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 unguiculatus. L. turanica parasitizes the macrophages in the interstitium of the testes of Cricetulus barabensis, 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 rodents 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 vectors of Leishmania, are described one of the volunteers; a reddish swelling developed below. with central dermal necrosis and lymphangitis, which disappeared 1 week later with the develop- Population and habitats of sandflies. A total of six ment of induration. This individual complained of species of sandflies were found in Karamay: P. mon- severe pain at the inoculation site. Microscopically, golensis, P. major wui, P. andrejevi, P. caucasicus, ulceration with parasites surrounded by infiltrating Sergentomyia minutus sinkiangensis and S. arpaklen- lymphocytes was observed on the 128th day; ulcera- sis. Being the principal species of the local sand- tion and necrosis were seen in some sections and flies, the first three constituted, respectively, 45.7% parasites were detected in all the prickle-cells and (4798/10 476), 28.2% (2956/10 476) and 13.1% the epithelial cells of hair follicles that were viewed. (1375/10 476) of the total sandfly population, where- A swelling measuring 5.0 cm x 7.0 cm was formed as the second three species represented only 0.3%, on the forearm of the second volunteer, which gradu- 7.3%, and 5.4%, respectively. P. mongolensis and P. ally decreased in size to reach 0.5 cm in diameter on andrejevi were closely associated with the ecology of the 18th day; no ulceration was visible (15). great gerbils, the major species in the rodent burrows. These results illustrate the high pathogenicity of The distribution of these two species of sandfly var- L. turanica in BALB/c mice, being equivalent to the ied with the soil zone, P. mongolensis accounting for lethal systemic infection produced by inoculating 67.3% of the sandflies in the burrows in the alluvial such mice with L. major isolated from the auricular zone and P. andrejevi for 71.6% of those in the sandy tissue of great gerbils in the former Soviet Union and zone. P. major wui was not an essential species in the the Islamic Republic of Iran (16-18). The size of the great gerbil burrows, accounting, respectively, for dermal ulcers that were formed in monkeys and 15.7% and 1.2% of the population in these two soil humans who had been inoculated with L. turanica, zones (25). was, however, smaller than that caused by L. major Homophilic habit of sandflies. P. mongolensis, P. (15). Also, the dermal lesions induced by L. gerbilli major wui, and P. andrejevi are anthropophilic but in BALB/c mice were confined to the inoculation also suck rodent blood. In contrast, the two species site and the number of parasites decreased and in of Sergentomyia discussed above only feed on cold- some instances vanished with time. In C. baraben- blooded animals. At night in residential quarters the sis, L. gerbilli parasitizes mainly the Sertoli's cells proportion of P. mongolensis and P. major wui in the of convoluted seminiferous tubules; L. gerbilli is not sandfly population always surpassed 70%, while P. pathogenic in humans (5, 19, 20). andrejevi was scarcely captured. Field surveys using human bait showed that P. mongolensis and P. major Location and content/activity of the wui accounted for 94.4% of the captured sandflies. histochemical components of L. turanica Multiplication and shift of L. turanica in the diges- A histochemical study of L. turanica indicated that tive tract of sandflies. After sucking blood from the the DNA, RNA, protein-combined a-amino acids, dermal lesions of experimental animals, engorged P. protein-combined tyrosine, tryptophan, and histidine, mongolensis, P. andrejevi and P. major wui had pro- as well as basic proteins, are mainly located in the mastigote infection rates of 37.6%, 52.9% and 62.8%, nucleus and kinetoplast of the promastigotes. Glyco- respectively, while the rates in those with completely gen is profuse in the cytoplasm of the parasite. In the digested blood were 8.3%, 6.9% and 55.6%, respec- nucleus and kinetoplast AKPase and ATPase are tively. A firm peritrophic membrane was formed to more active, while in the cell membrane and flagella limit the further reproduction of promastigotes in P. ACPase is comparatively active. The activity of mongolensis and P. andrejevi. Nevertheless, in about ACPase is comparable in L. major, L. turanica, and 25% of these two species this membrane had rup- L. donovani, and is more evident than that in L. ger- tured in the process of blood digestion, and the pro- billi (21). ACPase protects the proliferation of promastigotes were released into the stomach, where mastigotes in the digestive tract of sandflies, and further multiplication and shift to anterior parts of prevents the harmful action of neutrophils of the ver- the sandflies occurred. For P. major wui, the peri- tebrate host; its activity mirrors the pathogenicity of trophic membrane ruptured on the third day in all the parasite (22-24). those that had sucked blood and there was no con- straint on the multiplication and shift of promasti- Vectors of L. turanica gotes. The shift of the promastigotes to the oesopha- gus and pharynx occurred in all three of the The results of a series of surveys, conducted in above-mentioned Phlebotomus species. It was also accordance with the criteria stipulated by WHO for demonstrated that when sucking blood for the second

WHO Bulletin OMS. Vol 73 1995 669 Guan Li-Ren et al. time, the newly formed peritrophic membranes in could have some significance in the transmission of P. mongolensis and P. andrejevi could not envelop the latter. all the previously ingested promastigotes in the The L. turanica promastigote infection rates stomach, which were able then to reproduce and were rather low in both naturally and experimentally shift. This finding indicates that once infected with infected P. mongolensis and P. andrejevi, but the L. turanica, these two species of sandflies carry the pregnant females often sucked their hosts repeatedly parasites permanently and can transmit them repeat- so that the frequency of blood feeding was higher edly (26). than that of other species; hence the higher rate of transmission of L. turanica (30). Natural infection in sandflies and identification of Leishmania species. Naturally infected P. mongolensis and P. major wui were caught in field (man-bait, rodent burrows) and residential sites; naturally Discussion infected P. andrejevi could only be captured from Killina & Passova reported that of the Leishmania rodent burrows. Promastigotes were detected in the isolates collected from the auricular tissues of great pharynx of all the infected sandflies. The parasites gerbils in Uzbekistan, five displayed genic loci quite isolated from P. mongolensis and P. andrejevi repro- different from those of L. major, L. tropica, L. ger- duced readily in NNN culture (27). billi and L. donovani, as shown by enzyme electro- Promastigotes isolated from naturally infected phoresis (31). It was suggested that the parasite res- P. mongolensis and P. andrejevi were inoculated ponsible might be a new species of Leishmania (31). subcutaneously into BALB/c mice or intratesticularly In 1990, Strelkova et al. proved, using enzyme elec- into C. barabensis; the induced lesions were the trophoresis and DNA hybridization with a cDNA same as those caused by L. turanica (27). probe prepared from L. infantum, that the Leishma- The ultrastructure of the amastigotes in the nia isolates from great gerbils in the former Soviet experimental animals was also identical to that of L. Union consisted of L. major, L. gerbilli, and a new turanica (28). In contrast, promastigotes isolated species, L. turanica; and reported that this new spe- from naturally infected P. major wui did not induce cies occurred in Turkmen SSR, Uzbek SSR, southern dermal lesions in mice, but in both mice and C. bar- Kazakh SSR, as well as in Mongolia. Morphological abensis exhibited visceral involvement; also, growth studies using microscopy and experimental inocula- of the parasites in NNN culture was poor (27, 29), tions were also carried out. Dermal lesions without indicating that the species involved was different ulceration were detected in R. opimus and Mesocri- from L. turanica. cetus auratus after inoculation with L. turanica, but McAbs against the antigens of L. major, L. trop- in 2.5% of cases late ulceration was observed in M. ica, L. donovani and L. turanica were prepared and auratus. L. turanica was not pathogenic to Meriones used in dot ELISAs to detect 16 isolates from P. libycus and Arvicanthis abyssinicus. Of the three mongolensis and P. andrejevi. The results showed human volunteers inoculated with L. turanica, one that all the isolates reacted only to the McAb to the exhibited a dermal tubercle that vanished after 14 L. turanica antigen prepared from local great gerbil days, while the other two exhibited small ulcers isolates. 1.5-2 months later that spontaneously healed after P. mongolensis and P. andrejevi were estab- a further 2-2.5 months (32). lished to be the vectors of L. turanica (27). The Almost concurrently, after carrying out parasito- cloned 32P-labelled gene gp63 was used as a probe logical and morphological studies on Leishmania to detect the n-DNA of promastigotes isolated isolated from great gerbils in Karamay, Xinjiang, we from naturally infected P. major wui; genic typing suggested that the parasite concerned might be a new suggested that it was homogeneous with that of species (3). This was subsequently identified as L. L. infantum (29). turanica, the first time it had been recorded in China. Although both P. mongolensis and P. andrejevi Sufficient evidence has now accumulated to dis- were vectors for L. turanica, the latter sandfly sel- tinguish between L. turanica, L. major, and L. ger- dom flew into residential sites, mainly restricting its billi, indicating that each is an individual species. activities to rodent burrows, and was thus assumed Their differences have been characterized by not to play an essential role in the propagation of enzyme electrophoresis (31, 32), gene typing (32, L. turanica through a human-sandfly cycle (25). 11) and immunological assay (10), and by their natu- P. major wui can be naturally infected with L. infant- ral host predisposition and pathogenicity. A broad um, but not with L. turanica; none the less, experi- range of natural hosts for L. major has been found, mental infection studies revealed that it was an including R. opimus and several species of Meriones, appropriate vector for L. turanica (26), and hence Allactaga, Mus, Spermophilus (33), and Psammomys

670 WHO Bulletin OMS. Vol 73 1995 Leishmania turanica in China

(34); L. major also causes severe macroscopic der- mortelle, et elle provoque des lesions cutanees mal lesions in gerbils and acute dermal necrosis in chez Meriones unguiculatus. L. turanica parasite humans (33). In contrast, for L. turanica and L. ger- les macrophages de l'interstitium testiculaire de billi, the only natural host identified is R. opimus Cricetulus barabensis et d6truit entierement les (32, 35). Although no macroscopic dermal lesions cellules de Leydig des animaux fortement infec- were observed on the auricles of great gerbils infect- t6s. Son inoculation peut induire une leishmaniose ed with L. turanica, rather large necrotic areas of cutan6e chez le singe et chez l'homme. dermis were seen by microscopy (2) and small der- L'ACPase de la membrane cellulaire et des mal ulcers were formed in humans (32, 15). Never- flagelles des promastigotes de L. turanica est theless, L. gerbilli did not induce visible dermal relativement active. Les principaux vecteurs identi- lesions in R. opimus or in humans (5, 20, 35). fi6s dans la region ou a eu lieu l'6tude sont Phle- Currently, three species of Leishmania, i.e., L. botomus mongolensis et P. andrejevi. Ces travaux turanica, L. infantum and L. gerbilli, have been iden- apportent des informations sur les caract6ristiques tified in rodents or sandflies in Karamay, Xinjiang. biologiques de L. turanica qui seront utiles pour la The pathogenicity of L. turanica in humans has now poursuite de l'6tude de cette espece et pour les been verified (32, 15); human cutaneous leishmania- recherches 6pid6miologiques sur la leishmaniose sis caused by L. infantum has been confirmed in cutanee dans le nord du Xinjiang. eight countries in Europe, Asia and Africa.a Studies on the possible role of L. infantum and L. turanica in the prevalence of human cutaneous leishmaniasis in Xinjiang should be carried out. References 1. Guan Li-ren et al. Eight cutaneous leishmaniasis cases detected in Karamay, Xinjiang Uyger Autono- mous Asian of tro- a Desjeux P. Information on the epidemiology and control of the Region, China. Southeast journal leishmaniases by country or territory. Unpublished document pical medicine and public health, 1992, 23: WHO/LEISH/91.30, 1991. 803-804. 2. Yang Yuan-qing et al. [Leishmaniasis in Karamay 6. Pathological observations on the ear tissue of big gerbils naturally infected with Leishmania in Xiao- quai area]. Endemic diseases bulletin, 1991, 6: 17-19 (in Chinese). Acknowledgements 3. Guan Li-ren et al. [Leishmaniasis in Karamay 3. We are grateful to Dr D.A. Evans, Department of Medical Study on the morphology of Leishmania from the Parasitology, London School of Hygiene and Tropical big gerbil and its pathogenicity in laboratory ani- Medicine, for identifying nine isolates of Leishmania from mals]. Endemic diseases bulletin, 1990, 5: 54-62 (in rodents and to Dr P. Desjeux, World Health Organization, Chinese). for valuable suggestions and comments. 4. Kellina 01. [On the dimensions of Leishmania tropi- The study was supported by the National Natural Sci- ca major and L. tropica minor]. Meditsinskaia para- ence Foundation of China and by the World Health Organ- zitologiia i parazitarnye bolezni, 1962, 31: 727-733 ization. (in Russian). 5. Wang Jie et al. [A study on the Leishmania parasite of the big gerbil in northwest China]. Acta parasitologica Sinica, 1964, 1: 105-114 (in Chinese). Resume' 6. Shen Bing-gul et al. [Leishmaniasis in Karamay 2. Ultrastructure of amastigote of Leishmania in the big gerbil in Karamay, Xinjiang]. Endemic diseases bul- Leishmania turanica decouverte et etudiee letin, 1989, 4: 56-59 (in Chinese). pour la premiere fois en Chine 7. Shen Bing-gui, Guan Li-ren. [Leishmaniasis in Karamay 13. A record and description of the mor- Le present article rapporte les r6sultats d'6tudes phological characteristics of some amastigotes in multidisciplinaires men6es sur Leishmania turani- the big gerbil]. Endemic diseases bulletin, 1993, 8: ca a la suite de son isolement au Xinjiang (Chine) 15-17 (in Chinese). dans les tissus auriculaires de grandes gerbilles 8. Shen Bing-qui, Guan Li-ren. [Leishmaniasis in infectees naturellement. Ces 6tudes portent Karamay 7. Observations on ultrastructure of pro- notamment sur la biologie, la biologie moleculaire, mastigote of Leishmania in the big gerbil]. Endemic la pathogenicit6 pour les rongeurs et l'homme, diseases bulletin, 1992, 7: 23-25 (in Chinese). 9. Wang Jie et al. [Observations on the ultrastructure ainsi que sur les vecteurs. C'est la premiere fois of the promastigote stage of Leishmania gerbilli]. que L. turanica est signalee en Chine. Elle est for- Endemic diseases bulletin, 1989, 4: 57-60 (in Chi- tement pathogene pour la souris BALB/c, chez nese). laquelle elle entraine une infection g6neralis6e 10. Xu Yong-xiang et al. [Leishmaniasis in Karamay

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