The leishmaniases as emerging and reemerging zoonoses

International Journal for Parasitology 30 (2000) 1269±1281

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The leishmaniases as emerging and reemerging zoonoses R.W. Ashford* Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK Received 31 May 2000; received in revised form 7 August 2000; accepted 1 September 2000

Abstract The 20 or so species of Leishmania which have been recorded as human infections are all either zoonotic, or have recent zoonotic origins. Their distribution is determined by that of their vector, their reservoir host, or both, so is dependent on precise environmental features. This concatenation of limiting factors leads to speci®c environmental requirements and focal distribution of zoonotic or anthroponotic sources. Human infection is dependent on the ecological relationship between human activity and reservoir systems. Examples are available of the emergence of leishmaniasis from the distant past to the present, and can be postulated for the future. These emergences have been provoked by the adoption of new, secondary reservoir hosts, the adoption of new vector species, transport of infection in humans or domestic animals, invasion by humans of zoonotic foci, and irruption of reservoir hosts beyond their normal range. The leishmaniases therefore present an excellent model for emerging disease in general, and for the generation of the principles governing emergence. The model is, however, limited by gaps in our knowledge, usually quantitative, sometimes qualitative, of the structure of reservoir systems. q 2000 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Parasite ecology; Emerging diseases

1. Introduction The genus Leishmania comprises a growing number of species, currently some 30, of which around 20 cause disease in humans. There are ®ve main human diseases, collectively known as the leishmaniases. Two of the species can be maintained inde®nitely in human populations but there is no Leishmania species which appears to have become a human parasite through the coevolutionary pathway. All human infection is, therefore, either actively zoonotic or has been acquired by recent host transfer from a zoonotic source. The leishmaniases have been the subject of much recent secondary literature [1±7], in which reviews of most of the factual information in this article may be found. Many of the other statements are based on personal anecdotes or speculations, which are better not referenced in detail.

2. The leishmaniases as human diseases 2.1. Clinical diversity The main clinical varieties of leishmaniasis in humans are as follows. * Tel.: 144-151-708-9393; fax: 144-151-708-8733. E-mail address: [email protected] (R.W. Ashford).

² Cutaneous leishmaniasis (CL) or oriental sore, which is usually caused by Leishmania tropica, L. major, L. aethiopica, L. mexicana, L. amazonensis, L. panamensis, L. guyanensis, L. peruviana or L. braziliensis, but may be caused by any of the Leishmania species which infects humans. ² Visceral leishmaniasis or kala azar: this may be largely restricted to infants, caused by L. donovani infantum, or have little age-speci®city, caused by L. d. donovani. L. d. infantum affects people of all ages who are suffering from immunosuppressive disease. It has been suggested that L. tropica may cause visceral leishmaniasis but there is controversy over this. ² Mucocutaneous leishmaniasis, usually caused by L. braziliensis, following cure of the initial oriental sore. ² Post-kala azar dermal leishmaniasis, caused by L. d. donovani following cure of the initial visceral leishmaniasis. ² Diffuse cutaneous leishmaniasis, caused by L. aethiopica or L. amazonensis. The numerous other named species of Leishmania are mostly from Central or South America, rarely infect humans or domestic animals and, when they do infect humans, are usually responsible for uncomplicated CL.

0020-7519/00/$20.00 q 2000 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(00)00136-3

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2.2. Clinical symptoms and signs 2.2.1. Cutaneous leishmaniasis Oriental sore or CL ®rst appears as a persistent insect bite. Gradually the lesion enlarges, remaining red, but without noticeable heat or pain. Resolution of the lesion involves immigration of leucocytes, which isolate the infected area leading to necrosis of the infected tissues, and the formation of a healing granuloma in the ¯oor of the lesion. The necrotic process may be rapid, causing a large, open, wet sore (especially L. major, L. panamensis and L. braziliensis), or may be more indolent, without frank ulceration (L. tropica, L. aethiopica, L. peruviana). Natural cure without treatment is the rule, but the time taken varies greatly according to the identity of the parasite and the site of the lesion. Similarly, the size of the lesion may vary between a few millimetres and several centimetres in diameter. It is not unusual, especially with L. major infection, for numerous lesions to occur simultaneously, causing great dis®gurement and distress. Oriental sore is not usually associated with systemic signs or symptoms, but draining lymph nodes may become enlarged, and lesions may spread along lymphatic ducts (L. guyanensis). Oriental sore caused by L. tropica or L. major is not usually associated with any detectable serological response. A skin-test response develops prior to ®nal cure, and may be retained for life. Cured patients remain immune to the homologous infection for many years. 2.2.2. Kala azar Kala azar or visceral leishmaniasis is sometimes preceded by a dry or ulcerating lesion at the site of the infective bite. Systemic signs of intermittent medium-grade pyrexia, anaemia, splenomegaly, hepatomegaly and progressive cachexia develop at variable rates, between weeks and years following infection. Less constant signs include lymphadenopathy and persistent diarrhoea. The outcome of fully developed visceral leishmaniasis is death, said usually to be due to concomitant infection resulting from the weakened immunological state of the patient. There is, however, increasing evidence that many people who become infected never develop full-blown disease, and recover spontaneously [8]. The proportion of these subclinical cases varies from almost 100% with L. d. infantum infection in otherwise healthy adults to less than 25% during epidemics of kala azar in Africa. However, this resistance to infection may be destroyed by infection with the HIV virus, and L. d. infantum infection in AIDS victims may present in many and bizarre ways. Although full-blown kala azar is usually fatal, it is associated with very strong serological response, to the extent that the albumin/globulin ratio is reversed. The raised serum proteins are used diagnostically in a non-speci®c formol-gel test. Cured cases develop a positive skin-test reaction which is frequently used for retrospective epidemiological study, though there is evidence that this may be lost in a few years. In endemic areas or during epidemics, many apparently

healthy people may develop a positive serological response and, subsequently, skin-test reactions [8]. This is the best evidence for the subclinical cases mentioned above. 2.2.3. Mucocutaneous leishmaniasis Mucocutaneous leishmaniasis is occasionally reported from Sudan and other Old World foci. Here, it seems that the lesion commences at the site of a sand-¯y bite on or close to a mucosal surface. Occasional infections with L. d. infantum are reported from the tonsils or buccal mucosa. It is tempting to suppose that these originated from accidentally inhaled ¯ies. Classical mucocutaneous leishmaniasis or espundia is, however, restricted to L. braziliensis infections in which, following the apparently complete resolution of the initial oriental sore, sometimes many years later, metastatic lesions appear on the buccal or nasal mucosa. The mucosa and associated cartilage are gradually eroded till much of the face may be destroyed. The initial symptom is mild irritation of the tip of the nose, or other affected surface. Parasites are dif®cult to ®nd in these lesions, but, in appropriate geographical settings, a history of CL supported by positive serology (ELISA, IFAT, etc.) or skin test are important signs. 2.2.4. Post-kala azar dermal leishmaniasis Post-kala azar dermal leishmaniasis (PKDL) is normally a sequel to kala azar which has been cured by treatment. Occasional cases are reported with no history of kala azar. PKDL usually appears within 2 years of the complete cure of the visceral infection, and commences with the appearance of mottling of the skin, like freckles. 2.2.5. Diffuse cutaneous leishmaniasis Diffuse cutaneous leishmaniasis (DCL) is restricted to Venezuela and the Dominican Republic in the western hemisphere, and to Ethiopia and Kenya in Africa. It is usually a manifestation of infection with parasites which normally cause simple CL, associated with a speci®c anergy or immunological lack of response. The lesions may be restricted, perhaps to the border of an ear, or may be widespread all over the body. They are raised macules or patches of thin skin which, although painless, are grossly dis®guring. The initial cases described, both in Ethiopia and Venezuela, were at ®rst misdiagnosed as lepromatous leprosy. Typically, parasites are very numerous in the lesions. 2.3. Diagnosis In places where they are well known and endemic, or during epidemics, all three main forms of leishmaniasis, cutaneous, mucocutaneous and visceral, can be diagnosed with some reliability by clinical examination backed, in visceral leishmaniasis, by a blood count to show anaemia and leucopoenia. Con®rmation of the diagnosis of any of the leishmaniases generally depends on the demonstration of the amastigote

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parasites in infected tissue. Specimens examined are usually skin biopsies or aspirates from bone marrow or spleen. In any case the collection and examination of the specimen requires careful preparation and training. The parasites may be demonstrated in stained microscopical preparations or in culture. Various DNA and mab probes have been developed, but none of these has reached routine practice. A direct agglutination test is widely used for kala azar: this is valuable in clinical settings, but picks up numerous asymptomatic cases, so is less effective for active case detection in villages. In the formol-gel test, mentioned above, a drop of formalin is added to 1 ml of serum: a positive reaction is indicated by the rapid coagulation of the serum. Though still widely used, this test is insuf®ciently speci®c to be recommended. 2.4. Treatment Treatment of any of the leishmaniases is slow and expensive. The ®rst line drugs are compounds containing pentavalent antimony such as sodium antimony gluconate. These are delivered over a period of 20 or more days, either by i.v. injection (for visceral or severe CL) or by injection into the periphery of single lesions. Second line treatment, when antimony is ineffective or contraindicated, uses amphotericine B, allopurinol, pentamidine or paromomycin (monomycin) in diverse combinations and formulations. As each of these has unsatisfactory features, cheap, effective, rapid treatment for visceral leishmaniasis, and topical treatment for CL are urgent requirements. 3. The parasites 3.1. Morphology and life cycle Parasites of all species of Leishmania are morphologically similar and, while presumptive identi®cation may be made on circumstantial grounds, biochemical analysis is required for formal identi®cation at species level. All species inhabit the reticulo-endothelial cells of a vertebrate host and the gut of a phlebotomine sand-¯y. There are two main stages in the life history: the amastigote and promastigote. Amastigotes are intracellular, rounded or fusiform, some 5 mm in maximum diameter, containing a single nucleus, a kinetoplast, and a ¯agellar pocket with the rudiments of a ¯agellum. Amastigotes are the form found in the monocytes and macrophages of the vertebrate host. They divide repeatedly, and spread to further cells when the initial host cell bursts. In the sand-¯y host the amastigotes in the blood meal transform to promastigotes. These are longer, with a central nucleus and terminal kinetoplast, and with a well developed ¯agellum which is used either for propulsion or for attachment. Both amastigotes and promastigotes divide repeatedly by longitudinal binary ®ssion.

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Promastigotes may be cultured in various media, mostly containing inactivated serum. Amastigotes can be cultured in appropriate cell cultures. Identi®cation requires analysis of electrophoretic migration patterns of isoenzymes, though con®rmation of presumptive identi®cation can sometimes rely on monoclonal antibody or DNA probes, the latter with or without PCR ampli®cation. Neither DNA nor monoclonal probes have yet attained suf®cient reliability to replace isoenzyme analysis in identi®cation. 3.2. Transmission All forms of leishmaniasis are transmitted by phlebotomine sand-¯ies (Diptera: Psychodidae: Phlebotominae). The amastigote parasites are ingested with a blood meal, and proceed to divide in the sand-¯y gut. Very soon they transform to promastigotes which continue to multiply rapidly. Some 3 days following a feed, the sand-¯y defaecates the remnants of the blood meal. At this stage, any unattached parasites are evacuated. The parasites which are to survive attach themselves by their ¯agellae either to the microvillae of the mid-gut, or to the cuticular surface of the anterior part of the hindgut, the hind gut triangle or pyloric valve. In either case, following defaecation, the attached parasites are released. Again they divide repeatedly, eventually attaching themselves to the chitinous extension of the fore gut into the mid gut, the cardiac valve. Some parasites become differentiated as `metacyclic' forms, which are unattached, fast-swimming, with small body and long ¯agellum [9]. Transmission depends on the injection of these metacyclic forms when the sand-¯y next takes a blood meal. The suitability of a given mammalian host for the maintenance of Leishmania populations depends on many factors, the most important of which are the population density of the host, the duration of the infection (and longevity of the host), the location of the parasites within the host, and the immune status of the host following cure. The term `reservoir host' should be restricted to the species which sustain the reservoir system in which a parasite survives inde®nitely. Transmission may further be affected by the nature of the sugar meals taken by the ¯y [10], and by the response of the host to antigens in the ¯y's saliva. These are both areas of active and promising research. Transmission can readily be achieved by inoculation of infected material from one person to another. It was common practice in south-west Asia to deliberately infect young girls with material from an oriental sore so that their lesion would be in an inconspicuous place and they would subsequently be immune. More recently there is evidence that much of the visceral leishmaniasis associated with HIV infection in southern Europe is transmitted by sharing contaminated needles and syringes for the misuse of drugs [11]. The reported transmission, both by venereal contact and by blood transfusion, is of little, if any, general signi®cance.

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4. Distribution and strati®cation The leishmaniases occur in more than 100 countries, from warm temperate through subtropical to tropical climates [12]. Southeast Asia and Australasia are the only large areas with suitable climates where the diseases are absent. Visceral leishmaniasis is concentrated in eastern Africa, particularly Sudan and Kenya, and on the Indian subcontinent, in Bangladesh, north-east India and Nepal. Post-kala azar dermal leishmaniasis has a similar distribution, though the proportion of treated kala azar cases which develop this condition varies. Infantile visceral leishmaniasis is characteristically circum-Mediterranean in its distribution, extending east through south-west Asia to China, and west, to Central and South America where most cases occur in north-east Brazil. This last form is increasingly rare in its classical infantile form, but has expanded to affect adults who are infected with the HIV virus, or who are otherwise immunocompromised. Both anthroponotic and zoonotic CL, in the Old World, are mostly found in arid or semi-arid areas. The former occurs, usually as epidemics, in the densely populated cities of central and west Asia, from Aleppo in Syria, to Kabul in Afghanistan, while the latter is characteristic of semi-desert rural areas, both in Asia and North Africa, where colonies of the reservoir hosts are found. CL due to L. mexicana, L. amazonensis or L. braziliensis is widely distributed throughout much of South and Central America. The precise distribution is focal, and depends very much on the presence of suitable vectors and reservoir hosts. L. braziliensis appears to be expanding to urban foci, where it is dependent on domesticated dogs and, possibly, equines [13]. Each of the other forms of human leishmaniasis, such as CL due to L. aethiopica in the highlands of Ethiopia, or due to L. peruviana in the western Andes, has a focal distribution depending on the presence of reservoir hosts and/or vectors in suf®cient number and proximity to humans. The dependence of all forms of leishmaniasis on a limited choice of sand-¯y vectors, and of most forms on wild or domesticated reservoir hosts, leads to a strong correlation between leishmaniasis and environmental features, notably climatic and vegetation zones. The various combinations of vector and reservoir host which maintain the various Leishmania species in different habitats and geographical areas allow the strati®cation of the leishmaniases into `nosodemiological units', a simpli®ed table of which is given in Table 1.

5. Epidemiology and emerging issues Most of the leishmaniases are zoonotic, and those which are not clearly have recent zoonotic roots. While there is little correlation between the taxonomic classi®cation of the parasites and their pathogenesis in humans, there is very

good correlation between the recognized species and their ecology. It is therefore best to consider each species separately, sometimes breaking the account according to the different strata shown in Table 1. In the absence of effective surveillance in most, if not all, endemic countries, most information on the leishmaniases as emerging, re-emerging, or disappearing diseases is circumstantial and anecdotal. This account would be misleading (and very sparse), however, if such material were to be omitted. The bias in the account towards Old World examples re¯ects the author's knowledge and experience; doubtless there are numerous comparable stories from the New World. Despite these misgivings, there are numerous instances where the leishmaniases have apparently `emerged', and there are diverse causes for these emergences. This group illustrates, perhaps better than any other eukaryote parasites, many of the principles of emerging infection. 5.1. Leishmania donovani 5.1.1. The original home This species is conventionally divided in two subspecies, L. d. donovani and L. d. infantum. The considerable biological differences between the subspecies has led to their being considered by many authors as species, but no set of taxonomic character states has been identi®ed by which all strains can be allocated to one or the other. This, plus the almost complete allopatry between them justi®es their treatment as subspecies. Further, in southern Sudan, three strains have been described from a single outbreak, which corresponded to both subspecies, as well as an intermediate form [15]. We consider that the southern Sudanese forms represent an ancestral population in an ancestral homeland. This is where any zoonotic origin should be sought but, as yet, none has been adequately described. Comprehensive studies in the 1960s succeeded in isolating Leishmania parasites from various animals, among which the apparent maintenance host was the Nile grass rat, Arvicanthis niloticus. Methods were not then available for speci®c identi®cation of the parasites but they were identi®ed years later as L. donovani. This result has never been repeated despite considerable effort, and strains subsequently isolated from the viscera of rodents in East Africa have been identi®ed either as L. major or as a distinctive undescribed form. In view of this uncertainty, and the constant risk of mixing strains in the laboratory, the identity of any reservoir host in Sudan must be regarded as uncertain. There is, however, convincing evidence that L. donovani is zoonotic in Sudan: some of the earliest cases occurred in soldiers who had visited uninhabited areas; there is a serious risk of infection in some Sudanese game parks, and infected sand-¯ies have been found in uninhabited areas [16]. Assuming the ancestral home of L. donovani to be in Sudan, it must have emerged on numerous occasions, some more speculative than others.

Oriental sore (wet form)

Human infection unknown Human infection unknown Human infection unknown Chronic oriental sore; diffuse cutaneous leishmaniasis

L.(L.) major

L. (L.) gerbilli

L. (L.) donovani donovani

L. (L.) aethiopica

L. (L.) arabica

Kala azar or visceral leishmaniasis; post-kala azar dermal leishmaniasis

Oriental sore (dry form), leishmaniasis recidivans

L. (L.) tropica

L.(L.) turanica

Disease spectrum in humans

Leishmania species

Phlebotomus (Synphlebotomus) martini Phlebotomus (Euphlebotomus) argentipes

Northern Kenya, southwest Ethiopia North-east India, Bangladesh, Terai region of Nepal

Phlebotomus (Larroussius) orientalis

Phlebotomus (Larroussius) longipes and P.(Larroussius) pedifer P. (L) pedifer

Phlebotomus (Paraphlebotomus) spp P. (Paraphlebotomus) spp P. (P.) papatasi

Phlebotomus (Phlebotomus) duboscqi

P. (P.) papatasi

Phlebotomus (Paraphlebotomus) sergenti Phlebotomus (Larroussius) guggisbergi and others P. (P.) papatasi

Vector

Central and southern Sudan

Highlands of south-west Ethiopia

Highlands of Ethiopia and Kenya

Saudi Arabia

Eastern Russia, Mongolia Central Asia

Central Asia from Iran to Uzbekistan West Africa to Kenya, Sahel belt

Equatorial and southern Africa, Kenya and Namibia North Africa and southwest Asia, from Algeria to Saudi Arabia

Central to south-west Asia

Geographical distribution

Table 1 Strati®cation of the Leishmania species into `nosodemiological units'

Humans only

Presumably zoonotic but reservoir host unknown

Presumably zoonotic but reservoir host uncertain

Heterohyrax brucei

Great gerbil Rhombomys opimus Great gerbil Rhombomys opimus Fat sand-rat Psammomys obesus Rock hyraxes Procavia capensis and Heterohyrax brucei

Fat sand-rat Psammomys obesus (epidemic maintained by Meriones shawi) Great gerbil Rhombomys opimus Relative importance of different hosts to be determined

Probably rock hyraxes, Procavia capensis

Humans only

Reservoir host

Rodents: Arvicanthis niloticus, Acomys cahirinus, Praomys natalensis, Rattus rattus, serval cat Felis serval, genet Genetta genetta

Giant rat Cricetomys gambianus

Rodents: Arvicanthis spp, Praomys spp, Tatera robusta, Aethomys kaiseri, Taterillus emini, Xerus rutilus, Cercopithecus aethiops

Rodents: Gerbillus spp., Meriones shawi, M. libycus, M. crassus, Nesokia indica, cat? Numerous desert mammals

Dog (Cutaneous leishmaniasis)

Other mammal hosts a

Villages on alluvial plains

Semi arid bush, on laterite, with termitaria

Relict forest with giant ®g trees Ficus vasta, around 2000 m altitude Alluvial ¯at lands with forest of Acacia seyal and Balanites aegyptiaca

Saline depressions with Chenopodiaceae Cliffs and rocky areas, between 1500 and 2600 m altitude

Especially on loess soils

Especially on loess soils

Alluvial fans with loess deposits Sahel savannah

Saline depressions with Chenopodiaceae

Rocky places in semiarid areas

Densely populated cities

Habitat

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Human infection unknown

Cutaneous leishmaniasis

L (L.) amazonensis??

L. (L.) venezuelensis L. (L.) sp

Diffuse cutaneous leishmaniasis

Cutaneous leishmaniasis; diffuse cutaneous leishmaniasis

Texas

Cutaneous leishmaniasis

L. (L.) amazonensis (Syns: L. garnhami and L. pifanoi)

Central America

Cutaneous leishmaniasis; chiclero ulcer

L. (L.) mexicana

Towns in Lara State, Venezuela Dominican Republic

Trinidad

South America, mostly north of the Amazon

Unknown

Unknown

Lu. ¯aviscutellata

Lutzomyia (Nyssomyia) ¯aviscutellata

Lutzomyia anthophora

Lutzomyia (Nyssomyia) olmeca

Lutzomyia longipalpis; L. evansi

Dog (Viscerocutaneous leishmaniasis)

Various

Central and South America

Dog (Viscerocutaneous leishmaniasis)

Phlebotomus (Larroussius) perniciosus

Central and western Mediterranean basin, both Europe and N. Africa Through Mediterranean basin to Iran

Presumably zoonotic, but reservoir host unknown

Domestic cat?

Proechimys guyanensis, Proechimys cuvieri

Neotoma micropus

Forest rodents, Ototylomys phyllotis

Dog (Viscerocutaneous leishmaniasis)

Dog (Viscerocutaneous leishmaniasis)

Phlebotomus (Larroussius) ariasi

Southern France, Cevennes hills

Infantile visceral leishmaniasis; cutaneous leishmaniasis; AIDSassociated leishmaniasis

Reservoir host

L. (L.) donovani infantum (Syn. L. chagasi)

Vector

Geographical distribution

Disease spectrum in humans

Leishmania species

Table 1 (continued)

Opossums: Didelphis marsupialis, Metachirops opossum, Metachirus nudicaudatus, Marmosa cinerea, anteater Tamandua tetradactyla, kinkajou Potos ¯avus, fox Cerdocyon thous, rice rat Oryzomys capito, squirrel Sciurus vulgaris Opossums: Marmosa robinsoni, M. fuscata, Caluromys philander, rice rat, Oryzomys capito

Racoon dog Nyctereutes procyonoides (China), foxes Vulpes spp., jackals Canis spp. Fox Dusicyon thous (Records from Lycalopex ( ˆ Dusicyon) vetulus are probably misidenti®ed) Rodents: Heteromys desmarestianus, Nyctomys sumichrasti, Sigmodon hispidus

Fox Vulpes vulpes; black rat Rattus rattus

Fox Vulpes vulpes

Other mammal hosts a

Semi arid mesquite with prickly pear Opuntia

Villages and homesteads in semi-arid areas

Villages and suburbs, with calcareous outcrops, in sub-humid bioclimatic zone Various

Forested areas at middle altitude

Habitat

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Cutaneous leishmaniasis

Cutaneous leishmaniasis

Cutaneous leishmaniasis

Cutaneous leishmaniasis

Cutaneous leishmaniasis

L. (V.) lainsoni

L. (V.) naif®

L. (V.) panamensis

L. (V.) peruviana

L. (V.) shawi

Mammal names follow Corbet and Hill [14].

Cutaneous leishmaniasis

L. (V.) guyanensis

a

Cutaneous leishmaniasis; mucocutaneous leishmaniasis

L. (Viannia) braziliensis

Table 1 (continued)

Para and Acre States, Brazil

Arid valleys of the western Peruvian Andes

Panama

Amazonas State, Brazil; French Guyana

Northern Para State, Brazil

Guyanas

S. America from 198 N to 298 S

Lutzomyia (verrucarum) verrucarum, Lu (Helcocyrtomyia) peruensis, Lu (H) ayacuchensis Lutzomyia (Nyssomyia) whitmanni

Lutzomyia (Nyssomyia) umbratilis; (Lu. (N.) whitmani, Lu. (N.) anduzei Lutzomyia (Trichophoromyia) ubiquitalis Lutzomyia (Psychodopygus) ayrozai, Lu. (Ps.) paraensis Lutzomyia (Nyssomyia) trapidoi, Lu. (N) ylephiletor, Lu. (Lu) gomezi, Lu. (Psychodopygus) panamensis

Lutzomyia (Nyssomyia) whitmani

Lutzomyia (Psychodopygus) wellcomei, etc.

Main host uncertain

Transmission is thought mainly to be dependent on humans

Sloth Choloepus hoffmanni

Armadillo Dasypus novemcinctus

Cuniculus paca

Sloth Choloepus didactylus

Presumably zoonotic, but reservoir host unknown

Sloths: Choloepus didactylus, Bradypus griseus, primates: Cebus apella, Chiroptes satanus, coati Nasua nasua

Opossums: Metachirus nudicaudatus, Didelphis marsupialis, sloth Bradypus variegatus, rodents: Akodon sp., Rattus rattus, Proechimys semispinosus, Heteromys desmarestianus, tree porcupine Coendou sp, dog Dog (Cutaneous leishmaniasis)

Dog (Cutaneous leishmaniasis); horse, donkey Opossum Didelphis marsupialis, anteater Tamandua tetradactyla, spiny rat Proechimys sp.

Numerous forest animals

Forest

Western Andean valleys at 800 to 2000 m altitude

Primary forest

Primary forest, near water

Primary forest

Expanding in heavily populated suburbs

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5.1.2. Local emergences The incidence of human infection in Sudan is notoriously variable, with occasional epidemics and few sporadic cases in interepidemic years, usually concentrated in forested areas which constitute the speci®c habitat of the vector, Phlebotomus orientalis. The emergent outbreak which has best been described is that in the western Upper Nile region, previously free from the disease, in which it is estimated that half of the human population died between 1989 and 1994, and some 20% were treated and cured [17]. We have suggested that the infection was introduced by soldiers returning from training in areas well known to be infected, near the Ethiopian border, and that the regrowth of forest, which had been destroyed by ¯oods in the 1960s, allowed large populations of the vector to build up [18]. The current emergence of large numbers of human cases in the East of the country is less easily explicable. The infection has always existed in that area and there have been important environmental changes and movements of people in recent years. The main environmental change has been the replacement of forest with mechanized agriculture, which would have been expected to eliminate the vector. Outbreaks in neighbouring Kenya were thought to have originated with the return of soldiers from endemic areas in Sudan and Ethiopia, to central Kenya where a different vector, P. martini was able to maintain transmission for brief periods. Subsequent ®ndings that L. donovani, transmitted by P. martini, is focally widespread in northern Kenya, north-western Uganda and southern Ethiopia show that the explanation may not be so simple. It may be that L. donovani determines the distribution of people, at least on a local scale. The people of Aba Roba in southern Ethiopia live on steep hillsides overlooking the uninhabited Segen River valley. Plans by the Mengistu regime to populate this valley with people from another part of the country were mercifully shelved when it was pointed out that the area was uninhabited for the good reason that anyone sleeping there had a high chance of contracting kala azar. Although uninhabited areas are usually no-man's-land on the border between rival human populations, many are reinforced by the fear of disease, usually vaguely described as malaria. It is a reasonable guess that leishmaniasis, mistaken for malaria, is a frequent deterrent to human settlement, and that any settlement in such areas can spark off a serious emergence. 5.1.3. Out of Africa: L. d. infantum According to our scenario, the ®rst emergence of L. donovani out of Africa must have occurred shortly after the introduction of the domestic dog. The history of the dog in Africa is very poorly understood owing to the limited archaeological record (J. Clutton-Brock, personal communication). Carnivores normally live at population densities which are too low for them to maintain protozoa transmitted by free-¯ying vectors, but the arti®cially high density of this domestic species allows it to maintain L. d. infantum inde®-

nitely. A subset of strains capable of infecting dogs may have spread from Sudan, through the dog population, to North Africa, southern Europe and much of Asia, establishing populations wherever a suitable vector existed. Recent isolation of stocks from dogs in Sudan supports this conjecture [19]. Strains of this group were incapable of infecting most humans: just a small proportion of infants, and immunosuppressed adults are susceptible, and the parasites are not normally transmissible from humans. Much later, this form was introduced to dogs in South America, where another suitable vector allowed its permanent establishment. In much of the range of L. d. infantum, local carnivores, notably foxes (Vulpes vulpes in Europe; Dusicyon thous in S. America), and wild species of Canis become infected, but can probably not maintain parasite populations inde®nitely [20]. The suggestion that the dog may have acquired leishmaniasis relatively recently may be supported by the fact that it causes serious disease, eventually killing many infected animals. Having been quite common in Mediterranean Europe prior to the introduction of DDT, infantile visceral leishmaniasis almost disappeared in the 1950±1970s. This has been attributed to the use of insecticides for malaria control, but the disease remained a serious problem in dogs, so transmission was not, in fact, greatly affected. More probably, the increasing standards of living in rural areas following the Second World War led to improved nutritional status and better immunity in the children. Nutritional status has been shown to be a major risk factor in the progression from infection to disease [21]. 5.1.4. A second emergence in Europe The disease has re-emerged in recent years, both in Europe and South America, this time in immunosuppressed adults. Most of these have been made susceptible by infection with the HIV virus. Almost 1000 cases of co-infection were reported between 1990 and 1998 in southern Europe alone, so this represents a major emergence. The age distribution of cases corresponds precisely with that of intravenous drug abusers; this and other evidence strongly indicates that transmission occurs through the use of contaminated needles or syringes. The two infections, visceral leishmaniasis and HIV, appear to act synergistically, and patients rapidly develop AIDS-de®ning infections [11]. There is a clear parallel in the initial disappearance associated with improved living standards, followed by reappearance associated with HIV, between visceral leishmaniasis and tuberculosis. 5.1.5. A second emergence from Africa: L. d. donovani The appearance of what was probably L. donovani in India early in the 19th century must represent a second emergence from Africa, which can readily be explained by the slavetrade out of southern Sudan, through the Red Sea port of Sawakin, to the East. On arrival in India it found an ideal

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synanthropic vector, P. argentipes, widely distributed in the valleys of the Ganges and Brahmaputra, and caused a series of devastating epidemics of kala azar which persisted until the advent of DDT. The initial epidemics were in Bengal, and may have started in the 1820s; the subsequent spread to Assam was associated with the opening of that area for tea plantations, and the disease became known as `British Government Disease'. Fluctuating numbers of cases were attributed either to the cyclic development and loss of herd immunity or to natural disasters such as ¯oods and famines. There is scant evidence for either explanation, and it is dif®cult to explain herd immunity in a disease with a reported case-mortality approaching 100%. Following the cessation of malaria control, the disease reemerged in Bihar State around 1976 and spread again, occupying much of its former range in India, Bangladesh and Nepal, but not Assam, where it continues to spread despite (half-hearted) control measures. The infection in the Indian subcontinent is only known in humans and, as the vector, P. argentipes, does not feed on dogs [22], it is unlikely that a secondary zoonosis could be established. 5.2. Leishmania tropica 5.2.1. African origins? Until recently it appeared that L. tropica was a parasite of humans alone, and there was no evidence of a zoonotic origin. Strains isolated in Namibia and Kenya have now been identi®ed as belonging to the diverse complex which attaches to this name [23]. In addition, strains initially named L. killicki are very closely related. All these African strains of L. tropica s.l. are clearly zoonotic. Both the Namibian and Kenyan ones have been isolated from rock hyraxes Procavia capensis and there is evidence that the gundi Ctenodactylus gundi (a rodent with habits resembling those of hyraxes) may be the reservoir host of L. killicki in Tunisia [24]. These zoonotic African forms of L. tropica provide a possible source for the anthroponotic forms which circulate in the cities of west and central Asia. In addition, they provide a source of potentially emergent outbreaks in humans in Africa. L. killicki was the cause of a signi®cant outbreak of CL in Tunisia, and the high incidence of dis®guring lesions in the few people living in the Kenyan foci indicate the potential for serious problems [25]. 5.2.2. Asian anthroponosis Most L. tropica infection in humans occurs in the densely populated cities of west and central Asia, and is transmitted anthroponotically. Occasional infections in dogs certainly do not indicate a role for the dog in maintaining the parasite. Indeed, the epidemiology of urban CL illustrates the dif®culty of maintaining this disease in human populations. Each infected person carries a lesion for about a year and is then immune to further infection. This means that where transmission is intense, most people become immune in a few years, and the only susceptible people are new arrivals,

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by birth or immigration. In the absence of movement of people, the annual incidence of infection cannot exceed the annual birth rate for more than a short time. Maintenance of endemicity depends on transmission among a population most of whom are immune, so requires, in theory, an enormous density of vectors. We have calculated that 50 bites per person per night in the sand-¯y season would be required to maintain endemicity in Kabul, Afghanistan, in the absence of immigration of non-immune people. Such intense exposure to sand-¯y bites is inconsistent with the numbers of sand-¯ies observed: the continued and increasing incidence of the disease in that city since it was ®rst reported in 1960 is only explicable by immigration of large numbers of non-immunes [26]. Long-term endemicity is therefore very uncommon in any one place, and this disease is characterized by the emergence of epidemics that last a few years until most people are immune. Sometimes the epidemics last just long enough for the place to give its name to the disease: Delhi boil, Aleppo boil, Sart sore, Balkh sore, for example. Like measles and other viral infections, urban CL seems to require to be sequentially epidemic in non-immune populations. The bionomics of L. tropica means that control of the infection by reducing transmission may ®rst lead to an increase in the mean age at infection, so increasing its public health importance, then to the loss of herd immunity, followed by massive outbreaks. Measures to reduce transmission could easily do more harm than good! The epidemiology of L. tropica is by no means fully understood. In particular, sporadic cases occur in rural areas and small towns, notably in Jordan, Turkey and Morocco, which cannot readily be explained by local anthroponotic transmission; either these are dependent foci in which the parasite requires repeated introduction, or there may be unidenti®ed zoonotic sources. This idea is supported in Morocco by the greater diversity of L. tropica strains in sand-¯ies than in either dogs or humans [27]. 5.3. Leishmania major 5.3.1. Biblical origins? It is sometimes claimed that the biblical plague of boils could have been CL [28], in which case, L. major is the best candidate causative parasite. This form is almost unknown as an anthroponosis, and characteristically occurs in outbreaks associated with people moving into new areas, whether in response to natural disaster, for military purposes, or in pursuit of development projects. Numerous such outbreaks have been recorded through history, re¯ecting the propensity of this parasite to `emerge' in human populations when conditions are right. 5.3.2. Zoonotic sources In comparison with L. tropica, two features of the bionomics of L. major in man mitigate against it establishing anthroponotic populations: the lesions cure after about 3

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months, and the number of parasites in lesions is much smaller. The short duration of the lesions precludes survival of the parasites through any extended non-transmission season [29]. There are two main centres of zoonotic maintenance of this parasite, one in North Africa and south-west Asia, and the other in central Asia. Each is dependent on a single species of rodent reservoir host, each of which has speci®c ecological requirements which both determine its distribution locally and favour its role as a reservoir host. In North Africa the fat sand-rat Psammomys obesus depends on halophilic vegetation in saline depressions in semi-arid areas, in which high densities are associated with the abundant vegetation. In central Asia the great gerbil Rhombomys opimus lives in areas of highly seasonal climate where the rodents need to build deep complex burrow systems in order to survive the intense winter cold and summer heat. The maintenance of these burrow systems requires that many animals live together. Both rodents, then, occur in locally high densities on substrates suitable for burrowing, particularly on the loess deposits formed on the periphery of the Sahara in North Africa, and blown from the Pleistocene glaciers of central Asia. It is not possible to speculate usefully on which of these reservoir systems represents the origin of the parasite species nor, indeed, whether the ancestral system may have been dependent on one of the currently minor reservoir hosts such as Meriones spp. The outcome is that L. major is very widely distributed north and south of the Sahara, and extends far into central Asia. Its distribution in Asia is limited not by that of Rhombomys opimus, but by the northern limit of the vector, Phlebotomus papatasi.

A tempting explanation may be proposed for the many outbreaks of the second type which occurred in north Africa and Arabia since the ®rst-reported epidemic in Libya in 1974 [31]. The camel is the only competitor with P. obesus for the halophilic plants on which it depends. The replacement of the camel with motor vehicles in much of the relevant area may have led to the ¯ourishing of the vegetation, an increase in rodent numbers, and the extension of the range of both rodent and disease.

5.3.3. Two sources of epidemics In most of this range, human populations are thinly scattered, and residents become infected at an early age when the lesions are barely noticed. There are two main sources of emergence of the disease in large numbers of people. Travellers and immigrants entering zoonotic foci have a high probability of becoming infected, and outbreaks occur in explorers, military personnel and development workers. The other source of outbreaks is when unexplained irruptions of P. obesus extend the range of the rodent to normally unaffected permanent human settlements. The disease can then affect numerous people of all ages, and can be a serious public health challenge. Fortunately, epidemics of CL caused by L. major are self-limiting, due to the rapid build-up of herd immunity. The ®rst type of outbreak is typi®ed by that which affected Fijian troops of the Multinational Force of Observers which monitored the Israeli-Egyptian peace treaty in Sinai [30]. Many of the soldiers based in one particular camp became infected and there was grave concern that they would introduce the infection to Fiji, where sand-¯ies were known to be abundant. Fortunately, Fijian sand-¯ies are ceratopogonids not phlebotomines, and the problem did not arise.

5.4.1. Old World cryptic zoonoses There have been sporadic reports of CL, visceral leishmaniasis, and aberrant mixed forms from various places in Africa, including Tanzania, Zambia and Malawi. None of the causative parasites has been isolated or identi®ed. Supposing that these were not imported cases, it is clear that various forms of leishmaniasis are widespread in tropical Africa. These must be zoonoses, but the reservoir systems in which they survive are unknown. With changes in environment and human distribution, such zoonoses should be taken seriously as potentially emergent infections. The Gulf War produced a small number of mysterious cases of leishmaniasis in soldiers who presented with various atypical symptoms. The parasites, isolated from the viscera, were identi®ed as L. tropica [33]. There is, in this author's opinion, no conclusive evidence that L. tropica causes visceral infection in humans: each of the several reports is suspect for one reason or another. Further, L. tropica is not known to be endemic in the areas said to have been visited by the soldiers. This ®nding may, however, indicate the possible existence of populations of one or more Leishmania species, which must be zoonotic, which are capable of infecting humans, of whose ecology

5.3.4. Ephemeral anthroponosis? There has been one outbreak of L. major infection which can best be explained by anthroponotic transmission. This occurred in and around Khartoum in Sudan, in the late 1980s, with many thousands of cases [32]. CL had previously been unknown in the city, though the vector was known to be abundant. A potential reservoir host, the grass rat Arvicanthis niloticus, is abundant in the affected suburbs, but the parasite was apparently absent. The outbreak commenced at a time when large numbers of people were displaced by famine, from the west of the country where L. major is endemic, and many of these were employed as labourers on Tuti Island where the epidemic started. The most parsimonious explanation of this epidemic is that the parasite was introduced by these people and was spread by human-¯y-human transmission; it was then able to survive for a few years because of the short non-transmission season and the large number of nonimmune people. 5.4. Unidenti®ed Leishmania species

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we are completely ignorant. Again, it is important to be aware of these as potential sources of emerging disease. 5.5. Cutaneous leishmaniasis in the Americas Characteristically, L. braziliensis infection occurs sporadically in people whose activities take them to sparsely inhabited forested areas. The initial cutaneous lesion and subsequent mucocutaneous destruction are occupational hazards of military trainees, settlers, miners and explorers in much of the Amazon basin. Although this must be a disease of zoonotic origin, and the parasites have been isolated from several wild animals, the reservoir host(s) is unknown, as is the structure of the reservoir system. Both L. guyanensis and L. panamensis are similarly characteristic of forest habitats. These two species both seem to be maintained by sloths (Edentata: Bradipodidae), and have been isolated from various other wild animals of the forest. L. mexicana and L. amazonensis are less frequently isolated from humans, but are also predominantly forest species, in this instance maintained by forest rodents, though the former species extends into semi-arid `mesquite' scrublands in northern Mexico and Texas where it is maintained by the wood rat Neotoma micropus [34]. 5.5.1. Domestication of L. peruviana The possibility that some of these parasites have the potential to establish synanthropic populations is illustrated by L. peruviana. This is the cause of uta, the name given to CL in the high arid valleys of the western slopes of the Peruvian Andes. In the northern part of its range, where the two species are possibly sympatric, L. peruviana is barely distinguishable from L. braziliensis, but further south, where the two forms are separated by the high Andes, the differences increase [35]. It is supposed that L. peruviana was once a sylvatic zoonosis, but that with the deserti®cation of the hillsides, the original host has disappeared, and the parasite is restricted to humans and dogs, and is transmitted exclusively in peridomestic environments. The illustration of CL on ancient Peruvian pottery shows that these changes may have occurred long ago. 5.5.2. Domestication (and possible export) of L. braziliensis Similar changes are thought to be in progress currently, for L. braziliensis and possibly L. panamensis. Humans and domestic animals have been found infected with the former, in northern Argentina, southern Brazil, Bolivia, Colombia and Venezuela, in areas of extensive deforestation. The structure of these apparently new foci, in particular the relative importance of different mammalian hosts, is poorly understood. As mentioned below, forest clearance has been suggested as a measure to protect new settlements against both L. braziliensis and L. guyanensis infection, but this is hardly likely to be successful if peridomestic cycles can become established [36]. Numerous soldiers of the Ghurka regiment on jungle

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training exercise in Belize contracted CL, some caused by L. braziliensis and some by L. panamensis [37]. There is no evidence that these infections could be transmitted in their Nepalese homeland, but vector species of sand-¯y do occur there and if, as suggested above, the Leishmania species can be transmitted peridomestically, the risk should be considered. 5.6. Other New World scenarios In addition to these relatively well-known causes of CL, numerous other species have been described, mostly from forest animals, which cause occasional cases of CL in humans, whose ecological requirements are poorly known. A distinctive and as yet unnamed parasite, initially thought to be a Herpetomonas species, has been found twice in humans on the island of Martinique. At least one of the patients had concurrent HIV infection [38]. If it were true that HIV infection can lead to susceptibility to monoxenous parasites of insects, the possibilities would be endless for emergence of new disease in these people. The source of the small number of cases of diffuse CL in the Dominican Republic remains unknown: only two species of sand-¯y are known, and the endemic mammals are very scarce [39]. A cryptic zoonotic source must exist, from which further emergences are possible. In the remaining forests of Trinidad, a form close to L. mexicana is maintained in rodents, but human cases are unknown [40]. 6. Surveillance and control There has rarely been any systematic attempt to control transmission of any of the leishmaniases and even more rarely any effective evaluation, so there is no standard procedure for the control of any leishmaniasis. The minimal control measures recommended by WHO for all forms of the disease are the establishment of ef®cient passive case detection, and treatment where indicated. This must be accompanied by an ef®cient reporting and recording system. Active case detection is indicated in areas with poor health services, or during spreading epidemics [7]. Although CL is usually self-curing and not life-threatening, individual cases may be psychologically and socially damaging, and epidemics may be seen as a major public health priority. Attempts to control urban, anthroponotic, CL have used house spray with various insecticides. While this should be effective, and was effective in Soviet Azerbaijan in the 1950s, more recent efforts have been inadequately thorough, and have failed. Zoonotic CL has been controlled in Soviet Central Asia, by deep ploughing of colonies of the rodent reservoir host, or by poisoning. Attempts to do the same in Arabia and North Africa, where there is a different reservoir host have given ambivalent results [41]. In South American foci, control is even more dif®cult. Clearance of forest surround-

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ing new settlements has been effective, but contrary to earlier expectations, forest clearance has sometimes led to peridomestic transmission rather than to the elimination of the infections [42]. In the Indian region, visceral leishmaniasis should be controllable by house spray, as the vector is strictly synanthropic. Indeed, it is claimed that the disease almost disappeared during the 1960s as a result of malaria control measures. Half-hearted attempts to control more recent outbreaks of visceral leishmaniasis in this area have not been successful. Visceral leishmaniasis in Africa, on the other hand, is much more dif®cult to control as the vectors are sylvatic and the disease occurs in irregular epidemics in impoverished peripheral communities. The best that can be done in these circumstances is to ensure the availability of diagnosis and cure with, perhaps, the provision of ®ne-mesh bed nets for individual protection. There is no effective vaccine for any leishmaniasis, despite considerable effort. Trials have been conducted on vaccines against L. major infection in Iran with equivocal results [43], and a product has been tested for protection against L. braziliensis infection in Brazil. Deliberate infection, or leishmanisation is mentioned above. This procedure was abandoned by the Russian and Israeli armies due to the occurrence of a small number of aberrant, intractable lesions, but was carried out on millions of Iranian military recruits during the war with Iraq [44]. Improved methods of standardising both the strain of parasite and its stage of development could well eliminate the problem of aberrant disease, and it seems this method of protection could well be developed further. Control of transmission of canine leishmaniasis has two potential purposes: to reduce the likelihood of human disease and to protect the dogs themselves. The human disease is usually very rare and is closely dependent on age and nutritional state. Improved lifestyles in southern Europe since the 1950s have almost eliminated the infantile disease. It is normally preferable, and, in the long term, more economical to alleviate human deprivation, and to identify and treat patients, than to attempt to control transmission. The most important action in the protection of people from this canine zoonosis is to ensure that all human infections can be correctly and rapidly diagnosed and treated. This is a far from trivial question even in endemic areas and in exotic areas, correct diagnosis is the exception rather than the rule. Human infection with L. d. infantum was effectively eliminated in China in the 1950s by the almost complete elimination of all dogs. This has not been attempted elsewhere, and the detection and elimination of infected dogs has produced ambivalent results in Brazil. In areas of high transmission, protection of individual dogs is very dif®cult, and protection of the dog population is out of the question. Elimination of sick dogs is ineffective, as many dogs are infectious before they show symptoms, and valuable working dogs or pets will be withheld [45]. The relaxation of quarantine restrictions on animals enter-

ing UK is likely to lead to increasing numbers of dogs visiting endemic areas in southern Europe. This will inevitably lead to the occasional importation of cases of canine leishmaniasis. Increased awareness among veterinary practitioners in the UK is being promoted in advance of this problem [46]. 7. Conclusion The 20 or so species of Leishmania which have been recorded as causing human infections are all either zoonotic, or have recent zoonotic origins which can be postulated with some con®dence. The distribution of each one is determined by those of its vector, its reservoir host, or both. This concatenation of limiting factors leads to speci®c environmental requirements and focal distribution of zoonotic or anthroponotic sources. Human infection is dependent on the ecological relation between human activity and reservoir systems. Any change in the environmental factors is likely to lead to a change in the distribution of the parasite. Examples have been given of changes in distribution, and the emergence of leishmaniasis, from the distant past to the present, and can be postulated for the future. The leishmaniases therefore present an excellent model for emerging disease in general, and for the generation of principles governing emergence. The emergences have been provoked, or at least facilitated, by the adoption of new, secondary reservoir hosts, the adoption of new vector species, transport of infection in man or domestic animals, invasion by man of zoonotic foci, irruption of reservoir hosts beyond their normal range and, in one instance, by changes in the human host's susceptibility to infection. These factors have frequently been associated with environmental change, and have sometimes combined to establish quite new systems whose duration has ranged from ephemeral to inde®nite. The model is mainly limited by gaps in our knowledge, usually quantitative, sometimes qualitative, of the structure of reservoir systems. In all known primary reservoir hosts infection with Leishmania is chronic and without signi®cant morbidity. In humans and secondary reservoir hosts, none of the diseases is rapidly fatal, though epidemics can reduce populations, and even possibly limit their distribution. The chronic nature of the infections contrasts with most `emergent' bacterial or viral diseases. References [1] Cox FEG, Kreier JP, Wakelin D, editors. Topley and Wilson's microbiology and microbial infections, 9th ed., vol. 5. Parisitology, London: Arnold, 1998. [2] Dedet J-P, editor. Les leishmanioses. Paris: Ellipses, 1999. [3] Dowlati Y, Modabber F. Cutaneous leishmaniasis. Clin Dermatol 1986;14:417±546. [4] Gilles HM, editor. Protozoal diseases. London: Arnold, 1999.

R.W. Ashford / International Journal for Parasitology 30 (2000) 1269±1281 [5] Oumeish YO, Parish LC. Leishmaniasis. Clin Dermatol 1999;17:245± 344. [6] Palmer SR, Soulsby L, Simpson DIH, editors. Zoonoses: biology, clinical practice, and public health control. Oxford: Oxford University Press, 1998. [7] World Health Organization. Control of the Leishmaniases Report of a WHO Expert Committee. Geneva: World Health Organization, 1990. [8] Ali A, Ashford RW. Visceral leishmaniasis in Ethiopia. IV. Prevalence, incidence and relationship between infection and disease in an endemic area. Ann Trop Med Parasitol 1994;88:289±93. [9] Sacks DL, Perkins PV. Identi®cation of an infective stage of Leishmania promastigotes. Science 1984;223:1417±9. [10] Schlein Y, Jacobson RL. Mortality of Leishmania major in Phlebotomus papatasi caused by plant feeding of sand¯ies. Am J Trop Med Hyg 1994;50:20±27. [11] World Health Organization. Leishmania/HIV co-infection southwestern Europe 1990±1998, WHO/LEISH/2000.42. Geneva: World Health Organization, 2000. [12] Ashford RW, Desjeux P, de Raadt P. Estimation of population at risk of infection and numbers of cases of Leishmaniasis. Parasitol Today 1992;8:104±5. [13] Yoshida ELA, Correa FMA, Marques SA, et al. Human cutaneous leishmaniasis due to Leishmania braziliensis in the south-west region of Sao Paulo State. Brazil. Mem Inst Oswaldo Cruz 1990;85:133±4. [14] Corbet GB, Hill JE, editors. A world list of mammalian species. London: British Museum (Natural History), 1980. [15] Ashford RW, Seaman J, Schorscher J, Pratlong F. Epidemic visceral leishmaniasis in southern Sudan: identity and systematic position of the parasites from patients and vectors. Trans R Soc Trop Med Hyg 1992;86:379±80. [16] Elnaiem DA, Ward RD, Hassan HK, Miles MA, Frame IA. Infection rates of Leishmania donovani in Phlebotomus orientalis from a focus of visceral leishmaniasis in eastern Sudan. Ann Trop Med Parasitol 1998;92:229±32. [17] Seaman J, Ashford RW, Schorscher J, Dereure J. Visceral leishmaniasis in southern Sudan: status of healthy villagers in epidemic conditions. Ann Trop Med Parasitol 1992;86:481±6. [18] Ashford RW, Thomson M. Visceral leishmaniasis in Sudan: a delayed development disaster? Ann Trop Med Parasitol 1991;85:571±2. [19] Dereure J, Boni M, Pratlong F, et al. Visceral leishmaniasis in Sudan: ®rst identi®cations of Leishmania infantum and L. archibaldi from dogs. Trans R Soc Trop Med Hyg 2000;94:154±5. [20] Ashford RW, Snowden KF. Protozoan parasites. In: Meslin FX, Macpherson CNL, editors. The dog in health and disease. Wallingford: CABI, 2000 in press. [21] Badaro R, Jones TC, Lorenco R, et al. A prospective study of visceral leishmaniasis in an endemic area of Brazil. J Infect Dis 1986;154:639±49. [22] Addy M, Mitra AK, Ghosh KK, Hati AK. Host preference of Phlebotomus argentipes in different biotopes. Trop Geogr Med 1983;35:343±5. [23] Grove SS. Leishmaniasis in South West Africa/Namibia to date. S Afr Med J 1989;75:290±2. [24] Ben Ismail R. Rapport de fonctionnement: laboratoire d'eÂpideÂmiologie et d'eÂcologie meÂdicale. Arch Inst Pasteur Tunis 1994;71:86±107. [25] Sang DK, Njeru WK, Ashford RW. A zoonotic focus of cutaneous leishmaniasis due to Leishmania tropica at Utut, Rift Valley Province, Kenya. Trans R Soc Trop Med Hyg 1994;88:35±37. [26] Ashford RW, Kohestany KA, Karimzad MA. Cutaneous leishmania-

[27] [28] [29] [30] [31] [32]

[33] [34] [35] [36] [37] [38] [39] [40] [41] [42]

[43]

[44]

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