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Experimental infection of domestic sheep with culture-derived Leishmania donovani promastigotes
Veterinary Parasitology 74 Ž1998. 315–318
Short communication
Experimental infection of domestic sheep with culture-derived Leishmania donoÕani promastigotes C.O. Anjili a b
a,)
, C.K. Ngichabe b, P.A. Mbati a , R.M. Lugalia a , H.M. Wamwayi b, J.I. Githure a
Biomedical Sciences Research Center, Kenya Medical Research Institute, P.O. Box 54840, Nairobi, Kenya National Veterinary Research Center, Kenya Agricultural Research Institute, P.O. Box 32, Kikuyu, Kenya Received 20 December 1996; accepted 10 April 1997
Abstract Domestic sheep were intradermally inoculated with culture-derived stationary phase Leishmania donoÕani promastigotes. Sampling of site of inoculation, liver and spleen for 244 days showed that this parasite can stay alive in the skin for up to 28 days post-inoculation. Apart from pyrexia that was evident in all the animals for 42 days, no other symptoms of kala-azar were seen. No parasites were recovered from the visceral organs throughout the sampling period, suggesting that sheep are not susceptible to infection with L. donoÕani. It is therefore unlikely that sheep can be synanthropic reservoirs for this parasite. q 1998 Elsevier Science B.V. Keywords: Sheep protozoa; Leishmania donoÕani; Experimental infection
1. Introduction Visceral leishmaniosis caused by Leishmania donoÕani ŽKinetoplastida: Trypanosomatidae. is endemic in Rift Valley and Eastern provinces of Kenya. In these foci, L. donoÕani has been isolated from people ŽForbes, 1993. and its natural vector sandfly Phlebotomus martini ŽDiptera: Psychodidae. ŽPerkins et al., 1987.. Apart from the dog Ž Canis familiaris. from which L. donoÕani has been isolated ŽMutinga et al., 1980., this parasite has not been isolated from any other synanthropic canids or ungulates. While man to man transmission is thought to be important, undetermined animal reservoirŽs. for L. donoÕani probably exist ŽWHO, 1988.. Intracellular amastigote-like bodies have been reported in a biopsy section from the )
Corresponding author. Tel.: q254-2-722541; fax: q254-2-720030; e-mail: [email protected]
0304-4017r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 0 1 7 Ž 9 7 . 0 0 1 5 0 - 7
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C.O. Anjili et al.r Veterinary Parasitology 74 (1998) 315–318
pinna of a sheep in South Africa Žvan der Lugt et al., 1992.. These bodies were not cultured and it is therefore not possible to determine the authenticity of their identity as Leishmania. Sheep live in close proximity to people in L. donoÕani endemic foci especially in Rift Valley Province and it is not known what role sheep play in the epidemiology of visceral leishmaniosis in Kenya. In this study, we sought to determine the susceptibility of the blackhead sheep Ž OÕis aries . to infection with a Kenyan strain of L. donoÕani and hence its potential role as an animal reservoir for this parasite. 2. Materials and methods 2.1. Parasite cultiÕation L. donoÕani Žstrain MHOMrKEr82rLRC-L445s NLB-065., originally isolated from a Kenyan visceral leishmaniosis patient and which has been subsequently maintained by serial hamster to hamster intracardial inoculation of splenic homogenate, was used. A biopsy of the infected spleen was cultured in biphasic NNNrSchneider’s Drosophila medium supplemented with 20% heat-inactivated foetal bovine serum, 500 Urml penicillin, 500 m grml streptomycin and 250 m grml 5-fluorocytosine ŽHendricks and Wright, 1979; Kimber et al., 1981.. Promastigotes were incubated at 258C and grown to stationary phase to generate metacyclic forms. These were counted and the concentration adjusted to 1 = 10 6rml of culture medium. They were then washed at 3000 rpm for 15 min in sterile ice-cold phosphate-buffered saline ŽPBS. and resuspended in 40 m l PBS for sheep inoculation. 2.2. Animal inoculation and sampling Six adult male blackhead sheep were ear-tagged for consistency in sampling and individually bled intravenously prior to inoculation with Leishmania. Temperature of these sheep was taken on a daily basis before and after experimental infection throughout the sampling period. Each sheep was intradermally inoculated in the right-ridge of the nose with 1 = 10 6 parasitesr40 m l PBS. All the sheep were sampled at 14, 28, 42, 56, 70, 98, 140, 180 and 240 days post-infection ŽDPI.. During each sampling interval, a subcutaneous saline needle aspirate was taken from the site of inoculation and cultured in NNNrSchneider’s medium prepared as described above. This sampling was conducted to determine whether the parasite was still alive at the site of inoculation. Aspirates of liver and spleen were also taken to determine the time of visceralization of the parasite from the site of inoculation. At time 0, 40, 80, 100, and 244 DPI, all the sheep were bled and sera extracted from the blood and used to detect anti-L. donoÕani antibodies using the ELISA. At 244 DPI all the sheep were sacrificed and blood for serum taken. Portions of liver and spleen, blood, mandibular lymph nodes and nasal skin Žsite of inoculation. were cultured in NNNrSchneider’s medium. Impression smears from liver and spleen were made and stained with Giemsa for parasite examination and quantification. 2.3. Enzyme-linked immunosorbent assay ELISA for detecting L. donoÕani specific IgG antibodies was performed as previously described by Voller et al. Ž1976.. Briefly, U-well polyvinyl chloride microtiter
C.O. Anjili et al.r Veterinary Parasitology 74 (1998) 315–318
317
plates ŽDynatech Laboratories, USA. were coated overnight at 48C with 100 m l of buffer containing 50 m g of L. donoÕani antigen. The plates were washed three times, blocked with 5% bovine serum albumin in phosphate buffered saline containing 0.05% Tween 20 ŽPBST. and, coated with 200 m l of sheep serum at a dilution of 1:50 in PBST. Sera was tested in quadruplicate. Plates were incubated for 1 h at 48C and at room temperature, respectively, and were washed. Rabbit anti-sheep IgG Žwhole molecule. peroxidase conjugate ŽCappal w . was added at the recommended working dilution of 1:2000 and incubated for 2 h at room temperature. The plates were thereafter washed before the addition of 100 m l of 0.4 mgrml substrate O-phenylenediamine O-Diaminobenzene dihydrochloride ŽSigmaw . and 30% hydrogen peroxide ŽSigmaw . and incubated Žovernight for 40 min. in the dark at room temperature. The reaction was stopped by adding 25 m l of 1N HCl ŽBDH., and the optical density read using a Titertek w Elisa reader and a 492 nm filter. The means of quadruplicate absorbance values were considered positive if they exceeded twice the mean values of known negative sera. 3. Results 3.1. Temperature The highest temperature rise recorded was 408C at 22 DPI after which it stabilized between 38.58C Žnormal temperature. and 398C throughout the study. 3.2. Aspirate cultures Apart from 28 DPI when a nasal aspirate taken from one of the six sheep ŽNo. 944. revealed the presence of Leishmania after 12 days of incubation in culture at 258C, no other nasal aspirate had parasites. At no time were amastigotes seen in Giemsa-stained aspirate smears throughout the sampling period. None of the sheep developed nodules or lesions at the site of parasite inoculation. Cultures of liver and spleen aspirates were consistently negative for Leishmania throughout the sampling period. On sacrificing all sheep, cultures and smears of liver, spleen, blood, nasal skin and mandibular lymph nodes were also found not to have any parasites for up to 14 days in culture. 3.3. ELISA results L. donoÕani specific IgG antibodies were detected from only one sheep ŽNo. 944., which was the same sheep that had amastigotes at 28 DPI. The rest of the animals had no detectable humoral response to leishmanial antigens. 4. Discussion This study has shown that domestic sheep are able to harbor live viscerotropic L. donoÕani as amastigotes at the cutaneous site of inoculation for up to 28 DPI. These parasites were isolated from the only sheep that had detectable antileishmanial antibodies. It is therefore possible that intradermal inoculation of L. donoÕani does not elicit an appropriate humoral response in sheep or that most of the parasites are eliminated by the
318
C.O. Anjili et al.r Veterinary Parasitology 74 (1998) 315–318
sheep before they can elicit a substantial humoral response. In another study, in which Leishmania major, a parasite that causes cutaneous leishmaniosis in Kenya was intradermally inoculated in pinna of four male goats Ž Capra hircus ., these parasites could not be detected by either aspirate or skin culture after 42 DPI ŽAnjili et al., 1994.. The ability of the two species of ungulates to eliminate parasites after 28 and 42 DPI respectively suggests that sheep and goats may not be good reservoirs for L. donoÕani and L. major infection but are able to maintain transient skin infections for a short duration before the infection is aborted. L. donoÕani promastigotes inoculated intradermally in footpads and the nasal skin of golden hamsters Ž Mesocricetus auratus . have been shown to persist at the site of inoculation as amastigotes for at least 10 months ŽAnjili et al., 1996.. This rodent is highly susceptible to L. donoÕani and this susceptibility may explain why it is not able to eliminate all the parasites from the cutaneous site of inoculation unlike sheep that were used in this study. It is therefore possible that the amastigote-like bodies that were reported by van der Lugt et al. Ž1992. may have been Leishmania in a sheep with transient infection. From our observations, on the course of L. donoÕani in sheep, it is unlikely that these ungulates can serve as reservoirs for this parasite. Acknowledgements We would like to thank Mr. Edwin Njenga Kenda, the technical staff of KEMRI and KARI who helped us during the study. This study received financial support from KEMRI and KARI and has been published with the approval of Directors, KEMRI and KARI. References Anjili, C.O., Mbati, P.A., Mwangi, R.W., Githure, J.I., Koech, D.K., 1996. A simple method for maintaining, detecting and recovering Leishmania donoÕani in hamsters. Acta Trop. 60, 263–267. Anjili, C.O., Olobo, J.O., Mbati, P.A., Robert, L., Githure, J.I., 1994. Experimental infection of domestic goats with Leishmania major through bites of infected Phlebotomus duboscqi and needle inoculation of culture-derived promastigotes. Vet. Res. Commun. 18, 301–305. Forbes, J., 1993. A case of kala-azar from Elgeyo Reserve. East. Afr. Med. J. 10, 363. Hendricks, L.D., Wright, N., 1979. Diagnosis of cutaneous leishmaniasis by in vitro cultivation of saline aspirates in Schneiders Drosophila medium. Am. J. Trop. Med. Hyg. 28, 962–964. Kimber, C.D., Evans, D.A., Robinson, B.L., Peters, W., 1981. Control of yeast contamination with 5-fluorocytosine in in vitro cultivation of Leishmania spp. Ann. Trop. Med. Parasitol. 75, 453–454. Mutinga, M.J., Ngoka, J.M., Schnur, L.F., Chance, M.L., 1980. The isolation and identification of leishmanial parasites from domestic dogs from Machakos district in Kenya and the possible role of dogs as reservoirs of kala-azar in East Africa. Ann. Trop. Med. Parasitol. 74, 140–143. Perkins, P.V., Githure, J.I., Mebrahtu, Y., Kiilu, G., Anjili, C., Ngumbi, P.S., Nzovu, J., Oster, C.N., Leeuwenburg, J., Hendricks, L.D., Koech, D.K., 1987. Isolation of Leishmania donoÕani from Phlebotomus martini in Baringo District, Kenya. Trans. R. Soc. Trop. Med. Hyg. 82, 695–700. van der Lugt, J.J., Carlyon, J.F., de Waal, D.T., 1992. Cutaneous leishmaniasis in a sheep. J. S. Afr. Vet. Assoc. 63, 74–77. Voller, A., Bartlett, A., Bidwell, D.E., 1976. Enzyme immunoassays for parasitic diseases. Trans. R. Soc. Trop. Med. Hyg. 70, 98–106. WHO, 1988. Guidelines for leishmaniasis control at regional and subregional levels. Leishr88.25.
Short communication
Experimental infection of domestic sheep with culture-derived Leishmania donoÕani promastigotes C.O. Anjili a b
a,)
, C.K. Ngichabe b, P.A. Mbati a , R.M. Lugalia a , H.M. Wamwayi b, J.I. Githure a
Biomedical Sciences Research Center, Kenya Medical Research Institute, P.O. Box 54840, Nairobi, Kenya National Veterinary Research Center, Kenya Agricultural Research Institute, P.O. Box 32, Kikuyu, Kenya Received 20 December 1996; accepted 10 April 1997
Abstract Domestic sheep were intradermally inoculated with culture-derived stationary phase Leishmania donoÕani promastigotes. Sampling of site of inoculation, liver and spleen for 244 days showed that this parasite can stay alive in the skin for up to 28 days post-inoculation. Apart from pyrexia that was evident in all the animals for 42 days, no other symptoms of kala-azar were seen. No parasites were recovered from the visceral organs throughout the sampling period, suggesting that sheep are not susceptible to infection with L. donoÕani. It is therefore unlikely that sheep can be synanthropic reservoirs for this parasite. q 1998 Elsevier Science B.V. Keywords: Sheep protozoa; Leishmania donoÕani; Experimental infection
1. Introduction Visceral leishmaniosis caused by Leishmania donoÕani ŽKinetoplastida: Trypanosomatidae. is endemic in Rift Valley and Eastern provinces of Kenya. In these foci, L. donoÕani has been isolated from people ŽForbes, 1993. and its natural vector sandfly Phlebotomus martini ŽDiptera: Psychodidae. ŽPerkins et al., 1987.. Apart from the dog Ž Canis familiaris. from which L. donoÕani has been isolated ŽMutinga et al., 1980., this parasite has not been isolated from any other synanthropic canids or ungulates. While man to man transmission is thought to be important, undetermined animal reservoirŽs. for L. donoÕani probably exist ŽWHO, 1988.. Intracellular amastigote-like bodies have been reported in a biopsy section from the )
Corresponding author. Tel.: q254-2-722541; fax: q254-2-720030; e-mail: [email protected]
0304-4017r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 0 1 7 Ž 9 7 . 0 0 1 5 0 - 7
316
C.O. Anjili et al.r Veterinary Parasitology 74 (1998) 315–318
pinna of a sheep in South Africa Žvan der Lugt et al., 1992.. These bodies were not cultured and it is therefore not possible to determine the authenticity of their identity as Leishmania. Sheep live in close proximity to people in L. donoÕani endemic foci especially in Rift Valley Province and it is not known what role sheep play in the epidemiology of visceral leishmaniosis in Kenya. In this study, we sought to determine the susceptibility of the blackhead sheep Ž OÕis aries . to infection with a Kenyan strain of L. donoÕani and hence its potential role as an animal reservoir for this parasite. 2. Materials and methods 2.1. Parasite cultiÕation L. donoÕani Žstrain MHOMrKEr82rLRC-L445s NLB-065., originally isolated from a Kenyan visceral leishmaniosis patient and which has been subsequently maintained by serial hamster to hamster intracardial inoculation of splenic homogenate, was used. A biopsy of the infected spleen was cultured in biphasic NNNrSchneider’s Drosophila medium supplemented with 20% heat-inactivated foetal bovine serum, 500 Urml penicillin, 500 m grml streptomycin and 250 m grml 5-fluorocytosine ŽHendricks and Wright, 1979; Kimber et al., 1981.. Promastigotes were incubated at 258C and grown to stationary phase to generate metacyclic forms. These were counted and the concentration adjusted to 1 = 10 6rml of culture medium. They were then washed at 3000 rpm for 15 min in sterile ice-cold phosphate-buffered saline ŽPBS. and resuspended in 40 m l PBS for sheep inoculation. 2.2. Animal inoculation and sampling Six adult male blackhead sheep were ear-tagged for consistency in sampling and individually bled intravenously prior to inoculation with Leishmania. Temperature of these sheep was taken on a daily basis before and after experimental infection throughout the sampling period. Each sheep was intradermally inoculated in the right-ridge of the nose with 1 = 10 6 parasitesr40 m l PBS. All the sheep were sampled at 14, 28, 42, 56, 70, 98, 140, 180 and 240 days post-infection ŽDPI.. During each sampling interval, a subcutaneous saline needle aspirate was taken from the site of inoculation and cultured in NNNrSchneider’s medium prepared as described above. This sampling was conducted to determine whether the parasite was still alive at the site of inoculation. Aspirates of liver and spleen were also taken to determine the time of visceralization of the parasite from the site of inoculation. At time 0, 40, 80, 100, and 244 DPI, all the sheep were bled and sera extracted from the blood and used to detect anti-L. donoÕani antibodies using the ELISA. At 244 DPI all the sheep were sacrificed and blood for serum taken. Portions of liver and spleen, blood, mandibular lymph nodes and nasal skin Žsite of inoculation. were cultured in NNNrSchneider’s medium. Impression smears from liver and spleen were made and stained with Giemsa for parasite examination and quantification. 2.3. Enzyme-linked immunosorbent assay ELISA for detecting L. donoÕani specific IgG antibodies was performed as previously described by Voller et al. Ž1976.. Briefly, U-well polyvinyl chloride microtiter
C.O. Anjili et al.r Veterinary Parasitology 74 (1998) 315–318
317
plates ŽDynatech Laboratories, USA. were coated overnight at 48C with 100 m l of buffer containing 50 m g of L. donoÕani antigen. The plates were washed three times, blocked with 5% bovine serum albumin in phosphate buffered saline containing 0.05% Tween 20 ŽPBST. and, coated with 200 m l of sheep serum at a dilution of 1:50 in PBST. Sera was tested in quadruplicate. Plates were incubated for 1 h at 48C and at room temperature, respectively, and were washed. Rabbit anti-sheep IgG Žwhole molecule. peroxidase conjugate ŽCappal w . was added at the recommended working dilution of 1:2000 and incubated for 2 h at room temperature. The plates were thereafter washed before the addition of 100 m l of 0.4 mgrml substrate O-phenylenediamine O-Diaminobenzene dihydrochloride ŽSigmaw . and 30% hydrogen peroxide ŽSigmaw . and incubated Žovernight for 40 min. in the dark at room temperature. The reaction was stopped by adding 25 m l of 1N HCl ŽBDH., and the optical density read using a Titertek w Elisa reader and a 492 nm filter. The means of quadruplicate absorbance values were considered positive if they exceeded twice the mean values of known negative sera. 3. Results 3.1. Temperature The highest temperature rise recorded was 408C at 22 DPI after which it stabilized between 38.58C Žnormal temperature. and 398C throughout the study. 3.2. Aspirate cultures Apart from 28 DPI when a nasal aspirate taken from one of the six sheep ŽNo. 944. revealed the presence of Leishmania after 12 days of incubation in culture at 258C, no other nasal aspirate had parasites. At no time were amastigotes seen in Giemsa-stained aspirate smears throughout the sampling period. None of the sheep developed nodules or lesions at the site of parasite inoculation. Cultures of liver and spleen aspirates were consistently negative for Leishmania throughout the sampling period. On sacrificing all sheep, cultures and smears of liver, spleen, blood, nasal skin and mandibular lymph nodes were also found not to have any parasites for up to 14 days in culture. 3.3. ELISA results L. donoÕani specific IgG antibodies were detected from only one sheep ŽNo. 944., which was the same sheep that had amastigotes at 28 DPI. The rest of the animals had no detectable humoral response to leishmanial antigens. 4. Discussion This study has shown that domestic sheep are able to harbor live viscerotropic L. donoÕani as amastigotes at the cutaneous site of inoculation for up to 28 DPI. These parasites were isolated from the only sheep that had detectable antileishmanial antibodies. It is therefore possible that intradermal inoculation of L. donoÕani does not elicit an appropriate humoral response in sheep or that most of the parasites are eliminated by the
318
C.O. Anjili et al.r Veterinary Parasitology 74 (1998) 315–318
sheep before they can elicit a substantial humoral response. In another study, in which Leishmania major, a parasite that causes cutaneous leishmaniosis in Kenya was intradermally inoculated in pinna of four male goats Ž Capra hircus ., these parasites could not be detected by either aspirate or skin culture after 42 DPI ŽAnjili et al., 1994.. The ability of the two species of ungulates to eliminate parasites after 28 and 42 DPI respectively suggests that sheep and goats may not be good reservoirs for L. donoÕani and L. major infection but are able to maintain transient skin infections for a short duration before the infection is aborted. L. donoÕani promastigotes inoculated intradermally in footpads and the nasal skin of golden hamsters Ž Mesocricetus auratus . have been shown to persist at the site of inoculation as amastigotes for at least 10 months ŽAnjili et al., 1996.. This rodent is highly susceptible to L. donoÕani and this susceptibility may explain why it is not able to eliminate all the parasites from the cutaneous site of inoculation unlike sheep that were used in this study. It is therefore possible that the amastigote-like bodies that were reported by van der Lugt et al. Ž1992. may have been Leishmania in a sheep with transient infection. From our observations, on the course of L. donoÕani in sheep, it is unlikely that these ungulates can serve as reservoirs for this parasite. Acknowledgements We would like to thank Mr. Edwin Njenga Kenda, the technical staff of KEMRI and KARI who helped us during the study. This study received financial support from KEMRI and KARI and has been published with the approval of Directors, KEMRI and KARI. References Anjili, C.O., Mbati, P.A., Mwangi, R.W., Githure, J.I., Koech, D.K., 1996. A simple method for maintaining, detecting and recovering Leishmania donoÕani in hamsters. Acta Trop. 60, 263–267. Anjili, C.O., Olobo, J.O., Mbati, P.A., Robert, L., Githure, J.I., 1994. Experimental infection of domestic goats with Leishmania major through bites of infected Phlebotomus duboscqi and needle inoculation of culture-derived promastigotes. Vet. Res. Commun. 18, 301–305. Forbes, J., 1993. A case of kala-azar from Elgeyo Reserve. East. Afr. Med. J. 10, 363. Hendricks, L.D., Wright, N., 1979. Diagnosis of cutaneous leishmaniasis by in vitro cultivation of saline aspirates in Schneiders Drosophila medium. Am. J. Trop. Med. Hyg. 28, 962–964. Kimber, C.D., Evans, D.A., Robinson, B.L., Peters, W., 1981. Control of yeast contamination with 5-fluorocytosine in in vitro cultivation of Leishmania spp. Ann. Trop. Med. Parasitol. 75, 453–454. Mutinga, M.J., Ngoka, J.M., Schnur, L.F., Chance, M.L., 1980. The isolation and identification of leishmanial parasites from domestic dogs from Machakos district in Kenya and the possible role of dogs as reservoirs of kala-azar in East Africa. Ann. Trop. Med. Parasitol. 74, 140–143. Perkins, P.V., Githure, J.I., Mebrahtu, Y., Kiilu, G., Anjili, C., Ngumbi, P.S., Nzovu, J., Oster, C.N., Leeuwenburg, J., Hendricks, L.D., Koech, D.K., 1987. Isolation of Leishmania donoÕani from Phlebotomus martini in Baringo District, Kenya. Trans. R. Soc. Trop. Med. Hyg. 82, 695–700. van der Lugt, J.J., Carlyon, J.F., de Waal, D.T., 1992. Cutaneous leishmaniasis in a sheep. J. S. Afr. Vet. Assoc. 63, 74–77. Voller, A., Bartlett, A., Bidwell, D.E., 1976. Enzyme immunoassays for parasitic diseases. Trans. R. Soc. Trop. Med. Hyg. 70, 98–106. WHO, 1988. Guidelines for leishmaniasis control at regional and subregional levels. Leishr88.25.