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Isolation of Leishmania from the spleen of a dog in Iraq
859 TRANSACTIONS OFTHE ROYALSOCIETYOF TROPICALMEDICINEANDHYGIENE,VOL.
isolation
of Leishmania
6, 1981
from the spleen of a dog in Iraq
F. SUKKAR, S. K. AL-MAHDAWI,
Kala-Azar
75, No.
N. A. AL-DOORI AND J. A. KADHUM
Section, Endemic Diseases Institute, Alwiya, Baghdad, Iraq
Summary The isolation of Leishmania from the viscera of a mammal (dog) in an endemic area of infantile kalaazar in the central region of Iraq is reported. Introduction Leishmaniasis is endemic in Iraq and has probably been so from ancient times (PRINGLE, 1957; BRAY et al., 1967). The first parasitologically proven cases of kala-azar in Iraq were in Baghdad, as recorded by KIJLZ (1916); over 10 years (1971-80) a record of 8,407 patients with infantile kala-zaar was carefully collected from among the 13 million population of Iraq (Sukkar, unpublished). The search for Leishmania in the viscera of mammals started long ago (WENYON, 1911); Leishmania was seen in skin lesions of dogs (CHADWICK & MACHATTIE, 1927), isolated from the viscera of dogs from an imported pack of foxhounds near Baghdad (SHERIFF. 1957). and isolated from the viscera of a black rat colle&ed from the centre of Baghdad city where kala-azar had not been recorded (EL-ADHAMI, 1976). The Leishmania from the black rat was later characterized by isoenzyme studies as L. tropica (minor) by ALJEBOORI & EVANS (1980). Different workers have searched for Leishmania in the viscera of many mammals in Iraa (WENYON, 1911: PRINGLE. 1956; BRAY et al.. 1967;‘ BRAY 8~.DABB~GH, 1968; TAJLELDIN et al.; 1971; KADHIM et al., 1979). The kala-azar section has also surveyed several hundreds of animals including foxes, jackals, dogs, rodents, bats and other mammals (Annual Report, Endemic Diseases Institute, Baghdad (in Arabic), 1971-1980). None of these studies detected Leishmania in the viscera of any mammal. Materials and Methods Culture medium: 2 to 3 ml of semi-solid medium (ADLER & THEODOR, 1926), modified according to advice from Peters and Chance (personal communication) and which was later used successfully for primary isolation of L. donovani from hospital patients (RASSAM, 1979) was put into 5 ml screwcapped bottles. Dog: Two dogs shot in Shuhaimiya area (village No. 7), a tertiary political division belonging to Wasit (= Kut) Province in the Central Region of the country, longitude East 45”, latitude North 32” 70’ (about 20-30 meters above sea level). Isolation: Each of the shot dogs was kept in a separate plastic bag and brought immediately to a near-by dispensary where, after washing with soap solution and water, the abdominal wall was opened in three steps : skin, muscular layer and peritoneum
after free application of 70% alcohol to each layer before opening it; a fresh set of sterile surgical instruments was used for each layer; then about 20 mm3 from each spleen was triturated and added to one culture bottle-over the flame of a spirit lamp and 0.1 ml of crvstalline uenicillin in Locke’s solution (50,000 1.6. per mlj was added; another culture bottle was similarly inoculated but without any antibiotic. Culture inoculations were finished within two hours from the time the dogs were shot; the culture bottles were brought to the laboratory in a cooled thermos at about 4”C, reaching the laboratory after about six hours, where they were incubated at 25°C. Cultures were examined by making fresh preparations and smears stained with Leishman’s stain. Spleen, liver and skin from the nose of the dogs were brought to the laboratory in a cooled thermos at about 4” C for further studies but this material was lost through careless handling. Results On 25th May 1981, i.e. the fifth day after inoculation, promastigotes from antibiotic-free culture bottles of the spleen of one of the two dogs were clearly identified in both fresh preparations and stained smears; the parasite was maintained by subculturing in the same medium and in four hamsters each inoculated intraperitoneally with 10’ parasites in 1.0 ml Locke’s solution for future characterization. Discussion Preliminary studies enabling us to find a suitable medium for the primary isolation of local strains of L. donovani, together with full experience in the technique using hamsters and the rapidity with which the culture media were inoculated as soon as possible after collection of the animals to be investigated, were probably of great assistance in isolating this Leishmania successfully which, in contrast to L. tropica, is difficult to isolate. References Adler, S. & Theodor, 0. (1926). The identity of Lezshmania tropica Wright, 1903 and Herpetomonas papatassii Adler, 1925. Annals of Tropical Medicine and Parasitology, 20, 355-364. Aljeboori, T. I. & Evans, D. A, (1980). Leishmania spp. in Iraq. Electrophoretic iso-enzyme patterns. II. Cutaneous leishmaniasis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 74, 178-184. Bray, R. S. & Dabbagh, M. A. (1968). Investigations into the epidemiology of leishmaniasis. Unsuccess-
860
ISOLATION
OF
Leishmania
ful search for the reservoir host of kala azar in Baghdad. Journal of Tropical Medicine and
422-432.
El-Adhami, B. (1976). Isolation of Leishmania from a black rat in the Baghdad area, Iraq. American of
Tropical
Pringle, G. (1957). Oriental sore in Iraq. Historical and enidemioloaical nroblems. Bulletin ofI Endemic Diseases, Baghd;d, 2; 44-89.
Hygiene, 71, 46-47.
Bray, R. S., Rahin, G. F. & Taj-Eldin, S. (1967). The present state of leishmaniasis in Iraq. Protozoology, 2, 171-186. Chadwick, C. R. & Machattie, C. (1927). Cutaneous leishmaniasis in Iraq. Transactions of the Royal Society of Tropical Medicine and Hygiene, 20,
Journal
FROM DOG IN IRAQ
Medicine
and Hygiene,
25,
759-761. .-_ .--.
Kadhim, A. H., Sukkar, F. J., Al-Maghazachi, S. J. & Oawal. D. (1979). Evaluation of the role of fox& and jackals as’a possible reservoir host for visceral leishmaniasis in central Iraq. Bulletin of the Biological Research Centre, Baghdad, 2, 3-8.
Khairi, N. (1977). Search for possible existence of more than one strain of the parasite Leishmania donovani in Iraq. M.Sc. Thesis, Baghdad University. Pringle, G. (1956). Kala azar in Iraq. Preliminary epidemiological considerations. Bulletin of Endemic Diseases, Baghdad, 1, 275-294.
Rassam, Maysoon B. (1979). Leishmania and kala azar in Iraq. Biological, immunological and diagnostic studies. Ph.D. Thesis, Baghdad Uni-
versity. Sheriff, D. (1957). Canine visceral leishmaniasis in fox-hounds near Baghdad. Transactions of the Royal Society of Tropical Medicine and Hygiene, Rl. 467. --I
--..
Sukkar, F. (1976). Some epidemiological information from annual report on kala azar in Iraq 1975. Bulletin of Endemic Diseases, Baghdad, 17, 119-122. Taj-Eldin, S., Guigis, S. Y. & Al-Mashhadani, H. M. (1971). On the reservoir host of kala azar in Iraq. Iraqi Journal of Paediatrics, 1, 21-32. Wenyon, C. M. (1911). Oriental sore in Baghdad, together with observations on agregarine in Stegomyia fasciata, the haemogregarine of dogs and the flagellates of house-flies. Parasitology, 4, 273-344.
mm--
Accepted for publication 19thJune, 1981.
isolation
of Leishmania
6, 1981
from the spleen of a dog in Iraq
F. SUKKAR, S. K. AL-MAHDAWI,
Kala-Azar
75, No.
N. A. AL-DOORI AND J. A. KADHUM
Section, Endemic Diseases Institute, Alwiya, Baghdad, Iraq
Summary The isolation of Leishmania from the viscera of a mammal (dog) in an endemic area of infantile kalaazar in the central region of Iraq is reported. Introduction Leishmaniasis is endemic in Iraq and has probably been so from ancient times (PRINGLE, 1957; BRAY et al., 1967). The first parasitologically proven cases of kala-azar in Iraq were in Baghdad, as recorded by KIJLZ (1916); over 10 years (1971-80) a record of 8,407 patients with infantile kala-zaar was carefully collected from among the 13 million population of Iraq (Sukkar, unpublished). The search for Leishmania in the viscera of mammals started long ago (WENYON, 1911); Leishmania was seen in skin lesions of dogs (CHADWICK & MACHATTIE, 1927), isolated from the viscera of dogs from an imported pack of foxhounds near Baghdad (SHERIFF. 1957). and isolated from the viscera of a black rat colle&ed from the centre of Baghdad city where kala-azar had not been recorded (EL-ADHAMI, 1976). The Leishmania from the black rat was later characterized by isoenzyme studies as L. tropica (minor) by ALJEBOORI & EVANS (1980). Different workers have searched for Leishmania in the viscera of many mammals in Iraa (WENYON, 1911: PRINGLE. 1956; BRAY et al.. 1967;‘ BRAY 8~.DABB~GH, 1968; TAJLELDIN et al.; 1971; KADHIM et al., 1979). The kala-azar section has also surveyed several hundreds of animals including foxes, jackals, dogs, rodents, bats and other mammals (Annual Report, Endemic Diseases Institute, Baghdad (in Arabic), 1971-1980). None of these studies detected Leishmania in the viscera of any mammal. Materials and Methods Culture medium: 2 to 3 ml of semi-solid medium (ADLER & THEODOR, 1926), modified according to advice from Peters and Chance (personal communication) and which was later used successfully for primary isolation of L. donovani from hospital patients (RASSAM, 1979) was put into 5 ml screwcapped bottles. Dog: Two dogs shot in Shuhaimiya area (village No. 7), a tertiary political division belonging to Wasit (= Kut) Province in the Central Region of the country, longitude East 45”, latitude North 32” 70’ (about 20-30 meters above sea level). Isolation: Each of the shot dogs was kept in a separate plastic bag and brought immediately to a near-by dispensary where, after washing with soap solution and water, the abdominal wall was opened in three steps : skin, muscular layer and peritoneum
after free application of 70% alcohol to each layer before opening it; a fresh set of sterile surgical instruments was used for each layer; then about 20 mm3 from each spleen was triturated and added to one culture bottle-over the flame of a spirit lamp and 0.1 ml of crvstalline uenicillin in Locke’s solution (50,000 1.6. per mlj was added; another culture bottle was similarly inoculated but without any antibiotic. Culture inoculations were finished within two hours from the time the dogs were shot; the culture bottles were brought to the laboratory in a cooled thermos at about 4”C, reaching the laboratory after about six hours, where they were incubated at 25°C. Cultures were examined by making fresh preparations and smears stained with Leishman’s stain. Spleen, liver and skin from the nose of the dogs were brought to the laboratory in a cooled thermos at about 4” C for further studies but this material was lost through careless handling. Results On 25th May 1981, i.e. the fifth day after inoculation, promastigotes from antibiotic-free culture bottles of the spleen of one of the two dogs were clearly identified in both fresh preparations and stained smears; the parasite was maintained by subculturing in the same medium and in four hamsters each inoculated intraperitoneally with 10’ parasites in 1.0 ml Locke’s solution for future characterization. Discussion Preliminary studies enabling us to find a suitable medium for the primary isolation of local strains of L. donovani, together with full experience in the technique using hamsters and the rapidity with which the culture media were inoculated as soon as possible after collection of the animals to be investigated, were probably of great assistance in isolating this Leishmania successfully which, in contrast to L. tropica, is difficult to isolate. References Adler, S. & Theodor, 0. (1926). The identity of Lezshmania tropica Wright, 1903 and Herpetomonas papatassii Adler, 1925. Annals of Tropical Medicine and Parasitology, 20, 355-364. Aljeboori, T. I. & Evans, D. A, (1980). Leishmania spp. in Iraq. Electrophoretic iso-enzyme patterns. II. Cutaneous leishmaniasis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 74, 178-184. Bray, R. S. & Dabbagh, M. A. (1968). Investigations into the epidemiology of leishmaniasis. Unsuccess-
860
ISOLATION
OF
Leishmania
ful search for the reservoir host of kala azar in Baghdad. Journal of Tropical Medicine and
422-432.
El-Adhami, B. (1976). Isolation of Leishmania from a black rat in the Baghdad area, Iraq. American of
Tropical
Pringle, G. (1957). Oriental sore in Iraq. Historical and enidemioloaical nroblems. Bulletin ofI Endemic Diseases, Baghd;d, 2; 44-89.
Hygiene, 71, 46-47.
Bray, R. S., Rahin, G. F. & Taj-Eldin, S. (1967). The present state of leishmaniasis in Iraq. Protozoology, 2, 171-186. Chadwick, C. R. & Machattie, C. (1927). Cutaneous leishmaniasis in Iraq. Transactions of the Royal Society of Tropical Medicine and Hygiene, 20,
Journal
FROM DOG IN IRAQ
Medicine
and Hygiene,
25,
759-761. .-_ .--.
Kadhim, A. H., Sukkar, F. J., Al-Maghazachi, S. J. & Oawal. D. (1979). Evaluation of the role of fox& and jackals as’a possible reservoir host for visceral leishmaniasis in central Iraq. Bulletin of the Biological Research Centre, Baghdad, 2, 3-8.
Khairi, N. (1977). Search for possible existence of more than one strain of the parasite Leishmania donovani in Iraq. M.Sc. Thesis, Baghdad University. Pringle, G. (1956). Kala azar in Iraq. Preliminary epidemiological considerations. Bulletin of Endemic Diseases, Baghdad, 1, 275-294.
Rassam, Maysoon B. (1979). Leishmania and kala azar in Iraq. Biological, immunological and diagnostic studies. Ph.D. Thesis, Baghdad Uni-
versity. Sheriff, D. (1957). Canine visceral leishmaniasis in fox-hounds near Baghdad. Transactions of the Royal Society of Tropical Medicine and Hygiene, Rl. 467. --I
--..
Sukkar, F. (1976). Some epidemiological information from annual report on kala azar in Iraq 1975. Bulletin of Endemic Diseases, Baghdad, 17, 119-122. Taj-Eldin, S., Guigis, S. Y. & Al-Mashhadani, H. M. (1971). On the reservoir host of kala azar in Iraq. Iraqi Journal of Paediatrics, 1, 21-32. Wenyon, C. M. (1911). Oriental sore in Baghdad, together with observations on agregarine in Stegomyia fasciata, the haemogregarine of dogs and the flagellates of house-flies. Parasitology, 4, 273-344.
mm--
Accepted for publication 19thJune, 1981.