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A preliminary note on the relationship of the parasites of human and canine dermal leishmaniasis
418 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE.
VoI. XXIII.
A
No. 4.
PRELIMINARY
January, ]930.
NOTE
ON
THE
RELATIONSHIP
OF
THE
PARASITES OF H U M A N AND CANINE D E R M A L L E I S H M A N I A S I S . BY
E. A. MILLS, M.B, CH.B. (EDIN.), D.T.M. & H. (ENG.), Chief Pathologist to Government of Iraq, Director of the Anti-Rab,c and Vaccine Lymph Institutes, Baghdad, AND
C. MACHATTIE, M.R.C.¥.S., D.V.S.M., ,4sszstant Director and Veterinary Research Officer, Civil Veterinary Department of Iraq, IN
COLLABORATION WITH
MAIOR C. R. CHADWICK, M.R.C.V.S., Director, Civil Veterinary Department of Iraq.
Human cutaneous leishmaniasis is very prevalent in Baghdad, and careful investigation shows that the canine dermal infection with leishmania is very much more frequent than was originally supposed. It occurred to tile writers that it would be of interest to compare the human and canine leishmania from the morphological, cultural, epidemiological and serological points of view. The leishmania as found in the dermal lesions on the dog is morphologically indistinguishable from Leishmania tropica WRIGHT, 1903, as it occurs in the human sore. From the cultural point of view the morphological characteristics of the canine type appear indistinguishable from those of the human lesion. There appears to be no constant variation in the size of the flagellates as compared with those of the human type. The size depends on the age of the culture, and varies somewhat with different batches of media and the temperature at which growth takes place. (Plate No. 2, Figs. 1 to 6.) In common with the human variety the canine parasite appears to be more resistant to secondary infection than L. donovani.
414
]RELATIONSHIP OF PARASITES OF HUMAN AND CANINE DERMAL LEISHMANIASIS.
From the epidemiological standpoint certain interesting observations have been made. In Iraq, particularly in Baghdad city and its environs, there occur very definite areas in which human infection is common, and others in which human infection is rare. It is of interest to note that in those areas in which human cases are common canine infection has the same relative frequency, while in those areas in which human cases are rare canine cases are rarely seen. Further, in areas of scanty infection there would appear to be a definite association of the two types. In the British Cantonment of Alwiyah there is only one recorded case of human infection and only one canine case, both occurring in the same house. In Baghdad North there is a small British settlement, and there two human cases and two canine have occurred, both in the same household at the same period of the year. We find that the occurrence of human cases shows a very definite seasonal incidence. During the months of June, July, August and early September it is difficult to obtain a young lesion showing the typical papular characteristics, whereas from late September to January numerous cases are to be seen at the Central Laboratory. In the case of the dog, this seasonal incidence is even more marked. In late September, October and November numerous dogs in the areas of high infection are to be found with early sores. The general course of the infection in the dog would appear to be less prolonged than in the human being. Tissue destruction and erosion are generally comparatively slight, and the lesion heals usually within about six months, with the result that most of the canine cases are completely cured by June of the following year. Occasionally cases of the chronic ulcerative type are encountered in which leishmania can be demonstrated the whole year round. In Baghdad the average duration of the human sore is about nine months, but owing to the more benign character of the canine lesion its absence is most marked in the late summer, making the seasonal incidence even more apparent. As pointed out by WENYON (1911), the human infection is more a condition of the large towns than of the desert or villages. This is true also of the canine type. Both human and canine cases are certainly found amongst the tribes, but they are both rare as compared with the incidence amongst the urban population. It is most probable that infection in these cases occurs when tribesmen and their dogs periodically wander townwards with caravans and occupy infected caravanserais. It has been shown by other writers thatPhlebotomus sergenti andP.papatasii are in all probability vectors of L. tropica in the case of the human oriental sore, and the writers have demonstrated that both these sandflies can be infected by feeding on cultures of leishmania isolated from canine lesions. Both these species feed readily when placed on the dog's nose under experimental conditions. Plate No. 2, Figs. 8 and 9, shows flagellates in smears from the mid gut of _P. sergenti twenty-four hours after feeding on a suspension of cultures of canine leishmania.
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PLATE N o . 2. FIGS. 1 '1'O 5 , - - L e p t o m o n a d f o r m s f r o m canine leishmania as a p p e a r i n g in N . N . N . medium. F~c. 6 . - - R o s e t t e in L o c k ' s s e r u m agar. F~G, 7 . - - A g g l u t i n a t e d mass of flagellates from t u b e in Plate 1, Fig. 3 (b). FIGs. S AND 9 . - - F l a g e l l a t e s in smears f r o m m i d g u t of Ph!ebotomus sergenti t w e n t y f o u r h o u r s after feeding on cultures of canine leishmania.
E. A. MILLS,
G.
MACHATTIE AND
C. R.
CHADWICK.
415
In the h u m a n subject several observers have noted that the most c o m m o n sites of oriental sore are those to which sandflies have easy access. I n the dog the only portions of the b o d y uncovered by hair and exposed to attack are the internal surfaces of the ear, the nose, the margins of the eyelids and the junction of the pads with the hair of the legs, and these are precisely the areas in which the canine sores are to be found. I n c o m m o n with the h u m a n subject dermal leishmaniasis in the dog does not appear to be accompanied b y any severe constitutional disturbances. Infected animals in general appear to enjoy good health and manifest no signs of either debility or emaciation. In view of the observation of NELIGAN in Teheran (1913), a n u m b e r of dogs manifesting cutaneous lesions in various stages of development were killed, and careful search made for any evidence of concurrent visceral infection. Further, a n u m b e r of emaciated animals not necessarily showing evidence of cutaneous leishmaniasis were examined, but in all cases smears from blood, bone marrow, spleen and liver yielded entirely negative results. F u r t h e r it is of interest to note that up to the present no cases of endemic kala-azar have been encountered in the h u m a n subject in this country. I n view of the above points of similarity, experiments were undertaken to investigate the relationship of the h u m a n and canine parasites from the serological standpoint. These experiments were as follows : -
Experiment No. 1.--A strain of leishmania isolated in September, 1928, from a sore on a dog's ear was grown on Adler's modification of Noguchi's leptospira media made up as follows :--Locke's solution, .1.5 c.cm. with glucose, 0-2 per cent. ; nutrient agar, 0.5 c.cm. ; fresh rabbit serum, 0.5 c.cm. R~sult.--Growth was found to occur as a diffuse whitish film extending over the upper third of the medium. Microscopical examination showed the presence of numerous leptomonad forms by the fourth day, and by the seventh day the limit of growth was distinctly visible. The cultures appeared to be at their richest about the tenth day. No evidence of either clumping or flocculation was visible macroscopically. (Plate No. 1. Fig. 1.) Experiment No. 2 - - T h e serum from a pointer dog which had been under observation since September, 1928, and had recovered from non-treated sores on the ears, eyelids and pads in June, 1929, was employed in this experiment. Puncture of the saphenous vein was carried out with all sterile precautions. The above strain of leishmania was sown in Locke's serum agar, to which was added 0.5 c.cm. of this supposed immune dog's serum. Result.--No definite line of growth was obtained as in the previous experiment. By the sixth day a diffuse growth occnrred, and a number of whitish clumps were visible suspended throughout the upper portion of the media (Plate No. 1, Fig. 3b). Hanging drop preparations taken from areas between the clumps showed scanty leptomonad forms aggregated in clumps clearly distinguishable from the normal rosettes and agglomorated forms. (Plate No. 2. Fig. 7.) By the seventh day slow disintegration of these colonies began to take place presumably as a result of the lyric action of the dog's serum, while later no flagellates were visible on microscopic examination. Experiment No. 3.--In this case the blood serum of a man suffering from chronic
~16
RELATIONSHIP OF PARASITES OF HUMAN AND CANINE DERMAL LEISHMANIASIS
leishmaniasis of the hand and cheek of eleven months' duration was employed in place of the dog's serum in Experiment No. 2. Here a faint diffuse growth was obtained, and by the fourth day a number of flocculi were distinctly visible in the upper portion of the media (Plate No. 1, Fig. 2b), and these, on microscopical examination, showed numerous flagellates again in clumps showing feeble flagellar movement. By the fifth day disintegration set in, and by the seventh day no flagellates could be found. In Experiments Nos. 4 and 5 the method adopted by NocucHI was e m p l o y e d in w h i c h i m m u n e s e r u m was a d d e d to n o r m a l c u l t u r e s .
(1924)
Experiment No. 4 . - - A rich surface growth of the canine flagellates was obtained in Locke's serum agar as illustrated in Plate 1, Fig. 1. T o each tube was added 0"5 c.cm. of serum from the dog used in Experiment No. 2. Within fifteen minutes the turbid line of growth began to show signs of distinct granulation; after thirty minutes very distinct granulation; after a further thirty minutes very distinct signs of specific agglutination were visible, definite flocculant masses gradually appearing throughout the area of growth. (Plate No. 1. Fig. 5 b,) Microscopically, distinct agglutinated clumps were visible, some showing feeble movement, others being entirely motionless. Photo taken six hours after addition of dog's immune serum. Experiment No. 5 . - - I n this experiment all the conditions were as detailed in Experiment No. 4, with the exception that 0"5 c.cm. of the human serum used in Experiment No 3 was added to the normal cultures in place of the immune dog's serum. In this case the serum fell to the bottom of the tube, and it was necessary to employ gentle agitation to bring the serum into intimate contact with the growth in the upper portion of the medium. T h e final results were similar to those obtained in the previous experiment (Plate 1, Fig. 4). CONCLUSIONS.
1. I n t h i s c o u n t r y b o t h c a n i n e a n d h u m a n d e r m a l i n f e c t i o n w i t h l e i s h m a n i a show a similar seasonal incidence. 2. I n b o t h cases t h e r e are d e f i n i t e u r b a n areas s h o w i n g a h i g h a n d l o w i n f e c t i o n rate. 3. I n areas of l o w i n f e c t i o n t h e r e w o u l d a p p e a r to b e a d e f i n i t e a s s o c i a t i o n between the two types. 4. I n b o t h cases i n f e c t i o n o c c u r s in t h e u n p r o t e c t e d p o r t i o n s o f t h e b o d y . 5. T h e r e is s t r o n g e v i d e n c e t h a t i n cases o f i n f e c t i o n w i t h l e i s h m a n i a in b o t h m a n a n d d o g t h e i n s e c t v e c t o r s are sinfilar. 6. F i n a l l y t h e s e r o l o g i c a l r e a c t i o n s w o u l d a p p e a r to i n d i c a t e a v e r y close r e l a t i o n s h i p , if n o t t h e a c t u a l i d e n t i t y , o f t h e l e i s h m a n i a p r o d u c i n g c u t a n e o u s i n f e c t i o n in b o t h m a n a n d d o g as seen in I r a q . REFERENCES. NELIGAN, A . R . (1913). ffl. Trop. Med. & Hyg., xvi, 156. No~uenI, H. (1924). Action of certain Biological, Chemical and Physical Agents upon Cultures of Leishmania; Some Observations on Plant and Insect Herpetomonads.
Proceedings of International Conference on Health Problems in Tropical America. pp. 455 to 478. United Fruit Company, Boston, Mass.. 1924. (1911). Parasitology, iv, 273.
VV'ENYON, C . M .
VoI. XXIII.
A
No. 4.
PRELIMINARY
January, ]930.
NOTE
ON
THE
RELATIONSHIP
OF
THE
PARASITES OF H U M A N AND CANINE D E R M A L L E I S H M A N I A S I S . BY
E. A. MILLS, M.B, CH.B. (EDIN.), D.T.M. & H. (ENG.), Chief Pathologist to Government of Iraq, Director of the Anti-Rab,c and Vaccine Lymph Institutes, Baghdad, AND
C. MACHATTIE, M.R.C.¥.S., D.V.S.M., ,4sszstant Director and Veterinary Research Officer, Civil Veterinary Department of Iraq, IN
COLLABORATION WITH
MAIOR C. R. CHADWICK, M.R.C.V.S., Director, Civil Veterinary Department of Iraq.
Human cutaneous leishmaniasis is very prevalent in Baghdad, and careful investigation shows that the canine dermal infection with leishmania is very much more frequent than was originally supposed. It occurred to tile writers that it would be of interest to compare the human and canine leishmania from the morphological, cultural, epidemiological and serological points of view. The leishmania as found in the dermal lesions on the dog is morphologically indistinguishable from Leishmania tropica WRIGHT, 1903, as it occurs in the human sore. From the cultural point of view the morphological characteristics of the canine type appear indistinguishable from those of the human lesion. There appears to be no constant variation in the size of the flagellates as compared with those of the human type. The size depends on the age of the culture, and varies somewhat with different batches of media and the temperature at which growth takes place. (Plate No. 2, Figs. 1 to 6.) In common with the human variety the canine parasite appears to be more resistant to secondary infection than L. donovani.
414
]RELATIONSHIP OF PARASITES OF HUMAN AND CANINE DERMAL LEISHMANIASIS.
From the epidemiological standpoint certain interesting observations have been made. In Iraq, particularly in Baghdad city and its environs, there occur very definite areas in which human infection is common, and others in which human infection is rare. It is of interest to note that in those areas in which human cases are common canine infection has the same relative frequency, while in those areas in which human cases are rare canine cases are rarely seen. Further, in areas of scanty infection there would appear to be a definite association of the two types. In the British Cantonment of Alwiyah there is only one recorded case of human infection and only one canine case, both occurring in the same house. In Baghdad North there is a small British settlement, and there two human cases and two canine have occurred, both in the same household at the same period of the year. We find that the occurrence of human cases shows a very definite seasonal incidence. During the months of June, July, August and early September it is difficult to obtain a young lesion showing the typical papular characteristics, whereas from late September to January numerous cases are to be seen at the Central Laboratory. In the case of the dog, this seasonal incidence is even more marked. In late September, October and November numerous dogs in the areas of high infection are to be found with early sores. The general course of the infection in the dog would appear to be less prolonged than in the human being. Tissue destruction and erosion are generally comparatively slight, and the lesion heals usually within about six months, with the result that most of the canine cases are completely cured by June of the following year. Occasionally cases of the chronic ulcerative type are encountered in which leishmania can be demonstrated the whole year round. In Baghdad the average duration of the human sore is about nine months, but owing to the more benign character of the canine lesion its absence is most marked in the late summer, making the seasonal incidence even more apparent. As pointed out by WENYON (1911), the human infection is more a condition of the large towns than of the desert or villages. This is true also of the canine type. Both human and canine cases are certainly found amongst the tribes, but they are both rare as compared with the incidence amongst the urban population. It is most probable that infection in these cases occurs when tribesmen and their dogs periodically wander townwards with caravans and occupy infected caravanserais. It has been shown by other writers thatPhlebotomus sergenti andP.papatasii are in all probability vectors of L. tropica in the case of the human oriental sore, and the writers have demonstrated that both these sandflies can be infected by feeding on cultures of leishmania isolated from canine lesions. Both these species feed readily when placed on the dog's nose under experimental conditions. Plate No. 2, Figs. 8 and 9, shows flagellates in smears from the mid gut of _P. sergenti twenty-four hours after feeding on a suspension of cultures of canine leishmania.
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7b face page 414.
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PLATE N o . 2. FIGS. 1 '1'O 5 , - - L e p t o m o n a d f o r m s f r o m canine leishmania as a p p e a r i n g in N . N . N . medium. F~c. 6 . - - R o s e t t e in L o c k ' s s e r u m agar. F~G, 7 . - - A g g l u t i n a t e d mass of flagellates from t u b e in Plate 1, Fig. 3 (b). FIGs. S AND 9 . - - F l a g e l l a t e s in smears f r o m m i d g u t of Ph!ebotomus sergenti t w e n t y f o u r h o u r s after feeding on cultures of canine leishmania.
E. A. MILLS,
G.
MACHATTIE AND
C. R.
CHADWICK.
415
In the h u m a n subject several observers have noted that the most c o m m o n sites of oriental sore are those to which sandflies have easy access. I n the dog the only portions of the b o d y uncovered by hair and exposed to attack are the internal surfaces of the ear, the nose, the margins of the eyelids and the junction of the pads with the hair of the legs, and these are precisely the areas in which the canine sores are to be found. I n c o m m o n with the h u m a n subject dermal leishmaniasis in the dog does not appear to be accompanied b y any severe constitutional disturbances. Infected animals in general appear to enjoy good health and manifest no signs of either debility or emaciation. In view of the observation of NELIGAN in Teheran (1913), a n u m b e r of dogs manifesting cutaneous lesions in various stages of development were killed, and careful search made for any evidence of concurrent visceral infection. Further, a n u m b e r of emaciated animals not necessarily showing evidence of cutaneous leishmaniasis were examined, but in all cases smears from blood, bone marrow, spleen and liver yielded entirely negative results. F u r t h e r it is of interest to note that up to the present no cases of endemic kala-azar have been encountered in the h u m a n subject in this country. I n view of the above points of similarity, experiments were undertaken to investigate the relationship of the h u m a n and canine parasites from the serological standpoint. These experiments were as follows : -
Experiment No. 1.--A strain of leishmania isolated in September, 1928, from a sore on a dog's ear was grown on Adler's modification of Noguchi's leptospira media made up as follows :--Locke's solution, .1.5 c.cm. with glucose, 0-2 per cent. ; nutrient agar, 0.5 c.cm. ; fresh rabbit serum, 0.5 c.cm. R~sult.--Growth was found to occur as a diffuse whitish film extending over the upper third of the medium. Microscopical examination showed the presence of numerous leptomonad forms by the fourth day, and by the seventh day the limit of growth was distinctly visible. The cultures appeared to be at their richest about the tenth day. No evidence of either clumping or flocculation was visible macroscopically. (Plate No. 1. Fig. 1.) Experiment No. 2 - - T h e serum from a pointer dog which had been under observation since September, 1928, and had recovered from non-treated sores on the ears, eyelids and pads in June, 1929, was employed in this experiment. Puncture of the saphenous vein was carried out with all sterile precautions. The above strain of leishmania was sown in Locke's serum agar, to which was added 0.5 c.cm. of this supposed immune dog's serum. Result.--No definite line of growth was obtained as in the previous experiment. By the sixth day a diffuse growth occnrred, and a number of whitish clumps were visible suspended throughout the upper portion of the media (Plate No. 1, Fig. 3b). Hanging drop preparations taken from areas between the clumps showed scanty leptomonad forms aggregated in clumps clearly distinguishable from the normal rosettes and agglomorated forms. (Plate No. 2. Fig. 7.) By the seventh day slow disintegration of these colonies began to take place presumably as a result of the lyric action of the dog's serum, while later no flagellates were visible on microscopic examination. Experiment No. 3.--In this case the blood serum of a man suffering from chronic
~16
RELATIONSHIP OF PARASITES OF HUMAN AND CANINE DERMAL LEISHMANIASIS
leishmaniasis of the hand and cheek of eleven months' duration was employed in place of the dog's serum in Experiment No. 2. Here a faint diffuse growth was obtained, and by the fourth day a number of flocculi were distinctly visible in the upper portion of the media (Plate No. 1, Fig. 2b), and these, on microscopical examination, showed numerous flagellates again in clumps showing feeble flagellar movement. By the fifth day disintegration set in, and by the seventh day no flagellates could be found. In Experiments Nos. 4 and 5 the method adopted by NocucHI was e m p l o y e d in w h i c h i m m u n e s e r u m was a d d e d to n o r m a l c u l t u r e s .
(1924)
Experiment No. 4 . - - A rich surface growth of the canine flagellates was obtained in Locke's serum agar as illustrated in Plate 1, Fig. 1. T o each tube was added 0"5 c.cm. of serum from the dog used in Experiment No. 2. Within fifteen minutes the turbid line of growth began to show signs of distinct granulation; after thirty minutes very distinct granulation; after a further thirty minutes very distinct signs of specific agglutination were visible, definite flocculant masses gradually appearing throughout the area of growth. (Plate No. 1. Fig. 5 b,) Microscopically, distinct agglutinated clumps were visible, some showing feeble movement, others being entirely motionless. Photo taken six hours after addition of dog's immune serum. Experiment No. 5 . - - I n this experiment all the conditions were as detailed in Experiment No. 4, with the exception that 0"5 c.cm. of the human serum used in Experiment No 3 was added to the normal cultures in place of the immune dog's serum. In this case the serum fell to the bottom of the tube, and it was necessary to employ gentle agitation to bring the serum into intimate contact with the growth in the upper portion of the medium. T h e final results were similar to those obtained in the previous experiment (Plate 1, Fig. 4). CONCLUSIONS.
1. I n t h i s c o u n t r y b o t h c a n i n e a n d h u m a n d e r m a l i n f e c t i o n w i t h l e i s h m a n i a show a similar seasonal incidence. 2. I n b o t h cases t h e r e are d e f i n i t e u r b a n areas s h o w i n g a h i g h a n d l o w i n f e c t i o n rate. 3. I n areas of l o w i n f e c t i o n t h e r e w o u l d a p p e a r to b e a d e f i n i t e a s s o c i a t i o n between the two types. 4. I n b o t h cases i n f e c t i o n o c c u r s in t h e u n p r o t e c t e d p o r t i o n s o f t h e b o d y . 5. T h e r e is s t r o n g e v i d e n c e t h a t i n cases o f i n f e c t i o n w i t h l e i s h m a n i a in b o t h m a n a n d d o g t h e i n s e c t v e c t o r s are sinfilar. 6. F i n a l l y t h e s e r o l o g i c a l r e a c t i o n s w o u l d a p p e a r to i n d i c a t e a v e r y close r e l a t i o n s h i p , if n o t t h e a c t u a l i d e n t i t y , o f t h e l e i s h m a n i a p r o d u c i n g c u t a n e o u s i n f e c t i o n in b o t h m a n a n d d o g as seen in I r a q . REFERENCES. NELIGAN, A . R . (1913). ffl. Trop. Med. & Hyg., xvi, 156. No~uenI, H. (1924). Action of certain Biological, Chemical and Physical Agents upon Cultures of Leishmania; Some Observations on Plant and Insect Herpetomonads.
Proceedings of International Conference on Health Problems in Tropical America. pp. 455 to 478. United Fruit Company, Boston, Mass.. 1924. (1911). Parasitology, iv, 273.
VV'ENYON, C . M .