# The Indian Journal of Medical Research/Volume 8/The Vaccine Institute, Belgaum

THE VACCINE INSTITUTE, BELGAUM.
A RECORD OF ITS HISTORY AND WORK.
BY
R. W. FISHER, Esq., m.b., d.p.h.,
Director of the Institute.
[Received for publication, May 5, 1920.]
History of Vaccination in Bombay Presidency up to the opening of the new Institute.
The necessity for the introduction of animal vaccine lymph, to replace arm-to-arm methods of vaccination in Bombay Presidency, was brought to the notice of the Government of Bombay by the Government of India in the year 1889.
The Sanitary Commissioner for the Government of Bombay, after consultation with his Deputies, submitted certain proposals and reported to Government in 1903, that certain experimental trials with lanolinated preserved lymph, carried out by Captain* F. H. G. Hutchinson, i.m.s., Deputy Sanitary Commissioner, Southern Registration District, in the districts under his charge, had given encouraging results.
A scheme was accordingly prepared, and approved by Government, for preparing preserved lymph for use in the Southern Registration District and lanolinated lymph was supplied to the vaccinators in 1904-05.
Hutchinson had taken over the duties of Deputy Sanitary Commissioner, on 21st July, 1902, and continued to experiment with lanolinated vaccine.
Plan 1.
VACCINE INSTITUTE AT BELGAUM SITE PLAN SCALE 160FT TO 1 INCH
R. W. Fisher.—The Vaccine Institute, Belgaum. ​Captain W. F. Harvey, i.m.s., in the Punjab and Hutchinson in Bombay were in favour of the glycerine method of preserving lymph. From experiments, very carefully carried out, Harvey2 and Hutchinson3 showed that glycerinated vaccine lymph was to be preferred to lanoline. The arguments for and against lanoline may be stated:—
Lanoline has no effect in reducing the extraneous micro-organisms, which are always present, in large numbers, in lymph collected from the calf. Owing to the presence of these large numbers of extraneous germs, badly inflamed arms are very often met with after vaccination with lanolinated lymph, Lanoline merely forms a convenient vehicle in which the crude pulp may be ground-up and filled into tubes for distribution, so that, whatever extraneous organisms are present, when the pulp is collected from the calf, will remain alive and active when the lymph is used for vaccination. Further, lanoline tends to become acid in India, and acidity in vaccine lymph is fatal to the living virus. Emulsifying the crude pulp with 50 per cent glycerine solution, on the other hand, has a very powerful purifying effect and almost all extraneous germs, except those which form spores, are rapidly killed at tropical temperatures. It was further held, by the advocates of lanolinated lymph, that the vaccine principle was preserved intact, in the case of lanolinated lymph, for a much longer period than was possible, in the case of glycerinated lymph, but experience has not borne out their contention, and lanolinated lymph does not seem to give nearly such good results in India as were expected of it by its advocates. Further, it has been proved by Noguchi4, that tropical temperatures alone are sufficient to cause fairly rapid weakening of the vaccine principle, irrespective of the vehicle with which the lymph is mixed. In fact, he has shown, that vaccine lymph which has been dried, loses its potency in proportion to the increase of the temperature of the air. In the case of glycerinated lymph, if a strong strain of lymph is maintained and due care is taken in preparation and distribution, it has been proved that the lymph retains its potency sufficiently long to obtain very good results in the hands of vaccinators, working over very extensive areas of distribution, such as the Provinces of Bombay and the Punjab.
Proceeding in the light of these ascertained facts, Hutchinson in 1904 began gradually to replace the lanoline method by glycerine, and glycerinated lymph was supplied to all the vaccinators in 1907-08. ​ At this time Hutchinson's work was very much hampered; sufficient funds, staff and suitable buildings, in which to prepare his supplies of preserved lymph, were all wanting. The work was begun and carried on, for some time, in a stable in Belgaum town. When he was able to show that his lymph was proving a success, further funds were forthcoming and some kutcha buildings were rented about two miles from Belgaum in August 1904. The Institute was then called Belgaum Vaccine Institute and its staff consisted of a Superintendent, three vaccinators and two servants.
The new Institute, in this way, fulfilled the double purpose of preparing a reliable vaccine lymph and serving as a training school for Inspectors of Sanitation and Vaccination and vaccinators.
The result of this system of training the vaccinating staff was at once apparent in improved results in the towns and districts which were worked by the trained men. Every effort was made, accordingly, to get as many of the vaccinators as possible passed through the Institute for training and in the very distant districts, such as those of Gujerat, the Inspectors only were brought for training and the Deputy Sanitary Commissioner of the District was able, with the help of these trained Inspectors, to instruct his staff of vaccinators at convenient centres. The results obtained since 1906-07 are shown in the following table:—
Encouraged by the success already obtained, Hutchinson drew up, and sent in to Government, a scheme for the acquisition of 176 acres and 35 gunthas of land about 2½ miles to the south of Belgaum. Plans were drawn up for the erection of a Vaccine Institute on the most modern ​lines, at an estimated cost of Rs. 1,32,000, and the establishment of a suitable staff, at an estimated cost of Rs. 13,020 per annum in salaries. The scheme, with some minor modifications, was approved by Government,— funds were allotted, the land acquired and building of the new Institute was begun in 1907. The Institute was completed in 1910 and the work was transferred there in August of that year.
Method of Preparation and Purification of the Vaccine Lymph.
The supply of calves.—Calves are obtained from contractors of Belgaum. They supply all the calves required on a hire system. Rs. 4-8-0 per calf is paid for hire. The calves are carefully examined and selected on arrival at the dip enclosure. Unhealthy calves are rejected
Fig. 1.
Inoculated Calves' Stable.
Buffalo calves have been used for lymph production, from time to time, but their use has been given up. The lymph obtained from buffalo calves is very impure and difficult to free from extraneous organisms. The lymph, further, has not proved so reliable as calf lymph. Buffalo calves cannot be obtained on hire in Belgaum.
Method of Vaccination.
The calf having boon placed on the tilting operation table, the shaved area is thoroughly washed with soap and warm water, finishing off with ​sterile filtered water. The area is then dried and covered with a sterile cloth. No antiseptics are used in the cleansing of the calf's skin.
Vaccination is carried out, with an ordinary scalpel, dipped in the seed lymph; the scalpel is drawn lightly across the skin, making long, parallel incisions, into which the seed lymph is introduced by the back of the scalpel. The incisions should not be deep enough to draw blood, otherwise the resulting vesicles will not be so well formed. The lines of incisions are spaced about 3-8th of an inch apart. The vaccinated area is dried, by the aid of an electric fan, before removal from the table. The calf is then taken to the inoculated calves' stable, where it is kept for 5 days, while the vaccinia is maturing. Clean cloths are tied over the vaccinated part and renewed as required. Glycerine is applied to the developing vesicles 48, 72 and 96 hours after vaccination. This tends to prevent the premature drying of the vesiculation and helps its development. The calf is kept as dry and clean as possible, during this period.
Collection of Pulp.
At the end of 120 hours the vaccinated calves are again brought on the table and carefully washed with soap and warm water and sterile water and the area covered with a clean cloth. When thoroughly dry, the vesicles are inspected and a note made as to their quality. The quality is graded as good, fair or trial or rejected as unfit for collection. The pulp from those in good or fair class is not further tested as regards potency before issue. That from the 'Trial' class is tried on children before it is declared fit for issue.
The percentage of 'Rejected' calves is shown in the following table for a number of years:—
Table of Rejected Calves. :
| Year. | Percentage of rejected calves. |
| 1910–11.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. | 23.49 |
| 1911–12.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. | 06.35 |
| 1912–13.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. | 03.35 |
| 1913–14.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. | 01.75 |
| 1914–15.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. | 05.62 |
| 1915–16.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. | 15.07 |
| 1916–17.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. | 10.42 |
| 1917–18.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. | 02.58 |
| 1918–19.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. | 04.74 |
Fig. 2.
Matured Vesicles about to be collected.
to form a separate lymph number. This lymph number is dealt with, subsequently, so as to go through the mixing and purification processes without admixture with other lymph numbers and is passed for issue accordingly. The pulp from two or three calves usually forms one lymph number.
Mixture and Grinding of the Pulp.
The collecting bottles are now taken to the lymph mixing room and the pulp of each lymph number is carefully weighed and the requisite quantity of 50 per cent glycerine and water mixture is calculated, so that one part by weight of pulp is mixed with four times its weight of 50 per cent glycerine and water. The calculated quantity of glycerine and water is measured into a flask and. is set aside with its corresponding pulp. Pending grinding, the pulp bottles are kept in an ice-box.
Fig. 3.
Grinding the Pulp.
On the same day, on which the pulp is collected, grinding takes place. Doering's lymph machines are used. These are formed by four glass rollers revolving in contact. The pulp and glycerine and water are ​ground by passing through these machines four times, when a very uniform emulsion is obtained. Each machine is capable of grinding 20 grammes of pulp per hour. The glycerine and water mixture is prepared by mixing the best quality redistilled glycerine with sterile distilled water. The mixture is autoclaved, rendered faintly alkaline with soda bicarb., placed in a flask, which is held in a stand in an inverted position, connected by a 3-way tap to a graduated burette, and measured off into flasks as required. It is important that the lymph emulsion should be kept faintly alkaline, as any development of acidity is detrimental to the vaccine principle. The lymph emulsion, as it leaves the grinding machines, securely corked in suitable glass tubes, is found to contain a large number of extraneous micro-organisms. Ten milligrammes of this lymph, when mixed with agar culture medium and poured on plates, shows an uncountable number of these extraneous germs. They consist, for the most part, of staphylococcus albus, aureus and citreus from the calf's skin and seem to develop in the vaccinia vesicle at the same time as the vaccinia virus. They are not, as a rule, markedly pathogenic but, where crude unpurified lymph is used for vaccination, a number of abscesses are liable to be met with, and the inflammation, accompanying the development of the vaccine vesicle, is much more marked than in the case where lymph is used which has been rendered free from these extraneous organisms. Hence, the desirability of freeing the lymph from these germs. In many Vaccine Institutes, such as that at Hendon, London, reliance is placed on the purifying effect of glycerine, but this is found to be very slow, and may require many months' storage, where the lymph is kept at or below freezing point. Dr. Blaxall5 states, in this connection, that glycerine has no germicidal action of itself, but in conjunction with heat, it is a powerful germicide and this action varies directly with the temperature. Hence, to get the purifying effect of glycerine on the extraneous organisms, it is found necessary to store the lymph at temperatures considerably above freezing point. Blaxall5 showed this very clearly in his work at Hendon.
This method of purification was given a fair trial at Belgaum. I found it very uncertain in action, and a certain proportion of lymphs ontinue to show largo numbers of extraneous germs, necessitating prolonged storage and frequent bacteriological examinations, before the lymphs are pure enough for issue. Unfortunately, prolonged storage frequently reduces the activity of the virus, so that trial on children is advisable before despatch to the districts. ​ Blaxall6 has introduced the admixture of clove oil with glycerine and water mixture, in the proportion of l per cent. He finds that the lymph, containing clove oil, is much more rapidly purified, so that a much shorter storage is necessary. No reduction in the potency of the lymph has been noticed, in his hands, by this method.
A trial of the addition of 1 per cent clove oil was made at this Institute, but it was found that the action of the clove oil was very uncertain, in the case of the lymph produced here. The purification where clove oil was added, was more rapid than in the case of glycerine alone, but a certain number of lymph numbers proved very resistent and considerable numbers of organisms were still present after many weeks' storage. It seems a risky procedure to store a living vaccine containing an active disinfectant such as an essential oil. One would expect the active virus to be injured ultimately, and our experience of the method tends to confirm this to some extent, though, in view of Blaxall's work, its action must be very slow. Its use was, however, discontinued in favour of chloroformed lymph. Experiments were also made with glycerinated lymph, which was kept for a week or longer at about 50° F., till the extraneous germs were reduced sufficiently and then placed in cold store below freezing point. The results obtained by this method were not so good as in the case of chloroformed lymph.
In fact, the results obtained with glycerinated lymph, purified by chloroform according to the methods used at this Institute, have proved so excellent that all other methods have been given up for routine work. Rapidity of purification, by this method, is a great advantage, as experience has amply demonstrated that a certain proportion of vaccine lymphs, unless stored at very low temperatures, tends to decrease in potency the longer it is stored. So that, where lymph has been stored for any period over three months, it is found uncertain as regards its potency, and it cannot be issued, with safety, without previous trial on children to ascertain its activity.
Our experience at Belgaum is that, where a series of lymph numbers, collected from calves showing vesiculation of the same quality and treated in an identical way throughout the different processes, are stored under the same conditions, a small proportion of these lymph numbers will show marked deterioration, after four months' storage, while the remaining larger proportion will have retained its potency slightly, if at all, unimpaired. No cause, up to the present, has been discovered for this uncertain behaviour of stored lymph. It is probably due to the ​growth of certain moulds or other resistent growths, which give off, in their growth, some chemical agent which is inimical to the vaccine virus The trial of all lymph, to test its activity prior to issue, would be the ideal procedure, but lack of sufficient children in the neighbourhood of the Institute, and the labour entailed, renders this difficult to carry out. In the case of Sind, only, it has been found feasible to await the result of a trial issue before despatching the remainder of that lymph number to Sind vaccinators. In this case it is essential, that a vaccine of the highest potency should be supplied, owing to the time taken by lymph packets in the post, before they come to the hands of the vaccinators, and also, owing to the very high temperatures recorded in Sind during the hot season. Therefore a preliminary trial issue is made to the Superintendent of Vaccination at Karachi, who sees, personally, the results obtained and at once advises us if the vesiculation is of goo d quality. In this way lymphs which do not prove resistent to these adverse conditions are identified and excluded from issue.
The Chloroform Process of Purification.
The removal of extraneous organisms from vaccine lymph, by the agency of chloroform vapour, was first introduced by Dr. A. B. Green7 of the Lister Institute in 1902.
His method was to triturate the pulp with distilled water, one part pulp to three water. Chloroform-saturated air was then blown through this emulsion for two hours. The inlet and outlet of the vaccine tube were then clamped, and the chloroform retained in the emulsion for 18 to 42 hours. A current of sterile air was then passed through the lymph and the chloroform partially removed. The lymph was stored in an ice-chest at 10" C. and, immediately before issue, the remainder of the chloroform was blown off and glycerine was added, in the proportion of two parts of glycerine to the original weight of the pulp.
He found that all the extraneous organisms were killed off by this method and that the vaccine virus was still active, so that the lymph was ready for despatch without prolonged storage.
Blaxall informed me that this method of Green's was given a thorough trial at Hendon, but that it proved injurious to the vitality of the lymph in some cases, and he would only recommend it, where lymph was required so urgently as not to permit of storage.
The method seemed to oiler great advantages in India, where the facilities for cold storage were very limited, and Hutchiiison carried out a ​series of experiments, with the object of so improving on Green's original method, as to overcome its obvious shortcomings. He argued that the chloroform was probably left too long in contact with the lymph, thus endangering the vaccine virus. Therefore, after chloroform had been passed for 2 to 3 hours, air was bubbled through the lymph and all trace of chloroform removed. He found that the extraneous germs were killed off by this method, so that plates poured from the chloroformed lymph showed no growth of the usual flora found in the crude lymph. The lymph, on the other hand, proved quite active and gave excellent results in the hands of vaccinators. It was found possible, by using this method, to have a pure lymph ready for issue in two weeks from the time of collection of the pulp from the calf, so that prolonged storage was unnecessary for purification purposes. Modifications of the process have been introduced in the light of experience and the objections, which were valid against the original method of Green, have been entirely overcome. The results obtained, with our chloroformed vaccine lymph in a tropical country, will bear comparison with those obtained in temperate climates, where more conservative methods of purification are still carried out. The method has stood the test of experience, and it is claimed that a vaccine lymph is issued from Belgaum, of a very high standard of potency, free from the usual flora of extraneous micro-organisms. Where sporebearing germs and moulds gain access to vaccine lymph, no known method will eradicate them which will not, at the same time, destroy the vital virus. But where proper care is taken in all the details of preparing the calves for vaccination, and collecting and handling the lymph, and sufficient staff is employed to enable this to be insisted upon, the introduction of these spore-bearing organisms can be reduced to a minimum, and, with the exception of tetanus, none of these resistent germs are found to have any pathogenic effect. Harvey 1 has stated that, with care, pathogenic organisms, such as those of tetanus and malignant oedema, can be absolutely excluded from the lymph, and Dr. Anderson, 8 Director of the Hygienic Laboratory, U. S. A., Public Health Service, records that vaccine for 2,000,000 persons has passed through the Laboratory and in no case has the organism of tetanus been detected, although specially searched for ; and he further states that ' Where tetanus occurs after vaccination, the infection is received by a contamination of the vaccination wound such as may occur in the infection of any other surgical wound not properly cared for.' ​A statement showing the results obtained with Belgaum lymph is given in Appendix A.
It must be borne in mind, when considering these figures, that every effort is made, in the districts concerned, to check results. Inspectors of Sanitation and Vaccination are on duty in each district and they carefully check the work of the vaccinators. These Inspectors are officials trained
Fig. 4.
The Chloroforming Apparatus
in Bombay, who have had to pass a searching examination and receive good pay. Their reports are, therefore, to be relied on. A Deputy Sanitary Commissioner is in charge of a number of districts and, during his tour of seven months of the year, he checks, personally, the vaccination work; and I am informed by these officers, that every reliance can be placed on the figures sent in. There are four Deputy Sanitary Commissioners ​on duty in the Presidency proper and one in Sind, so that a very efficient supervision of the vaccinating staff is maintained. Further, it must be emphasised, in considering these results obtained with Belgaum lymph, that vaccination work is carried on throughout the hot season and not only in the cold weather, as obtains in some parts of India. A very severe test is thus thrown on the potency of the lymph, as, in many of the northern districts of the Presidency, very high temperatures are recorded during the hot months of the year, and the heat in Sind during the summer is notorious, shade temperatures of over 120° F. being often recorded.
Detailed Description of the Chloroform Process of Purification as practised at Belgaum.
The apparatus used:—
The apparatus consists of a water blast pump A, which is connected to a steady head of water, so that pressure varies as little as possible. The pump is connected by rubber tubing to a large empty bottle B, which receives any water accidentally driven over from the pump. The current of air then passes through an air filter C, consisting of a brass tube filled with cotton wool, the whole being sterilized in the autoclave. After passing through the filter, the air enters the gas washing bottle D, containing pure chloroform ( B. W. and Company, anaesthetic ). The chloroform-saturated air is now led through a Woulff's bottle E, containing dry sterile sand designed to interrupt any chloroform which might be driven over by accident. The air then passes into a chest F, which contains the tubes of lymph. This chest has a double jacket, the inner, made of brass, is so arranged that ice can be introduced into the box, ice cold water circulates round the jacket and a temperature of 10° C. can be maintained. The tubes G are large 1 1/4 bore and arranged in series, with rubber tubing, so, that the air is bubbled through each tube in succession and escapes at the end of the series through a tube led outside the box at H. At the beginning of the series an empty tube intervenes, so that any condensed chloroform is retained and does not gain access to the lymph-containing tubes. One-third of each tube is filled with lymph, and the glass tube, carrying the entering air, passes down close to the bottom of the tube, which causes the chloroform-saturated air to bubble up through the lymph and pass on to the next tube of the series. The pressure of air is so regulated that the air bubbles gently through the lymph without​carrying over to the next tube. Where more than one lymph number is dealt with at the same time, an empty tube intervenes. The temperature of the chest is important and great care should be taken to see that the correct temperature is maintained. The chloroform vapour should be passed while the interior of the chest and tubes are at 20° C. ; at this temperature the chloroform acts in such a way that a passage of vapour through the lymph for 20 minutes to half an hour is sufficient to remove most of the extraneous organisms from calf-lymph. Our object is not to remove all the extraneous germs and show no growth on agar plates, when subsequently the lymph is plated out. A few colonies should always remain, and these colonies furnish an accurate indication that the chloroforming has not been pushed so far as to injure the vaccine virus. When all colonies are killed, one cannot be certain that too much chloroform has not been used. If clean plates result from the plating of the chloroformed lymph, then the time of passing the vapour should be reduced, until a small number of colonies, say under 15, are shown on the plates. These few organisms are easily dealt with by the glycerine in the lymph, especially at the temperatures obtaining when the lymph is despatched through the post to the vaccinators in Indian districts. Experience has fully demonstrated that, if some organisms are found in the lymph after chloroforming, the vaccine virus remains uninjured. When the time necessary for passage of chloroform vapour has been determined by this method, such that a few colonies remain alive, as shown on agar plates, the vapour is passed for half that period and the blast pump is then closed and the tubes of lymph are reversed in the chest, so that the first tube becomes the last in the series and the last tube becomes the first. The pump is againt umed on and chloroform passes for the remainder of the time. This method of reversing the tubes prevents the lymph, receiving the vapour first, from being more acted upon than the end of the series, and tends to a uniform purifying process for all the tubes. When chloroform has passed for the correct time the gas-washing bottle is removed and air alone is blown through the lymph, the temperature of the chest being maintained for 10 minutes at 20° C. After this, ice is added to the chest and the temperature is lowered to 10° C. and maintained at this temperature while the air is being blown through. It is found that the passage of air for three hours removes every trace of chloroform from the lymph. The object of keeping the temperature at 20° C. for ten minutes is to prevent the possibility of the chloroform, dissolved in the lymph, condensing out as liquid chloroform, which might occur if the temperature ​were at once lowered while the lymph was fully saturated. Liquid chloroform, coming in contact with the lymph, tends to kill the vaccine virus. The lowering of the temperature to 10° C. has for its object the prevention of injury to the virus by the action of glycerine at warm air temperature, in fact, all through, from the collection of the pulp until the lymph is finally sent to the post, every effort is made to keep the lymph as cool as possible, so as to retain the potency unimpaired by the effect of temperatures above freezing.
The great advantage of this method of removing the extraneous micro-organisms from lymph are obvious. The method, the details of which must be carried out with great care, has proved, after years of trial, perfectly safe, and, if the safeguards which have been described are attended to, no injury results to the vaccine virus. Lymph, by this method, can be rapidly and cheaply freed from extraneous germs, so that a pure product can be in the hands of vaccinators within three weeks from the date of collection. The results of vaccination on children, with chloroformed vaccine, are very satisfactory. Inflammation is reduced to a minimum, and, on the seventh day after vaccination, firm regular vesicles are obtained with a marked absence of inflammation in the surrounding tissues, the latter condition being the rule before the introduction of a germ-free lymph. An interesting experiment, with results bearing on the heat-resisting qualities of our chloroformed lymph, compared with lymph prepared by a number of other methods is worth putting on record at this point.
Three calves, showing vesiculation of fairly good quality, were selected and the pulp collected from each separately. These three pulps were each divided as follows:—
Two grammes labelled A, 2 grammes B, 2 grammes C and the remainder labelled D.
A was emulsified with four times its weight of normal saline containing 1 per cent phenol.
B was emulsified with four times its weight of normal saline containing 5 per cent phenol.
C was emulsified with four times its weight of normal saline solution.
D was emulsified with four times its weight of 50 per cent glycerine and water.
After emulsification 10 c.c. of D was taken and labelled D. The remainder was treated with chloroform vapour for 30 minutes and the ​chloroform then blown off. This portion was labelled E. The five lymphs, now prepared as above, were examined by plating on agar and colonies counted.
The five lymphs, A, B, C, D and E, were then divided into three parts and labelled as follows : —
A 1, A 2, A 3, B 1, B 2, B 3, and so on.
The A 1, B 1, CI, D 1 and E 1 were placed in the incubator at 37° C.
A 2, B 2, etc., were kept at room temperature, average about 24° C.
A3, B 3, etc., were placed in cold-store, average temperature about 5° C.
After seven days' storage, as above, calves were vaccinated with each of these emulsions in separate lines of 3" in length, so that calf No. 681 had three lines, representing the A series, three lines of B series, etc., in all 15 lines. This was repeated on two other calves. The calves were examined after 120 hours and the resulting vesiculation carefully compared and recorded; vide Appendix B.
The emulsions were then returned to the incubator, room and coldstore, as before, and kept for a further seven days and again a series of calves was vaccinated in a similar way and the results recorded. This was again repeated in further series of calves after 21 and 28 days' storage, but those emulsions, which gave no vesiculation, after 21 days, indicating the death of the virus, were not carried on further.
A study of the results, recorded in the Table, will show the very definite effect of temperature on the vitality of the vaccine principle. Whatever the diluent used, the virus is much weakened, and in many cases killed outright, after 14 days at blood heat. The very marked lethal effect of 1 per cent phenol at 37° C. is noticeable. Noguchi4 found that his strain of vaccine was more resistant in dilutions of .5 to 1 per cent phenol than in 40 per cent glycerine. My experiments do not confirm this; in 40 per cent glycerine the virus retained its vitality almost unimpaired for seven days at 37° C. and was still alive after 28 days. In 1 per cent phenol, on the other hand, the virus was killed after seven days and was barely alive after 14 days in 5 per cent phenol.
The effect of heat on the lymph, which was prepared in saline solution, is very unexpected. The virus was killed after seven days at 37° C, except for one or two points which survived. The lethal effect must be accounted for by the marked multiplication of extraneous organisms which took place in the case of the saline-diluted lymph. The bacteriological results have not been included in the Table but are being worked out separately ​it may be stated, however, that a very great increase of germs, as shown on agar plates, occurred in the case of the saline-diluted lymph, while in 1 per cent phenol, and the glycerinated lymphs, the organisms were much reduced after seven days' storage at 37° C. Evidently, the growth of these germs is very injurious to the virus, and, if lymph is not prepared containing an active germicide, such as phenol or glycerine, the virus will suffer when the temperature of storage is above freezing point. In Noguchi's experiments, already referred to, he worked with a germ-free lymph obtained by his intratesticular method. Such diluents, as saUne distilled water and Ringer's solution, did not prevent the continued vitality of his virus at blood-heat; so it is evident, that the injurious factor is the growth of extraneous organisms, which are always present in lymph prepared from the skin. This would account, also, for the rapid deterioration, which has been shown to occur in the case of lanolinated lymph; as the lanoline exerts no germicidal action and the extraneous germs are free to grow and, by the action of their toxins, in Cure the virus. These experiments are all in favour of glycerine as a diluent for vaccine lymph, as neither the glycerinated nor glycerinated and chloroformed lymphs showed the deterioration which was so marked in the phenolated and saline diluted lymphs. The vesiculation, in the case of the chloroformed lymph, was rather better than that given by the glycerinated lymph, while the vitality was very similar in both. The chloroformed lymph was rendered practically free from extraneous germs, the agar plates only showing 10 colonies, while in the case of the glycerinated sample, many organisms were present, but became rapidly reduced at blood-heat and room-temperature. In cold-store, the glycerinated lymph showed a slow but steady reduction of extraneous organisms 12 colonies being present in 10 milligrammes after 28 days' storage. The experiment, therefore, confirms the experience gained in the districts with chloroformed glycerinated lymph and ratifies the opinion, previously-formed, that glycerinated lymph, carefully treated with chloroform vapour, has very satisfactory heat-resisting qualities and can be depended upon to retain its potency, at blood-heat, for two weeks and at room-temperature for four weeks.
Purification by the chloroform method is under complete control, as the bacteriological test is a sure guide to the amount of choroform necessary to reduce the organisms always present in crude lymph. When dependence is placed on glycerine alone to purify the lymph, it is impossible to determine how long it will require for the glycerine to ​reduce the extraneous germs sufficiently, to permit of issue. Certain lymphs purify quickly, while others are most resistant, and many months are often necessary before the germs are got rid of. In the meantime, the potency of the lymph has been endangered by prolonged storage, and such lymphs must be tried, to test their activity, before they can be issued with safety. Such uncertainty is very unsatisfactory and repeated bacteriological tests are required, entailing labour and expense. I am, therefore, strongly in favour of the method I have described for freeing vaccine lymph from extraneous micro-organisms and I can strongly recommend it to workers in the same field.
Storing, Tubing and Despatch.
After the lymph has passed through the chloroform process, office copies of each lymph number are taken in small 2 c.c. glass tubes, each corked with a sterile cork and dipped in melted paraffin. The rest of the lymph is filled into test tubes, corked securely and paraffined, leaving as little air between the lymph and the cork as possible. These separate lymph numbers are placed in wire baskets and kept in cold-storage with their corresponding office copies. When convenient, the office copies are taken out and agar plates are poured from each. Two loopfuls of lymph, weighing 10 milligrammes, are taken from each lymph number and mixed in a tube of melted agar culture medium at 42° C, well shaken up and poured into petri dishes. The plates are incubated at 37 °C. and colonies are counted after four days. Those plates showing 15 or less colonies are marked as passed for issue and entered in the despatch register accordingly. When more than 15 colonies are found, the lymph is stored for another week and again plates are poured from it. When colonies are reduced sufficiently, the lymph is passed for issue. It is rarely necessary to store lymph longer than a few weeks, as the large majority are ound pure enough at the first pouring to be passed.
Filling.
Lymph numbers are selected from the despatch register and the tubes taken from store and carefully decanted into a filling funnel. This is a glass funnel with a cover; a piece of rubber tube is attached, which has a spring clip and glass nozzle. The glass funnel containing the lymph is put into an outer copper container (A), mounted on a tripod stand (B) and surrounded by crushed ice and the cover fixed. The rubber tube, with clip attached, projects from the bottom of the copper container and the Assistant, who has charge of the filling, is provided with a copper box (C) ​containing the vials to be filled. These vials have been very carefully cleansed and plugged with cotton wool. They are placed in the copper box and sterilised by dry heat at 175° C. They measure 27 m.m. by 8 m.m. A vial is taken from the box and the cotton wool plug removed and c.c. of lymph is run into the vial from the glass nozzle by easing the spring
Fig. 5.
Filling the Lymph.
clip. The charged vial is then placed quickly in a wooden block, having holes to receive the vials. Another Assistant takes the vial and securely corks it. The corks are also kept in a copper box which has a perforated false bottom, each cork being placed head up in a perforation of the false bottom. They have been sterilised in position, by autoclaving, at a temperature of 125° C. for 45 minutes. The Assistant grasps the cork ​by the head and introduces it into the vial and passes the corked vial on to a third Assistant who dips the cork and head of the tube in melted paraffin. The vial is then placed in a copper box with perforated false bottom and this box is pushed in gradually into a special metal box like a drawer (D). This metal box contains crushed ice. So the vials are always kept cold. These vials, containing 1/2 c.c, lymph, are issued as sufficient for 10 primary vaccinations, each child under one year receiving two insertions on each arm. Above one year, three insertions on one arm and two on the other is the rule with Government vaccinators. In the case of re- vaccinations, three insertions are given on one arm only. Capillary tubes were tried by Hutchinson but given up, as it was found that lymph did not retain its potency as well in capillary tubes, owing to the lymph being more exposed to the effect of temperature in the capillary tube.
Packing and Despatch.
The vials are now ready for packing for despatch through the post. Each vial is put into a rectangular piece of wood which has a hole bored in its long axis from one end, large enough to receive the vial. A piece of wool is placed at the bottom of the cavity of the wooden case, the vial is dropped in and the case is corked. The wooden cases containing the vials are packed in stout cardboard shding boxes, each box large enough to contain four wooden cases. Along with the wooden cases a result statement form is packed in the cardboard box. The cardboard box is now placed in a large envelope, made of reinforced paper, and tied with string. The packet is then stamped and is ready for posting. A sub-post office, on the Institute premises, receives the packets. The result statement is returned to the Institute by the vaccinator correctly filled up and showmg the insertion and case success. Cardboard boxes, wooden cases, and empty vials are also returned to the Institute.
While describing the method of despatch, something must be said regarding the difficulties which were encountered in dealing with Sind. Owing to the long distance of Sind from Belgaum, postal packets requiring a minimum of four days and maximum of nine to ten days to reach their destination in the Sind districts, together with the intense heat encountered on the railway journey from Bombay to Sind, in passing through the sandy tracts of that part of India, and the very high temperature recorded in Sind itself, it was felt that, under such adverse conditions, it would be asking too much of the keeping ​qualities of the lymph to expect it to retain its activity when finally it reached the hands of the vaccinators. During three months of the winter season lymph could be safely transmitted, without encountering extremes of temperature, and would retain its potency long enough to give good results with Sind vaccinators. This short period, however, was not sufficient to allow vaccinators time to vaccinate the children
Fig. 6.
Packing and Despatching the Lymph.
in the areas for which they were responsible, and the objectionable and expensive practice of calf-to-arm vaccination would have to be resorted to during the remainder of the year, if some method could not be devised for counteracting the evil effects of the high temperatures on the potency of the supply of lymph from Belgaum. Thermos flasks were tried, but found too expensive in postage and difficult to pack to prevent ​breaking, — also lymph despatched by this method would have to be sent in bulk to a distributing centre, — an unworkable arrangement. I have to thank Dr. Blaxall, Director of the Lymph Establishment at Hendon, for very kindly suggesting the method which was adopted and proved very satisfactory. This method consists of packing the wooden cases, containing the lymph vials, in small cane baskets. The wooden cases are surrounded by cotton wool, which has been saturated with water, and packed in the cane baskets. The lids are fitted and the whole securely tied with strong twine. A cardboard label is sewn on to the lid to receive the address and stamps. These baskets are made for me in the Ahmedabad Jail. They measure 5" square, fitted with a lid. They are made of stout cane work and permit of evaporation of the moisture from the wet wool. It was found, by experiment, that a mass of wool of this size retained its moisture for about nine days in the hot weather, and during that time the contained lymph vial is kept quite cool by evaporation of the moisture. The hotter and drier the air, the cooler the packet becomes.
The improvement in the results from the Sind districts, when this method was introduced, was marked. Instead of an insertion and case success of 86"89 and 95- 63, respectively, the results went up to 97 and 98.
In the case of despatch to Sind, it was found, further, that a certain small proportion of lymphs, which were classed as fair or good and which would return good results in the nearer districts, proved weak when exposed to the long journey to Sind. A system of trial was then introduced, as pointed out earlier in this paper. In addition, the Superintendent of Vaccination at Karachi came to Belgaum and studied, thoroughly, the methods of handling preserved lymph and was instructed in the precauticjns which are essential to success in carrying out vaccination. He, on his return, held a class for vaccinators of Sind, and with the co-operation of the Deputy Sanitary Commissioner, the vaccinating staff was well trained in modern methods. Much credit is due to the then Deputy Sanitary Commissioner, Major W. O'S. Murphy, i.m.s., for the way he persevered with the introduction of Belgaum lymph into Sind, and the results obtained to-day are largely due to his efforts. The insertion and case success, returned from Sind for the year 1918-19, were 97 and 98, respectively, and vaccination was carried on right through the hot season. It is claimed that the problem of carrying out successful vaccination with preserved lymph in the very hottest tropical climate, has been solved. ​
Further Remarks on Methods.
In the preparation of animal vaccine lymph, one of the many difficulties met with, in a tropical country, is the maintenance of a potent strain of seed lymph. If vaccinia is carried on from calf to calf, after a time the strain weakens in potency and the vesiculation obtained, after from six to eight moves on the calf, becomes very poor and many calves have to be rejected as unfit for collection. In the old days, in Europe, efforts were made to renew the stock by obtaining a fresh strain of vaccinia from natural outbreaks of the disease among cattle. This was an uncertain method, as the occurrence of natural vaccinia among cattle is a rare occurrence and hard to discover. Retro-vaccination, or the passage of the virus through children and back from the child to the calf, was found fairly efficient in maintaining the activity of vaccinia. The objections to this are obvious. Parents object to the collection of lymph from their children's arms, especially in a country, like India, where most of the population is uneducated and very suspicious of modern scientific procedures. The passage of calf lymph through buffaloes and back to calves, was found to keep the activity of the virus at a fairly high level, and this method is in use in many of the Vaccine Institutes of India. At Hendon, Local Government Board Lymph Establishment, the use of rabbits, for this purpose, was demonstrated to me by Dr. Blaxall and introduced in Belgaum. I find it an excellent method and, since rabbits have been used, no difficulty has been found in maintaining a seed lymph of high potency. The method in use is as follows:—
Some of the best vesicles, found on one or two calves at a morning's collection, are chosen and collected for use as seed lymph. The pulp is treated in the routine manner, being chloroformed and examined bacteriologically. This seed is used for vaccinating a further batch of calves, and again seed is collected from a calf or two which shows the best vesicles. After four or five moves on the calf, the seed is passed through a pair of rabbits. An area on the rabbit's side is carefully shaved and a non-pigmented area is selected for vaccination. Grey rabbits, so-called Belgian hares, are found the most suitable animals. The skin having been well washed and dried, the lymph is spread over the area selected and gently scratched with a piece of wood, such as a match. If incisions are made, with a lancet or scalpel, the results are very poor on the rabbit, but if the skin is well shaved, a few gentle linear scratches are sufficient. After 72 hours, vesicles will have formed and are removed by scraping over the surface of the skin with a scalpel. ​The vesicles obtained on the rabbit are not formed like the vesicles found on a calf or child and often only a number of raised red papules are obtained. These, however, contain the active virus and must be scraped away with the sharp scalpel. The admixture of some blood in the scrapings does not matter and the material is rubbed up in an agate mortar with 50 per cent glycerine and water. The emulsion is then stored for use, and, when required, is used to vaccinate a calf. The result of the first move, on. the calf, is usually good enough to be used for despatch. The best of the vesicles are chosen and collected for seed for calf vaccination. It is found that, after passage through the rabbit, the vesiculation on the calf is greatly improved, and gives firm, unbroken vesicles after 120 hours.
APPENDIX A.
Statement showing the results obtained with Belgaum lymph during the years 1914-15 to 1918-19 inclusive.
REFERENCES.
1. King, Col. W. G.. c.i.e., l.M.s. (Retired) 2. Harvey, Capt. W. F., i.m.s. 3. Hutchinson-, Capt. F. H. O. I.M.S. 4. NOGUCHI HiDEYS, M.D. 5. Blaxall. F. R., M.D. 6. Blaxall, F. R., m.d. 7. Greex, a. B., Dr. 8. Anderson, John
The cultivation and preservation of animal vaccine under tropical conditions. Berlin Congress of Royal Institute of Public Health, 1902.
Vaccine lymphs in the Punjab. Indian Medical Gazette. Vol. XL, No. .3. March, 1905.
Preparation, distribution and use of calf vaccine in the tropics. Transactions of The Bombay Medical Congross 1909.
Further studies on the proiieilies of pure vaccine virus cultivated in ^^vo. Journal of Experimental Medicine, Vol. XXVII, No. 3, March 1st, 1918, p. 425.
On glycerine as a germicide. Local Government Board Reports. 1902-03. Appendix C. No. 1.
Report on the use of oil of cloves in the preparation of glycerinated calf lymph. Local Government Board Reports. 1911-12. Appendix C.
On the use of chloroform in the preparation of vaccine. Local Government Board Reports, 1902-03. No. 5.
United States Public Health Reports, 1915, July, p. 2112.
* ↑ * Now Lieut. Col. F. H. G. Hutchinson, i.m.s., Sanitary Commissioner with the Government of India.
* ↑ Now Lieut.-Col. W. F. Harvey, i.m.s., Director of the Central Research Institute, Kasauli.