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190 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ II, II
A castor-oil mineral-oil mixture, as used in experiment 4, also failed to provide
adequate purgation, and apparently this mixture is not advantageous for use as a
purgative in connection with n-butylidene chloride. The results against cylicos¬
tomes were disappointing in this experiment and also in experiment 5, in which
aloes was used as a purgative, although respective efficacies of 52 per cent. and 60
per cent. were obtained for Strongylus spp. N-butylidene chloride in these two
experiments removed nearly all worms from the caecum but failed to have an
adequate effect on those in the double colon, a fact which suggests that the pur-
gative used was not of a nature suitable to carry the anthelmintic into the double
colon in sufficient quantity or concentration to act effectively on the worms located
there. It may be assumed from a theoretical standpoint that most of the n-buty-
lidene chloride was absorbed before it reached this portion of the gut.
It will be noted that extremely variable results were obtained with n-buty-
lidene chloride against Strongylus spp., the percentage of efficacy ranging from 0 to
100. As these parasites wander as larvae into the tissues, develop to fully grown
agamic froms at points outside the lumen of the large intestine, and ultimately
return to the lumen and promptly assume the appearance and character of mature
worms, it cannot be said with certainty that strongyles found post mortem 5 to 10
days after treatment were present in the gut at the time of treatment. No doubt
this return of strongyles to the lumen of the gut is a more or less continuous
process, especially in the case of S. vulgaris, the agamic forms of which are found
in aneurisms usually present in the anterior mesenteric artery or its branches. As
such aneurisms were present in nearly all the test animals used in these experi-
ments, it is possible, and even probable, that the ineffectiveness of n-butylidene
chloride against S. vulgaris in some of the tests was in part due to the fact that
adult forms found in the large intestine at necropsy had reached that location after
the administration of the anthelmintic.
Of one thing we can at least be sure, that n-butylidene chloride is as effective
as indicated in these tests ; probably it is much more effective against Strongylus
spp., than some of the tests would indicate. In this connection Hall (4, p. 521)
reports :
*****
We are, therefore, safe in con-
cluding that the drugs which remove all or almost all of these worms are
effective, that those which remove few or none and leave many behind
are ineffective, and that those removing any given fraction based on the
number collected from the manure and the number found post-mortem
are substantially as effective as the indicated fraction stated in percen-
tage figures, with the qualifying statement that they may be somewhat
more effective than the findings indicate.
N-BUTYLIDENE CHLORIDE
191
ASCARIDS.
The colt used in experiment 1 was the only test animal infested with ascarids.
Only 11 per cent. of these worms were removed. However, as all ascarids found
post-mortem were immature and the animal was not killed until 17 days after the
administration of the anthelmintic, it is possible that many of these worms reached
the small intestine after the treatment was given. In this test, n-butylidene
chloride was given in raw linseed oil, a method which probably greatly reduced its
efficacy. Evidently this test does not afford a basis for drawing final conclusions,
as in Dr. Bardwell's test an efficacy of 100 per cent. was apparently obtained
against ascarids.
BOTS.
The results against bots were very unsatisfactory. In experiment 1, only 1 per
cent. of the bots were removed and in experiment 6, 46.7 per cent. In five other
tests, n-butylidene chloride failed to remove any of the bots present. It is apparent
that the drug possesses little efficacy against these parasites.
HABRONEMA SPP.
N-butylidene chloride failed to kill the one specimen of H. muscae present in
one animal but showed an efficacy of 55.5 per cent. against this species in another
animal. In a third test, the drug proved to be 97.2 per cent. effective for the
destruction of H. microstoma. In this test, a greater period of time elapsed between
treatment and killing of the animal, a circumstance which no doubt permitted the
drug more time to act on the parasites. Probably the use of a preliminary gastric
lavage would increase the efficacy of n-butylidene chloride for Habronema spp.
STRONGULUS SPP.
In experiments 1, 2, 3, and 6, n-butylidene chloride failed to remove any
specimens of S. vulgaris; the drug removed 60 per cent. of this species in experi-
ment 4 and 100 per cent. in experiment 5. The efficacy of n-butylidene chloride
against S. edentatus varied from 44.4 per cent. in experiment 4 to 100 per cent. in
experiments 2, 6 and 7. In experiment 4, the drug removed 50 per cent. of
S. equinus and in experiment 8, 100 per cent. The efficacy against all species of
Strongylus varied from 0 to 100 per cent., the most consistent being in those cases
where n-butylidene chloride was followed in five hours by a dose of raw linseed oil
adequate for the individual animal.
CYLICOSTOMES.
In experiments 2, 6, 7 and 8, where the anthelmintic was followed in five hours
by an adequate dose of raw linseed oil, 100 per cent. effectiveness was obtained
192 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ II, II
against cylicostomes. In experiment 3, where the dose of linseed oil was not
sufficient, the treatment removed 97 per cent. of these forms. The administration
of n-butylidene chloride in raw linseed oil gave only 80.2 per cent. efficacy in
experiment 1 In experiment 4, where a castor-oil mineral-oil mixture was used,
and in experiment 5, where aloes was used, the results against cylicostomes were
decidedly inferior. When properly administered, n-butylidene chloride apparently
can he depended upon to remove all or nearly all cylicostomes.
TRIODONTOPHORUS
SPP.
These parasites were present in only four of the eight test animals. In experi-
ment 2, n-butylidene chloride removed all of the T. tenuicollis present. It failed to
remove any of this form in experiments 1 and 7 or any of the specimens of
T, serratus present in horse No. 466 (experiment 5).
POTERIOSTOMUM SPP.
In experiments 1, 2, 6, and 8, n-butylidene chloride removed all Poteriosto¬
mum spp. present. It was only 75 per cent. effective in experiment 3, where an
adequate dose of raw linseed oil was not given. With a castor-oil mineral-oil
mixture in experiment 4, the drug removed only 37 per cent. of these forms, and in
experiment 5, where aloes was used as a purgative, the results were negative.
PROBSTMAYRIA VIVIPARA.
Probstmayria vivipara was present in only one animal (experiment 1); n-buty-
lidene chloride administered in raw linseed oil failed to remove any of these forms.
CRATEROSTOMUM
SPP.
The drug removed all Craterostomum spp. present in test animals in experi-
ments 2 and 3.
OESOPHAGODONTUS ROBUSTUS.
N-butylidene chloride removed the one specimen of O. robustus present in the
test animal in experiment 3.
OXYURIDS.
In experiment 1, n-butylidene chloride removed 2-oxyurids but left hundreds
of larvae, being less than 1 per cent. effective. In experiment 3, the drug was 43
per cent. effective, and in experiment 5 it was 50 per cent. effective. Followed by
a castor-oil mineral-oil mixture in experiment 4, n-butylidene chloride failed to
remove the one larva present. These results are not conclusive, as this species
N-BUTYLIDENE CHLORIDE
193
was not present in any of the animals in which, n-butylidene chloride was followed
by a dose of raw linseed oil sufficient for adequate purgation, a method of treat-
ment which might have been productive of better results of the efficacy of the drug
can be judged by its effects against other parasites when it was administered in this
way.
MICROSCOPICAL PATHOLOGY.
The microscopical pathology of the liver and kidneys of three of the animals
used in these tests was studied. Portions of these organs were obtained immediate-
ly after slaughter and fixed in 10 per cent. formalin solution. The sections were
stained with haematoxylin and eosin. A study of these sections indicated the
following :—
Mule No. 430 (dosed with 0.3 c. c. of the drug per kilogram of weight).—
Liver : Chronic interstitial hepatitis ; generalized cloudy swelling of more than
moderate intensity; a few large and small fat droplets in the cells of some lobules
but distribution not uniform and no marked fatty degeneration. Kidney : Con-
siderable hyperemia ; other lesions subacute or chronic, consisting of interstitial
nephritis with extensive round-cell infiltration and beginning fibroblastic formation ;
glomerulonephritis with thickening of the glomerular capsule.
Horse No. 466 (dosed with 0.3 c. c. per kilogram of weight).-—Liver : Slight
cellular hepatitis ; generalized cloudy swelling of only moderate intensity and less
in degree than that exhibited by the previous animal; a few small fat droplets in
cells of some lobules, these droplets being situated mainly in the periphery of the
lobules ; slight eosinophilia. Kidney : Considerable hyperemia ; marked chronic
interstitial nephritis.
Horse No. 452 (dosed with 0.3 c. c. per kilogram of weight).—Liver : Cloudy
swelling of about the same degree as that exhibited by mule No. 430 ; cytoplasm
of liver cells granular and poorly stained ; considerable accumulation of large and
small fat droplets not uniformly distributed but more numerous in some lobules
than in others ; in some lobules fatty inclusions from the centre to the periphery of
the lobules ; slight cellular hepatitis and slight leucocytosis. Kidney : Severe
generalized cloudy swelling ; cytoplasm of tubular epithelium granular and disin-
tegrated, the lumen of the tubules containing epithelial debris, some of which
seemed to be partially degenerated into hyalinelike material; marked hemorrhages
into the tubules and glomeruli; slight chronic interstitial nephritis.
From the above study it may be said that n-butylidene chloride, in doses of
0.3 c. c. per kilogram of body weight, produces in the horse a moderately severe
cloudy swelling of the liver with a slight deposition of fat, which in no case is well
194 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ II, II
marked or extensive. The acute kidney lesions in the first two animals were con-
fined to a hyperemia, but in horse No. 452 the lesions were more pronounced. The
liver of this animal also had a greater accumulation of fat than did the livers of the
other two animals. Horse No. 452 was apparently suffering from constipation at
the time of treatment; no feces were passed on the day after treatment, and it
was necessary to give an additional dose of purgative on the afternoon of that day.
It seems reasonable to assume that the inadequate elimination in this animal
resulted in an increased absorption of the anthelmintic from the intestinal tract
with the production in the liver and kidneys of lesions more severe than those in
the two animals in which proper elimination was obtained. It is probable, there-
fore, that in those cases where the n-butylidene chloride would be rapidly elimina-
ted from the intestinal tract by the action of the purgative, damage to the liver
and kidneys would not be sufficient to militate against the use of this drug as an
anthelmintic for the horse. In fact, in this respect n-butylidene chloride seems to
possess some advantage over some anthelmintics now in use.
SUMMARY.
N-butylidene chloride administered to four animals at a dose rate of 0.207 to
0.333 c. c. per kilogram of body weight and followed in five hours by 960 c. c. of
raw linseed oil for a 1,000-pound animal resulted in efficacies of 50 per cent., 94 per
cent. and 100 per cent, respectively, against Strongylus spp., and of 100 per cent.
for cylicostomes in all four animals. The anthelmintic removed all of the Poterios¬
tomum spp. present in the three animals that harbored these parasites.
In other tests the efficacy of n-butylidene chloride for the abovementioned
parasites was considerably lowered where the drug was administered in raw linseed
oil or where it was followed by aloes or by a castor-oil mineral-oil mixture.
When administered in raw linseed oil, n-butylidene chloride was relatively
ineffective for the removal of ascarids, but when given without oil the drug was
apparently effective for the removal of this species.
N-butylidene chloride, administered to three animals weighing from 800 to
1,200 pounds, in doses of 60 c. c. in capsules, failed to destroy one Habronema
muscae in one animal; was 55.5 per cent. effective for this species in a second
animal; and was 97.2 per cent. effective for H. microstoma in a third animal. It
seems probable that a preliminary gastric lavage would add to the efficacy of
n-butylidene chloride against Habronema spp.
The drug was relatively ineffective against bots. Species of parasites other
than those mentioned above were not present in sufficient numbers to warrant final
conclusions.
N-BUTYLIDENE CHLORIDE
195
N-butylidene chloride was well tolerated and produced no gross pathological
alterations. Microscopical pathology in the livers of treated animals was confined
to cloudy swelling with some fatty degeneration but no central necrosis. In two
of three animals, the drug produced only a hyperemia of the kidneys, but in the
third animal cloudy swelling and degeneration of the tubular epithelium were
present. The severity of the lesions in this animal was apparently associated with
increased absorption of the anthelmintic due to insufficient purgation.
LITERATURE CITED.
(1) Blieck, L. De, and Baudet, E. A. R. F. 1923. Tetrachloorkoolstof als middel tegen Gastrophiluslar-
ven, Ascaris, strongyliden on oxyuris bij hot paard. Tijdschr. Diergeneesk. 50 (1): 140.
(2) Demnitz. A. 1923. Uber die cignung des chlorkohlenstoffes als wurmmittel beim pferde. Deut.
Tierarzll. Wchnschr. 31: 573-575.
(3) Grassnickel, W. 1930. Der Bilirubinspiegel des blutes nach Tetrachlorkohlenstoffgabe beim pferde.
Arch. Wiss. u. Prakt. Tierbeilk 62 (1): 76-88.
(4) Hall, M. C. 1921. Treatment for removing the Gastro-intestinal parasites of horses, with some tests
of carbon tetrachlorid and of mixtures of carbon tetrachlorid and carbon bisulphid. North Amer.
Vet. 2 : 512-524, 550.
(5)—
1922.
Carbon Tetrachloride as an anthelmintic. Amer. Jour. Troup. Med.
2 :373-379.
(6) Lamson, P. D., Robbins, B. H., and Ward, C. B. 1929. The Pharmacology and Toxicology of
Tetrachlorethylene. Amer. Jour. Hyg. 9 : 430-444.
(7) Minot, A. S. 1927. The relation of calcium to the toxicity of carbon tetrachloride in dogs. Soc.
Expt. Biol. and Med. Proc. 24 : 617-620.
(8) Neumann-Kleinpaul, K., and Pelckmann, K. 1930. Ueber Tetrachlorkohlenstoff-vergiftun-gsers¬
cheinungen bei pferden, deren zusammenhang mit dem blutserumcalciumspiegel and aber
versuche, die vergiftungsercheinungen durch erhohen des blutkalkgehaltes abzuschwachen. Arch.
Wiss. u. Prakt. Tierheilk, 62 (1): 1-75, illus.
(9) Robbins, B. H. 1929. The absorption, distribution and excretion of carbon tetrachloride in dogs
under various conditions. Jour. Phrmacol. and Expt. Ther. 37 : 203-216, illus.
(10) Schlingman, A. S. 1926. Miscellaneous tests of Tetrachlorethylene, a new anthelmintic. Jour.
Amer. Vet. Med. Assoc, (n. s. 21) 68 : 741-754.
(11) —
1929. Further miscellaneous tests of tetrachlorethylene as an anthelmintic.
Jour. Amer. Vet. Med. Assoc. (n. s. 28) 75 : 74-85.
(12) Shul'ts, R. E. S., and Raevskaia, Z. A. 1929. (Experiments on the treatment of equine strongylosis
with carbon tetrachloride). Vestnik Sovrem. Vet. Moskva. 5 (24): 620-624. illus. (Original in
Russian).
(13) Wright, W. H. Bozicevich, J., and Underwood, P. C. 1931. Critical experiments with carbon
disulphide in the treatment of habronemiasis. Jour. Roy. Army. Vet Corps. 2 (2) : 3-7.
(14) Wright, W. H. and Schaffer, J. M. 1929. Anthelmintic tests of chlorinated alkylhydrocarbons and
a correlation between the anthelmintic efficacy and the chemical composition (Abstract). Jour.
Parasitol. 16: 107-108.
G
RESEARCH WORK ON FOOT-AND-MOUTH DISEASE.
[The conclusions of the research work appearing on pp. 8-31 of the Fourth Progress Report of the
Foot-and-Mouth Disease Research Committee are reprinted with the kind permission of the Ministry
of Agriculture and Fisheries, England.]—Ed.
Since the publication of the Third Progress Report, research on foot-and-
month disease has been carried on intensively in this and in other countries;
amongst the latter the work at the Island of Riems has been particularly instruc-
tive.
Three comprehensive treatises on foot-and-mouth disease have appeared
within the. last 2 years. Professors, Waldmann and Trautwein, of Riems, Pro-
fessor K. Trautwein, and Professor Maitland, who has done much research for the
Committee in past years, have each summarized the knowledge on the subject
up to the dates of their publications.
The present era of research may be said to have begun with the discovery of
Waldmann and Pape in 1921 that guinea-pigs were susceptible to inoculation with
great regularity and that by this means the presence of virus could be readily
detected in inanimate substances or in animals suffering from infection of an un-
certain nature.
Since that time and indeed since the commencement of research by this
Committee in 1924, the whole outlook on this disease has changed. Knowledge
of the infective virus itself, its means of dissemination, the resistance of suscepti-
ble animals, the progress of the disease in them, and the nature of immunity and the
processes by which it is produced in the animal body, have made much growth.
The earlier foundations laid by Loeffler and the many important contributions to
knowledge by Vallee and other workers in France, have been confirmed and ex-
tended. Nor are the additions to knowledge merely academic, though much of
the information is so recent and the problems involved so complex that practical
application is only being gradually found possible. It is necessary that each step
should be securely based and fully confirmed before practical measures can be
devised.
As in previous reports, a brief summary of the results arrived at by the
investigators in this country and elsewhere, since the Third Progress Report in
1928, will be reviewed in the Introductory pages and the detailed experiments and
conclusions of the scientific individual workers for the Committee will be set out
in the Appendices. A bibliography of the literature on foot-and-mouth disease
published since the last Report has been added as Appendix V. A good deal of the
( 196 )
RESEARCH WORK ON FOOT-AND-MOUTH DISEASE
197
work has of necessity consisted in the examination more exactly and with a view
to practical application of principles and hypotheses enunciated in former years.
At the experimental Station at Pirbright only has work on the large farm
animals been carried out, and preliminary, parallel or confirmatory experiments
have also been made there on small animals.
In the laboratories of the Lister Institute of Preventive Medicine and the Nation-
al Institute for Medical Research at Hampstead, much essential work has been
made possible by the use of guinea-pigs and other small animals. By these means
aspects of the problem have been submitted to test, for which large animals could
not have been used on account of the much greater space required and the relative-
ly longer time and greater labour involved. The more important conclusions have
been confirmed by all our workers in the different laboratories as far as the con-
ditions permitted.
A.—THE VIRUS.
The invisible infective agent in foot-and-mouth disease can still only be
recognised for practical purposes by its effect when introduced into susceptible
living animals, but more has been learnt about the conditions of its ability to
survive in the outside world and the substances which can be used to destroy it or
make it inactive.
B.—CULTURE OF THE VIRUS.
Among the most baffling of the problems which arise in the study of foot-
and-mouth virus has been the failure till quite recently to propagate the virus
outside the bodies of animals. Search has been made in all centres of research
for methods by which multiplication in vitro could be produced in order to estab-
lish more firmly the exact nature of the infective agent and also with a view to
further attempts at special means of protecting and curing infected animals.
Although growth of the virus in completely artificial media has not yet been
obtained, a method has been found by Professor H. B. and Dr. M. C. Maitland with
which propagation results with considerable regularity in small flasks containing
pieces of the living tissues of embryo guinea-pigs to which filtered virus has been
added. In these experiments the virus has increased often one thousand and
sometimes 100,000 times in three or four days. From the first flasks containing
such a growth 16 further subcultures had been made in series between March and
December, 1929, from one flask to another without a break. In the final flask the
virus could be diluted 10,000 times and still infect guinea-pigs. It was calculated
that the original virus introduced into the first flask, if any of it were still present,
would have been diluted many million million times—actually 1039 times—
making the evidence of real growth and multiplication incontrovertible.
H
198 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ II, II
Somewhat similar results have also been obtained by Hecke (1930) working
on the island of Riems, but his work and that of the Maitlands has been entirely
independent.
The Maitlands reported to the Committee that they had arrived at the tenth
culture in series in October. 1929. At this stage the work was unavoidably in-
terrupted, but after the virus from the flask containing the tenth culture had been
kept for three months in the cold room it was found to be still active and the
series of cultures was resumed from the stored contents.
In some cases a greater bulk of virus was obtained by culture in larger flasks.
The animal tissues used in the culture flasks were derived from embryo
guinea-pigs. Chicken embryo which had been used for similar propagation of
vaccinia virus was not found to be suitable for foot-and-mouth virus. For purposes
of culture the skin of the guinea-pig appeared to be the best tissue, but the hairy
skin was found to be as good a culture medium as that from the sole of the
guinea-pig's foot, though the latter is the only part of the external skin in which
the virus has been observed to multiply during life.
C—CONODITIONS OF OXIDATION AND REDUCTION WHICH FAVOUR SURVIVAL OF
VIRUS.
Attempts at cultivation of foot-and-mouth virus in artificial media have
shown that it dies rapidly after the addition of certain substances—not obviously
harmful in themselves—to liquid media in which it was hoped that propagation
might take place. Bedson and Maitland (1927) found that the addition of 10 per
cent. blood serum to broth definitely cut short the life of the virus although
excess of acidity or alkalinity was excluded by careful testing. This observation
was remarkable, because the addition of serum usually favours the culture of
pathogenic micro-organisms and has always been recommended by previous
workers who have essayed the propagation of foot-and-mouth virus. They found
too, that when small pieces of sterile skin or other tissues were also added to the
medium longer survival was again possible, although the acidity was then greater
than that generally found to be the optimum. It is known that in the case of
certain bacteria, especially such as grow best when oxygen is absent or only present
in restricted amount, the best culture media owe their favourable qualities largely
to their possessing a certain definite potentiality for causing oxidation or reduction,
i.e., hindering or promoting the action of oxygen on other constituents of the
mixture. Dr. Edwards has examined the oxidation-reduction and acid-base
properties of various liquids by means of indicator dyes.