Further Observations on Epizootic Cerebrospinal Nematodiasis:

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This anresthetic was the most difficult to manage because of the lengtl,l ,of )proP.uced by an anresthetic dose and the <;iifficulty of ju.dging depth. It was found that a dose of o.6 ml. of the I per cent solution often produced sleep lasting well over half an hour, while less than this dose frequently did not anresthetise the rat at all. An inducing dose of o.6 ml. was therefore given to all except the lightest rats (under I 20 gm.), which were given 0.5 ml. A much ye&er degree of reflex movement on stimulation than after nembutal was ta~en as the indication for injecting a supplementary dose. After anresthesia these animals r,emained sleepy for several hours, although responding actively to m:imulation. It was noted that at post-mortem on rats dying during pentothal anresthesia, t,h~r.e W.<.lS fnequently a considerable amount of fluid in the peritoneal cavity. ~ar.c~is

Acknowledgments

I am indebted to Messrs. Bengers, Ltd., Holmes Chapel, for allowing me the facilities of their 'laboratory and for providing the experimental animals used. lly thanks are also due to Dr. F. Fletcher, of Bengers Laboratories, for much useful advice, and to Prof. J. G. Wright for helpful criticism. BIBLIOGRAPHY 0) Swerdlow, M. : Brit. fl. AntEsthesia., 24, 25, 1952. 0) Swerdlow, M . In the Press. (3) Dreyer, G., and Walker, E. W. A.: Proc. Roy. Soc., 1?87, 319, 1914.

'FURTHER OBSERVATIONS ON EPIZOOTIC CEREBROSPINAL ·NEMATODIASIS: ,(. Ch.e motherapeutic Control of the Disease bjy I....Diethylcarbamyl-4-Methylpiperazine .Citrate: ~Preliminary 1Field Trial By CHUSABURO SHOHO, D.V.M., Dip.Agric.,* Nakayma Soen Post Yamamoto, J(awabe-Gun Hyogo Ken, Iapan.

Introduction introduction of the cotton rat (Sigmodon hispidus), naturally infeCted with filaria (Litos.omoides carinii), in I 945, for the evaluation of drug activity ~pplanted all other in vivo methods in the search for new antifilarial chemical r~gents. A comprehensive account of all work done in the U.S.A., by many :~rJceFS, leading to the discovery of a I -diethylcarbamyl-4-methylpiperazine bydrooliloride, as an unrivalled active filaricide, was presented at a conference ,pn " The Chemotherapy of Filariasis" in 1947 (published by New York Academ,y of Sciences, I 948). Since then further evidence has accrued and the early promise of efficacy against Wuchereria bancrofti has fulfilled expectations. In (Japan, ·the citr-ate salt of the same compound has been tried ·by some workers ·trHE

'*'Formerly working 1at ·Institute of Infectious Diseases, University, Tokyo.

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against ascariasis-Hano, et al. (I95o), and Mitsufuji, et al. (I950)-and against human filariasis (Wuchereria malayz}, in which remarkable results were obtained by Hayashi, et al. (I95I), on Hachijo Kojima Island. There was thus a lead to test this same drug against epizootic cerebrospinal nematodiasis in sheep, goats - and horses, caused by Setaria digitata, concerning which many previous papers have now appeared (Yamagiwa and Shoho [I944], Tanaka, et al. [I945]), Shoho [I952], Innes [I95I], Innes and Shoho [I952], and Innes, Shoho and Pillai [I952]). From all the latter no further doubt should remain that (a) this neuroparalysis is a common disease (perhaps in many parts of the world remote from the Orient, as Innes, Shoho and Pillai indicate), (b) the pathology is unique and distinctive (a focal encephalomyelo-malacia), and (c) that nematodes play the primary causative role. The following is a summary of the results of a pilot type of field trial, undertaken to ascertain the value of the drug in a prophylactic and curative direction-the latter at the very early onset of nervous signs. Publication at this stage, tentative though it may appear to be, may stimulate work along similar lines in other parts of the world concerning this helminthic neuroparalysis and its control, for it may be proved in time (as Innes and Shoho believe) to have an importance far beyond that at present imagined. Field Experiments and Methods The trial was made in the Date district of Fukushima prefectu\re, with the active interest and financial aid of the local Corporation of Animal Husbandry. All sheep and goats in this district are kept in shelters with small compounds, for the animals are not grazed freely, as in other countries. In the summer season they are fed on grass, and sometimes on the remnants of mulberry leaves--primarily used for silk-worm feeding. All foals and yearlings (Arabian or thoroughbreds) are kept separately in stables, with outside grazing. In nearly all farms, cattle (native breed crossed with Brown Swiss) are kept adjacent to sheep, goats and horses. The cattle, as described elsewhere, are carriers of the adult Setaria digitata, and in the summer and autumn the cattle are the hosts for transmission of the microfilaria through mosquitoes to sheep, goats and horses (unnatural hosts), which in tum may become victims of the neural nematodiasis. Clinical experience against human filariasis indicated that repeated small daily doses of the drug seemed the ideal method, but with large numbers of animals in the field this was impossible. In any case, the first concern was to determine whether the drug had any activity against the Setaria wandering from subcutaneous tissue to, and in, the craniovertebral cavity, for microfilaria are not present in the blood of the unnatural hosts---sheep, goats and horses. Yet, consideration must be given to the possible coincidental occurrence of microfilaria of Setaria equina. A preliminary note by Sasa, .et al. (I 95 I), indicated that a daily dose of IOO mg./kg. body-weight for five successive days did not produce toxic signs in goats. Thus, a single dose of 20 mg./kg. was chosen arbitrarily for prophylactic trials, but in some animals 40 mg./kg. was given. For therapeutic work 40 mg./kg., on one or two successive days, was given

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to sheep and goats, and up to So mg./kg., on two successive days to valuable foals (in the latter after blood examination revealed an absence of microfilaria of S. equina). The drug was dissolved in an adequate quantity of water, and given by stomach tube. It was first thought that in ruminants, and in horses (with such an anatomically large ca:cum), slow absorption of the drug might be obtained by one large dose. Later it was found in carrier calves that rapid absorption occurs from the ruminant stomach, for a filaricidal action on microfilaria of S. digitata in the blood was noted within three hours after dosing. The reports of the Japanese Commission on Research of Lumbar Paralysis in Kcrea (I 939-44), in which the original discovery of nematodal cause for the disease was reported, showed that in sheep (imported into Korea from Australia) signs of neuroparalysis appeared about one month after being landed in Korea. A comparable latent period was noted in their transmission experiments, while Itagaki, et al. (I946-47) noted a range of I6 to 66 days in the case of experimental horses; hence it was assumed that the shortest incubation period might be about I 6 days. For our purposes in assessing activity of the drug as a prophylactic it was decided to use a period of 20 days after dosing, after w:hich a second or third medication might be given. Results of the Trials (A) PROPHYLACTIC. Four separate groups of prophylactic trials were made, and the results are tabulated below. Comments on each trial are made and the results thereafter discussed. TABLE I. Trial. I

2

3

4

Village.

Numbers of Sheep Treated. No. of Cases. Nos.

Danzaki Handa Total

161 58 219

Ookido (Aug. 31) Handa (Sep. I) Danzaki (Sep. 2) Ishido (Sep. 3) Total

78 30 87 36 231

Ishimoda (Sep. 10) Morieno (Sep. 12) Total

I9 68 87

Handa (Sep. 21) Danzaki (Sep. 22) Total

29 67 g6

0

633

5

Grand Total

0 I

0

3 0 0 0

Untreated Controls. Nos. Cases.

119 62 181

0 0 0

85 go 193 236 604

? 3

50 I82 232 9I 231 322 1,339

II ? 14+

3 ?

3+ 3 4 '21+

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The first trial was made between August 1o and 13, but the relatively cool early and middle summer did not predispose to a large number of paralytic cases occurring. In the second trial, the sheep used included those in the Danzaki and Handa locality treated in the first trial. Forty-eight of the 78 sheep in Ookido area received 40 mg./kg.; some of the control animals in addition had been previously treated by intravenous antimonial compound by others, which rather vitiated the result of no cases ( ?) being found. Further, the one case noted in the treated group (Ookido) showed signs of slight inco-ordination and paralysis on the day treated; two daily successive treatments of 20 mg./kg., and 40 mg./kg., resulted in dramatic recovery, and it was able to stand on the fourth day; no further .signs were thereafter noted. The case in the treated group (Danzaki) showed signs on the seventeenth day ,and again similar improvement occurred by further dosage of 40 mg./kg. In the sheep in Danzaki and Handa taken together there was thus a total of I 4 cases o~ paralysis in the control sheep within 20 days, as opposed to only one in the treated, which might speak in favour of the value of repeated drug administration within two to three weeks. In the third trial the results were somewhat confusing, as some of the untreated sheep had also been given previous antimonial preparation by ·the farmers. In the fourth trial the only case in the treated group occurred on the seventh day after dosing, and this animal recovered completely after being given 40 mg./kg. Comment.-All field trials of this nature, without adequate data on pharmacology of the drug, and without the constant supervision of the animals as can be effected in Experimental Field Stations, can never give wholly satisfactory results. Yet it is contended there is sufficient evidence to indicate that the drug has some prophylactic activity~ certainly to justify a further, wider, and more carefully-controlled experiment. The prompt disappearance of nervous signs in the four cases treated therapeutically with 40 mg./kg. might indicate that this dose would be more effective when given also prophylactically. Perhaps the first need, therefore, is for adequate pharmacological studies of the drug on ruminants and horses. If the results of the second and third trials in Handa, Danzaki and Ishimoda are separated from the whole and considered, perhaps they take on more significance in view of what was known (by personal investigation) regarding the incidence of the disease. In this area from about I 942 it has been estimated that about 6-7 per cent of sheep were affected annually, occurring from about the middle of August to the middle of O ctober. Hence, in this second and third trial, out of a total of I 36 treated sheep only one case was found, as against I 7 .cases in 333 untreated control sheep, and some of the latter had also been given antimonials; the percentage in the latter (5. I per cent) thus corresponds to what was known about annual natural incidence (6-7 per cent). E xperiments with Foals Cerebrospinal nematodiasis due to immature Setaria digitata in horses and foals is relatively common in Japan. In adult animals, however, neurological signs may not appear, or may not be discernible, as was shown first by the

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important findings of Tanaka, et al. (1945); in a routine neuropathological ~urvey of the brains of I oo old serum horses, they found minute lesions with parasites in the brains of seven animals which had shown no neurological signs i,n life. As Innes, Shoho and Pillai point out, this may not be considered astonishing, for in adult animals with a voluminous nervous system such as in the equine tribe-as the lesion of nematodiasis is about the same magnitude as in other affected species, sheep and goats-much would depend on the location of the nematode (and ensuing lesion) in the brain or cord. Affected foals and yearlings conversely show unmistakable neurological signs-noticeable to laymen-which have an exact clinical counterpart in the descriptions of both wobblers of foals and yearlings in the U.S.A., and so-called Kumri of horses in India and Burma. Innes, et al., have also pointed out that, in effect, these may be identical diseases, and that the idea of nematodal cause in the case of Kumri can be traced back to antiquity, though accurate pathological studies have still to be made and reported. The equine form of cerebrospinal nematodiasis usually occurs at a slightly later part of the season than in the case of the disease in sheep and goats-i.e., usually around the middle of September. Further, ocular filariasis of horses in Japan (mostly due to Setarw digitata, according to the recent work of Kume, et al. [I 95 I]) appears even later still, about the last weeks of October, when the incidence of neural nematodiasis has begun to subside. All this might be understandable, for in horses it might take the infective larva: much longer to undertake the " I o-day Cook's tour" (referred to by Chandler to describe larval tissue migration) from subcutaneous inoculation to nervous system or eye. (In Itagaki's work a period of 66 days occasionally elapsed.) Obviously, the actual seasonal date of infection by the mosquito ve<;tors must be the ,same for all domestic .animals. Thus, for prophylactic purposes it was decided to administer the drug .o n September 20. Seven valuable foals were given the drug at a dooage varying from 40-60 mg./kg. on September 20. No toxic effects were obser-Ved. No signs of either neural or ocular nematodiasis were observed in any animal by the end of -November. The small number of animals used makes any conclusions almost worthless, except for two facts, (a) the results of the trials in sheep, and (b) more important still, the dramatic curative effect of the drug in two unmistakable cases of neural nematodiasis in foals described below. (B) THERA'PEUTIC. Before discussing the results of therapeutic treatment it is necessary to refer readers to a previous paper by Innes, Shoho and Pillai (I952), in which the neuropathological nature of the disease is fully described and illustrated. The damage in the nervous system produced by the wandering, developing, immature nematode is essentially traumatic disintegration, sometimes hremorrhagic, leading to liquefaction, and perhaps cavitation. It is also known that there is a variable involvement of the meninges and nerve roots, shown by the presence of inflammatory cellular exudate in which eooinophils may predominate. We know that there is a latent period of some weeks between the time the m05quito bites

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the animal, transmits infective larvre; and appearance of nervous signs. What we do not know is the minimal amount of damage or irritation (meningeal, radicular or nervous) required before animals show neurological signs, other than those of complete flaccid paralysis. This complex question of correlation 'Of clinical neurological signs and nervous lesions is indeed a constantly recurring complex one in the whole field of comparative neuropathology. It might, how~er, be imagined that the early presence of the immature worm in the craniavertebral cavity might produce, initially, inflammation of the meninges and nerve roots, and consequent irritation, with an insidious onset of inco-ordination and ataxia, and which might not be succeeded by actual nematodal invasion of nervous tissue, with its resultant malacic process, for a few days. This might, therefore, be one reason why it should be possible to cure affected animals, -provided the cases are in a very early stage. We know from other directions in experimental neurophysiology that in animals such as sheep even a massive lesion may be compensated in time, particularly if such a lesion is not located in vital parts. Lesions in the lower part of the lumbar spinal cord would ·certainly not cause such permanent severe neurological impairment as at higher levels. Obviously, once the lesion has reached the malacic liquefactive stage no complete cure by the drug would be possible, i.e., in an animal with a history ·of paralysis of many days' or weeks' duration. (I) Sheep and goats.-A total of 64 sheep and four goats have been treated, and in no case was any toxic action, due to the drug, observed. In :general, the efficacy of the drug was such that nervous symptoms disappeared within one or two days. Out of I I ovine cases treated by a single dose of -40 mg./kg., IO showed prompt recovery and with no residual signs; it is manifest that a single dose at this level does kill the wandering nematode. In the remainder, more than one dose was employed with no better results. In a few, repeated doses did not prevent residual neurological symptoms, but in these cases the course of the disease before treatment had been longer. Experience in goats was less, but in three animals affected and obtained in an early stage ccomparable dramatic effects were obtained by 40 mg./kg. In a fourth caprine case, 40 mg. / kg. was given for six successive days with increased appetite and obvious locomotor improvement within two days, but this animal again showed nervous signs on the tenth day. The question arose as to the possibility of the animal having been reinfected, for it is known that this phenomenon does occur. A second course of treatment on the tenth day resulted in complete recovery. (2) Foals.- The therapeutic results were equally as striking as in the case of the sheep. . In the first case, nervous symptoms were noted on September I 4 (many years ago this foal would certainly have been diagnosed as virus encephalitis). The animal was seen personally on September I 6 and a diagnosis of cerebrospinal nematodiasis made. On September 16, 20 mg./kg. was given orally, IO mg./kg. on the qth and 18th, and 30 mg./kg. on the 1gth and 2oth. The foal was still ataxic on the 18th, but it was able to stand, while by the 26th it had recovered, except for a slight residual weakness of the right hind leg. The second case, found to be markedly ataxic on September 22, was given so mg./kg.

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on the 23rd, and 8o mg./kg. on the 24th; some improvement was seen on the 25th, and by the 28th the animal was normal, without any neurological signs. whatever. General Discussion

Viewed in the cold light of science, or in that of those who will only believe statistical evidence, the results given above may not appear of profound significance. Yet it must be reiterated that the object of such a pilot trial was to obtain merely preliminary indication of some activity of this new filaricide, already proved to be efficacious against human filariasis. In the opinion of my· collaborator in the U.S.A. (Dr. Innes), the results are just as promising, and rest on the same insecure statistical basis, as in the case of the very early work on phenothiazine as an anthelmintic, or on copper as an antiswayback measure. The very nature of the disease and its filarial cause should be remembered, for· then dramatic .t herapeutic and prophylactic results, provided sufficient dosage was given, cannot be viewed as extraordinary. In Japan, for several years other chemotherapeutic agents have been tried, especially arsenical and antimonial preparations, but all were found to be toO> toxic. The advent of the compound used in these trials, which was discovered' in Lederle Laboratories in the U.S.A., opens up an entirely new vista in the problem of the control of this disease in Japan, Korea and Ceylon-and else-. where if the convictions of Innes, Shoho and Pillai are confirmed. Theoretically, a.c; in other fields of parasitic disease, perhaps the ideal method of control would reside in treatment of the natural hosts (cattle), harbouring the adult Setaria and acting as carriers and reservoirs of infection. Personal observations have already indicated that a marked drop in the microfilarial count in carrier cattle can be achieved within a matter of hours by a single dose of 100 mg./kg., and' no doubt might be completely eliminated by adequate dosage; considerably more work is necessary, first in a pathological direction and then with a sufficient number of "microfilaria carrier" cattle. At this stage there is no need t<> consider the most suitable mode of administration, either as a prophylactic for sheep, goats or horses, or a drug to eliminate the cattle reservoirs of infection;: for practical field purposes individual dosing would be a great drawback. Conclusions

Data are presented which indicate that I -diethyl-carbamyl-4-methylpiperazine citrate possesses a filaricidal activity iri the disease of domestic animals (sheep, goats and horses) now known as epizootic cerebrospinal nematodiasis. In the dosage employed in the field trials no toxic reactions were noted. The results of prophylactic trials are certainly of sufficient significance to support ideas for comparable projects on a far more ambitious scale in Japan, Ceylon,. and elsewhere. The therapeutic results in the light of personal experience of a large number of cases are considered to be dramatic. Acknowledgments

The paper is based on a paper to be presented to the Japanese Society of Veterinary Science in April, 1952, by Shoho, Nirasawa (Sr.), Nirasawa (Jr.),.

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Inoue, C., and Kubo, Z. The author is deeply indebted to Dr. M. Sasa, Institute for Infectious Diseases, Tokyo University, for the initial suggestion regarding the use of the drug, and for other information. Thanks are also due to the Local Corporation of Animal Husbandry, Fukushima, for their interest and financial aid. Between the author and his long-distance collaborator, Dr. J. R. M. Innes, in U.S.A., an immense volume of information has been exchanged by correspondence since 1 950; it is partly due to this that we have been able to bring to light in the English language Japanese investigations, and to link them with other work by Dr. Innes, ~ well as with studies of obscure nervous disease of animals still of unknown retiology, in other parts of the world, and which we think may be cerebrospinal nematodiasis. REFERENCES Hano, et a/. (1950): Folia Pharnwcol. lap ., 45, 132. (In Japanese.) Ibid. (1950): Folia Pharnwcol. lap ., 46, 1. (In Japanese.) Hayashi, S. M., Kano, R., and Sato, K. (1951) : Nisshinigaku, 38, 19. (In Japanese.) Innes, J, R. M. (1951): Brit. Vet. ll., 107, 187. Innes, J. R. M., and .Shoho, C. (1952): Arch. Neural. Psychiat. (In press.) Innes, J. R. M., Shoho, C., and Pillai, C. Perumal (1952): Brit. Vet. J/., March, 71. ltagaki, S., Taniguchi, M., and K awata, M. (1946-47): J/. lap. Med. Vet. Assoc., 3, 79, and 4, 73 and 105. (In Japanese.) Kume, S. (1951) : "Recent Advances in Veterinary Science." Edited by S chool of Veterinary Medicine, University of Tokyo. (In Japanese.) Mitsufuji, et a/. (1950): Fol. Pharmacal. lap., 45, 135. Sasa, M., Hayashi, S., and Sato, K. (1951) : U npublished data. .Shoho, C. (1952): lap. fl. Exper. Med . (In press.) Tanaka, T., Suu, S., Shoho, C., and Yamagiwa, .S. (1945) : lap. fl. Vet. Sci., i, 117. (In Japanese with German summary.) Yamagiwa, S., and Shoho, C. (1944): lap. fl. Vet. Sci., 6, 413. (In Japanese with German summary.)

RECENT TREATMENT OF A CASE OF BURSATTEE By M. F. SAID, D.V.M.S. (Egypt), A. H. MAHMOUD, M.V.Sc.(Liverpool), M.R.C.V.S.(England), D.V.M.S.(Egypt), and SH. H. KAMEL, B.V.Sc.(Egypt) Faculty of Veterinary Medicine, Fouad I University, Giza, Egypt.

Subject ON 25.1. 1951, a she-donkey about 5 years old was brought to the Faculty's hospital for treatment. History

On questioning the owner, it was revealed that the animal had been brought to the same hospital the previous year suffering from large ulcerated wounds in "the shoulder and neck. Those lesions healed up after two months of ordinary treatment. This year, in the exact time, the animal showed symptoms of ·similar lesions on the hock, eyelid, and fetlock. It began as swellings, which later on became harder. In about two weeks' time the skin covering the diseased areas. sloughed, exposing an extensive ulcerated surface. The owner ·stated that the lesiorui were a fortnight old and he had not tried any treatment ~xcept ordinary antiseptics. ·