Discovery and Study of Leishmania Turanica for the First Time in China* Guan Li-Ren,1 Yang Yuan-Qing,2 Qu Jing-Qi,3 & Shen Wei-Xia4
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Discovery and study of Leishmania turanica for the first time in China* Guan Li-Ren,1 Yang Yuan-Qing,2 Qu Jing-Qi,3 & Shen Wei-Xia4 Reported are the results of multidisciplinary studies on Leishmania turanica, which was isolated from the auricular tissues of naturally infected great gerbils in Xinjiang, China. Discussed are the biology of the parasite, its molecular biology, its pathogenicity in rodents and humans and its vectors. This was the first time that L. turanica had been reported in China. L. turanica is highly pathogenic in BALBIc mice, with the resulting systemic infection being lethal, and it causes dermal lesions in Meriones ungui- culatus. L. turanica parasitizes the macrophages in the interstitium of the testes of Cricetulus baraben- sis, and entirely destroys the Leydig's cells of severely infected animals. Inoculation of L. turanica can induce simian and human cutaneous leishmaniasis. The cell membrane and flagella of the promastigotes of L. turanica have rather active ACPase. The major vectors of L. turanica were Phlebotomus mongolensis and P. andrejevi. In 1988, we detected a species of Leishmania in the in Karamay. Of the animals examined, 17 (63.0%) auricular subcutaneous tissue of great gerbils cap- exhibited Leishmania infection in auricular tissues, tured in Karamay, Xinjiang; later, eight human cases and the isolated promastigotes grew well and replica- of cutaneous leishmaniasis were confirmed parasito- ted in NNN culture; whereas visceral smears and logically (1). Over the period 1989-92, multidiscipli- cultivation gave negative results (1). Although nary studies of the Leishmania species were carried macroscopically there was no visible dermal lesion out to characterize it and to determine its pathogen- on the auricles of the infected great gerbils, hyper- icity in humans. This is the first identification of aemia and diffuse infiltration of inflammatory cells Leishmania turanica to be reported in China. L. in the dermis of the auricles were found upon histo- turanica was found to be pathogenic in both logical examination-some with necrotic lesions monkeys and humans, with Phlebotomus mongolen- showing the parasites in the macrophages and sis and P. andrejevi being its major vectors. prickle-cell layer. Also, the parasites were present in the cytoplasm of the epithelial cells of hair follicles (2). Results Leishmania parasites were not detected upon microscopic examination of specimens of auricular Natural infections in great gerbils tissue and visceral smears taken from 46 other ro- dents including Meriones tamariscinus (15 animals) Over the period 1988-90, superficial examinations M. meridianus (11), Euchoreutes naso (10), Dipus and necropsies were performed on 27 great gerbils sagitta (4), Mus musculus (3), M. erythrourus (2). (Rhombomys opimus) that had been captured from Cricetulus migratorius (1) and five hedgehogs: all Xiaoguai Farm and Baijiantan, oil-production areas the animals examined were caught at or close to the site where the infected great gerbils were found (Guan Li-Ren et al, unpublished data, 1992). From: Institute of Parasitic Diseases (IPD), Chinese Academy of Preventive Medicine (CAPM), 207 Rui Jin Er Lu, Shanghai 200 025, China. Morphological study I Associate Research Fellow, Department of Epidemiology, IPD, Microscopically, the amastigotes of L. turanica were CAPM, Shanghai, China. Requests for reprints should be sent to this author. 4.71 ± 0.71 ,m in length and 2.35 + 0.44 ,um in 2 Senior Research Fellow, Pathology Unit, Department of Phar- width, the size index being 11.09 ± 2.92 ,um (3); macology, IPD, CAPM, Shanghai, China. these parameters were significantly different from 3Senior Research Fellow, Department of Immunology, IPD, those of L. major and L. gerbilli isolated from the CAPM, Shanghai, China. auricular tissues of great gerbils in the Old World 4Senior Research Fellow, Department of Scientific Information, (4,5). IPD, CAPM, Shanghai, China. An ultrastructural study revealed that the aver- Reprint No. 5646 age perimeter of the L. turanica amastigotes was Bulletin of the World Health Organization, 1995, 73 (5): 667-672 © World Health Organization 1995 667 Guan Li-Ren et al. 9.98 ± 2.01 pm and the mean number of subpellicu- degeneration and necrosis of muscle cells occurred. lar microtubules, 92 ± 13 (6). Interestingly, 20.6% of Infiltration of lymphocytes and plasma cells, and to a the flagellae of the amastigotes collected from some lesser extent neutrophils, occurred in lesions of the vertebrates extended from the flagella pockets dermis; concurrently, an increase in the number of beyond the main body of the organisms, the mean fibroblasts and a marked decrease in collagenous and length of the extruded portion being 0.55 pm reticular fibres occurred. Degeneration and necrosis approximately a third of the length of the parasite of peripheral nerve fibres occurred 5-6 months after (7). The number of subpellicular microtubules in the infection. Infiltration of lymphocytes and plasma L. turanica promastigotes was 80 ± 9, considerably cells still persisted 7-months' post-infection, while fewer than the average number (113) for L. gerbilli collagenous and reticular fibres had proliferated and (8, 9). encircled the parasites, causing reduction or even disappearance of Leishmania in the lesion; neverthe- Identification of the Leishmania species less, metastastic lesions were still present on the skin and cartilage of the auricle, nose, and tail, which Monoclonal antibodies (McAbs) against L. donovani, exhibited identical histopathological changes to L. major, L. tropica and the Leishmania species col- those on the primary lesion (12). The dermal histo- lected from the great gerbils in Karamay (KGL) were pathological changes in L. turanica-infected BALB/c used in a dot enzyme-linked immunoassay (ELISA) to mice were the same as those in cases of human cuta- detect the antigens from 10 isolates of Leishmania neous leishmaniasis (13, 14). from rodents in the pilot area. The antigens from the In outbred Kunmin-strain mice, L. turanica only 10 isolates reacted only with the McAb prepared caused dermal swelling at the inoculated site; from KGL (10). Typing the n-DNA gene of KGL although the parasites could be detected, there was and L. gerbilli using the gene gp 63 (1.8 kbase) as a no evidence of visceral invasion (3). probe proved that each retained its specific bands Subcutaneous inoculation of L. turanica into the (11). auricles of Meriones unguiculatus induced the for- Isoenzymatic (ALAT, GPI, 6PGD, SOD) assays mation of small ulcers and scabs. Tissue sections on the genic loci of eight Leishmania isolates from revealed a large number of Leishmania that had been Xiaoguai Farm and an isolate from Baijiantan were engulfed by macrophages in the dermal layer and carried out using isoenzyme electrophoresis. The abscess formation in the prickle-cell layer; focal eight isolates from Xiaoguai were identified as necrosis was present in some animals. There was no L. turanica and the one isolate from Baijiantan as evidence of spontaneous healing over a 14-month L. gerbilli (D.A. Evans, unpublished reports 1992- observation period (3). 93). This was the first time that L. turanica had been Intraperitoneal inoculation of L. turanica into found in great gerbils from north Xinjiang, China. Cricetulus barabensis failed to establish infection; however, intratesticular inoculation of the parasites Pathogenecity of L. turanica and pathology gave rise to local infection. L. turanica mainly para- in infected rodents, monkeys and humans sitized the macrophages in the interstitium, some parasites being found in the giant cells and a few in Subcutaneous inoculation of L. turanica into the Sertoli's cells in the convoluted seminiferous footpads of in-bred BALB/c mice induced ulcerative tubules; many of the lymphocytes that surrounded and necrotic lesions and resulted in metastasis at the parasites had been invaded; hyperaemia and plas- several sites. There was no tendency for spontaneous ma retention occurred in the interstitium, all the healing. Some of the parasites invaded the viscera of Leydig's cells were destroyed, and abscesses of dif- the mice. A large number of Leishmania were found ferent sizes were formed. Despite the severity of the in Kupffer's cells and the reticuloendothelial cells in pathological changes in the testes, no ulceration was the lymphatic sinus of the spleens of the infected observed (3). mice. Eventually all the mice with systemic infection Subcutaneous inoculation of L. turanica on the died (3). face and forearm of a monkey produced cutaneous Histopathological studies on mouse skin over a tubercles and ulcers which spontaneously healed period of 1 year revealed that at the preliminary after 3-4 months. Upon autopsy 1 year later, a few stage of infection, hyperaemia, oedema and infiltra- parasites were still present in lesions, lymphocytes in tion of inflammatory cells (largely neutrophils and the vicinity of parasites had been invaded, while vis- eosinophils) occurred at the inoculation site; many ceral smears and cultures were negative (15). Leishmania were visible in the macrophages, and Two healthy volunteers were subcutaneously some in the neutrophils. At the abscess stage, the inoculated with L. turanica on the flexion side of the parasites were detected in the prickle-cell layer, and forearm. A dermal reaction similar to that resulting 668 WHO Bulletin OMS. Vol 73 1995 Leishmania turanica in China from leishmanin injection occurred 48 hours later in discriminating