth ed, New York, 1951 Levinson, S O , Mitzer, A. Shaughnessy, 11 ), Neal, J L & Oppenheimer, F (1945) A new method for the production of potent inactivated vaccines with ultraviolet erradiation II Sterilization of bacteria and immunization with rabies and St Louis encephalitis vaccines, Immunol 50, 317 Poindexter, H A (1949) Experimental animal colony in tropical West Africa, Pudl Hlth Rep, (Wash 1 64. 57 Rivers, T Med Varal and nicketrial infechons of man, 2nd ed. Philadelphia, London, Montreal, 1952 Rooyen CE van & Rhodes, AJ Virus diseases of man, 2nd ed. New York, 1948 Simmons, J S & Gentzkow, C Laboratory methods of the United States Army, 6th ed, Philadelphia, 1946 Snell, GD ed (1941) Biology of the laboratory mouse, Philadelphia Terkel, E S (1948) A modified Habel-Sockrider ultraviolet trradiation apparatus for use in serum and vaccine production, Amer vet med Ass 112, 18 Webster, L T Rabies, New York, 1942 Worden, A Ned The UFAW handbook on the care and management of laboratory animals, London, 1949 149

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·WORLD HEALTH ORGANIZATION: MONOGRAPH SERIES No 1 PSYCHIATRIC ASPECTS OF JUVENILE DELINQUENCY – Lucien Bonet (1951) 619 $100 Sw, fr. 4 - 2 MATERNAL CARE AND MENTAL HEALTH – John Bowlby (2nd edition, 1992) 10/ $200 Sw fr. 8- (paperbound) 17/6 $250 Sw fr. 10 - (clothbound) 3 LUTTE ANTIPALUDIQUE PAR LES INSECTICIDES A ACTION RÉMANENTE E Pampana (1951) $100 Sw fr 4 - French edition only (out of print) 4 EXPERIMENT IN DENTAL CARE - John T Fulton (1951) 5- $100 Sw fr 4 – 5 ATLAS OF FRAMBOESIA - 5- $100 Sw ft. 4 - Kenneth R Hull and others (1951) 6 CARDIOLIPIN ANTIGENS - 57 St 00 Sw fr 4 - Mary C Pangborn and others (1951) 7 THE COST OF SICKNESS AND THE PRICE OF HEALTH -- CE 94 inslow (1951) 7/6 $150 Sw. fr. 6- 8 KWASHIORKOR IN AFRICA - J Broch & M Autres (1952) 57 $100 Sw. fr. 4 - 9 BIOLOGY OF ANOPHELES GAMBIAE - AH Holstein (1954) 13/6 $200 Sw fr 8 - 10 FIRST INTERNATIONAL SYMPOSIUM ON CHEMICAL MICROBIOLOGY (1952) 15/ $300 Sw fr 12 - Mired-language edition 11 EPIDEMIOLOGY AND CONTROL OF ENDEMIC SYPHILIS - EI Gr (1953) $100 Sw fr 4 12 BCG, VACCINATION 15/ $300 Sw fr. 12 Lydia B Edwards and others (1953) 13 MALARIA TERMINOLOGY 51 - $100 Sw fr 4- Sir Gordon Corell and others (1953) 14 MILK PASTEURIZATION - HD Kay and others (1953) 1276 Sw fr 10 -- $250 15 FIRST INTERNATIONAL SYMPOSIUM ON YAWS CONTROL (1953) S+ SO Sw fr 18 - Mixed-language edition 2216

·150
ADDITIOVAL REFERENCE SOURCES
French
Babes, V. Trante de la rage, Paris, 1912
Dumas, J., Bordet, P., Laporte, R., Lépine, P. Pochon. J. & Prérot, A R. Buterblogle
médicale, Paris, 1951
Lépinc, P. & Sohiet, R. Techniques de kiboratoire appliquers ou diagnostic des malades
durus, Paris, 1954
Levaditi, C. & Lépine, P. Les ultrailrus des maladies humaines, 2nd ed , Paris, 1948
Levaditi, C., Sanchis-Bayarri, V. & Schoen, R. (1928) Neuro-infections auto-sienilo.
sables (cncéphalite, herpes, rage) C. R. Soc. Biol (Paris), 98, 911
Mane, A L'érude expérimentale de la rage (Collection de l'Encyclopédie scientifique).
Paris, 1909
Remlinger, P. & Bailly, J. Etudes sur la rage, Paris, 1938
Romlinger, P. & Bailly, J. La rage, Paris, 1947
German
Doerr, R & Hallauer, C. ed Handbuch der Virusforschung, Wien, 1954
Kolle, W & Wassermann, A., ed. Handbuch der pathogenen Ifikroorganismen, Jena,
1930
Kraus, R , Gerlach, F. & Schucinburg. r. Lyssa hei Mensch und Tier, Berlin & Wien,
1926
Lubinskı, II & Prausnitz, C (1936) Lyssa, Ergebn 11g Buhi 8,1
Italian
Termi, C La rahblu, Siena, 1950

·VETERINARY PUBLIC-HEALTH PUBLICATIONS OF WHO - WORLD HEALTH ORGANIZATION: MONOGRAPH SERIES This series contains important contributions to public health literature which give guidance on questions of international significance, and provide a source of information not otherwise readily available No 14 MILK PASTEURIZATION Planning, Plant, Operation, and Control H.D KAY J R CUTTELL.H S RALL, A TR MATTICK & A ROWLANDS This monograph 19 a joint FAO/WHO publication designed for all those concerned with the planning of pasteurization schemes or with pasteurization legislation It is an abundantly illustrated work on modern techniques, in which the authors discuss the definition and background of modern pasteurization, the buildings, plant, and methods that should be used, and the problems of laboratory control Anneres list the necessary laboratory equipment and deal with pasteurization legislation This monograph should also be valuable in the training of plant foremon and milk-laboratory workers English edition, 1953, 204 pages, 93 figures, 3 tables French edition, 1954, 222 pages, 93 figures, 3 tables 12/6 $2 SO Sw fr 10 - No 19 ADVANCES IN THE CONTROL OF ZOONOSES Bovine Tuberculosis - Brucellosis - Leptospirosis - Q Fever - Rabies WHO/FAO Seminar on Zuonuses, Vienna, Novemher 1952 Zoonoses is a relatively new term designating those diseases which are naturally transmitted between vertebrate anuals and man Their control is one of the major fields of veterinary public health in November 1952, FAO and WHO invited to a seminar in Vienna about 50 medical and veterinary specialists, from 20 countries, to consider the problems raised by the fise zoonoses which are most often met with in Europe The papers read and the discussions which followed are recorded in this jountly published monograph Veterinarians, physicians, and public health officials should find here ·16 TOYIC HAZARDS OF CERTAIN PESTICIDES TO MAN - J. V Auto (1441) 7.6 $1.50 Sw. fr. 6 - 17 THE AFRICAN MIND IN HEALTH AND DISEASE - Corchers (1931) 10, $200 SH fr. 8 - 18 DESIGN AND OPERATION OF SEPTIC TANKS (1951) 10' $I $0 Sw fr 6 -- 19 ADVANCES IN THE CONTROL OF ZOONOSES (1953) 15/ $300 Sw.fr. 12 --- 20 INFLUENZA: A REVIEW OF CURRENT RESEARCH (1954) 17/6 $250 S# fr. 10 - 21 L'HOPITAL RURAL - R. F. Bridgman (1954) 13/6 $2.00 Sw fr 8 - English edition in preparation 22 PLAGUE - R Polhuser (1954) £3 59 00 Sw, fr. 36 - (paperbound) E3 Ss SIO 00 Sw, fr, 40(clothbound) 23 LABORATORY TECHNIQUES IN RABIES (1954) $300 Sw. fr. 12- (paperbound) £ISS $100 Sw fr 16 - (clothbound) 24 DONOVANOSIS - R V. Rajam & P. N. Rangiuh (1954) 10 - $1 30 Sw fr 6 BULLETIN OF THE WORLD HEALTH ORGANIZATION From time to time articles of interest to seterinary public health personnel are published in the Bulletin, which is largely desoted to internationally signifikant studies of the distribution of diseases, and of the results achiesed by specific control methods A number to he issued shortly comprises a selectwn of the paper presented at the suhe Kitton on rabies of the Sisth International Congress for Macrobiology, held in Rome in kopiemher 1951 Contnbutions to this issue come from mine different country, and Lunct the epidemiology and epizootiology of rabies, measures for its control in animals and man methods of vaccine production, and recent experimental research Putin *ining original articies in either English or french with a det" 1 language Two volumes are published annually, caule vrtupne 4 numbers summutir + const inx nu Su fr 14 SU Sub 10. £: 175 ES 10 $50 $40 $16 ne olume olumes ·No. 67 BRUCELLOSIS Second report of the Joint FAO/WHO Expert Committee 34 pages, first impression, 1933 The second report, like the first, summarizes the latest knowledge in human and animal brucellosis Diagnostic problems and advances in the therapy of human brucellosis are considered; indications and contra-indications for the vaccination of cattle are reviewed; and the various diagnostic tests carrted out on milk, as well as the most reliable methods for the isolation, culture, and identification of Brucella, are described, The activities of the tuelve FAO/WHO brucellosis centres established over the world within recent years are summarized This report, which represents a clear statement of the problem in its scientific and practical aspects, will stimulate the research worker and will be of great value to those responsible for the organization of the control of animal and human brucellosis 2/ $0 23 Sw fr 1 - No 40 ZOONOSES Report on the first session of the Jout WHO/FAO Expert Group 47 pages, second impression, 1933 Zoonoses are defined as "those diseases which are naturally transmitted betweca ertebrate animals and man" A list of the zoonoses, comprising more than 80 diseases, is appended, but the report is mainly concerned with five-boyine tuberculosis, Q fever, anthrax, psittacosis, and hydatidosis These diseases are covered from the standpoint of training, prevention, and control in man and animals The responsibilities of the public health veterinarian are outlined on the basis of a definuxon of veterinary public health as comprising all the community efforts influencing or influenced by the veterinary medical arts and sciences applied to the prevention of disease, protection of life, and promotion of the well-being and efficiency of man". The training of veterinary public health specialists is detailed in an annex 2/3 $0 30 Sw fr. 1 20 ·much valuable technical information, and fresh ideas for a joint attack on their common problems. English edition, 1933; 275 pages, 32 figures, 24 tables French sdition, 1984; about 300 pages, 32 figures, tables 15 $300 Sw.fr. 1! WORLD HEALTH ORGANIZATION: TECHNICAL REPORT SERIES This series contains the reports of WHO ctpers committecs, sub committees, joint committees with other specialsred agencies, and other study and advisory groups The following issues are of special interest in the icterinary public health field. No. 28 RABIES Report on the first session of the Expert Committee 26 pages, third impression, 1953 This report summarizes the advances in the prevention and control of rabies in man and animals, and contains pertinent recommendations, and suggestions for future research 1/6 $0 20 Sw.fr. 0 0 No 82 RABIES Second report of the Expert Committee 27 pages, first impression, 1954 The second report of the WHO Expert Committee on Rabies should I read in conjunction with the first in it, the committee considers the results of termenis! studies and field trials of hyperimmune scrum-saccine combinations in man, new chalen embryo vaccines for use in animals, control wildlife, and modifications indicated for the prevention and control of this disease under varying circumstances throughout the world 1/9 50 S S. fr. 1- No 37 BRUCELLOSIS Report on the first session of the point 110/11 NO Erent l'unel 14 puges, second impression, 1933 ouders the twofold aspect of brucellosis as a xorld problem - (a) 19 rubin has a disease transmitted from animals to man, and threat to tb} in animal industry, since it produces abortion, decreased milk yelj. and is or permanent inftruity among Insestock Detaiko recomend). tions for huma Ips and for control of the discute in cattle, sheep, and man) "" given In annetos to the panel s report, the control of brucellosis 19 imine is Will rhus icity and a wd in the bacteriological diagnosis of the diseak pre deti $0:5 od pro

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