2

2.7. Incubation Period and Transmission :
Leishmania spp. is usually transmitted indirectly between hosts by sand flies of the genera Phlebotomus and Lutzomyia, which are biological vectors. Each species of Leishmania is adapted to transmission in certain species of sand flies.
Sandfly activity occurs when it is humid, and there is no wind or rain. These insects are usually most active at dawn, dusk and during the night, but it will bite if they are disturbed in their hiding places (animal burrows, holes in trees, caves, houses and other relatively cool, humid locations) during the day. They are attracted to light and may enter buildings at night (70).
(71) revealed that the other arthropods including ticks (Dermacentor variabilis and Rhipicephalus sanguineus) and canine fleas may also act as mechanical vectors for Leishmania. Where sand flies transmit Leishmania spp., ticks and fleas are probably not important in the epidemiology of the disease; however, they might be involved in rare cases of dog-to-dog transmission in other locations.

Mammals can be infected asymptomatically for long periods, and they often remain chronically infected even after clinical cure. Subclinically infected animals can transmit Leishmania spp. to sand flies. These parasites have also been transmitted via blood transfusions in people and dogs, and by transplacental transmission in dogs, mice and humans (72). In canine leishmaniasis caused by L. infantum, the parasites can sometimes be found in saliva, urine, semen and conjunctival secretions, as well as in blood (73).

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Epidemiological investigations in the U.S.A. also suggested that L. infantum has been transmitted directly from dog to dog, although sand fly mediated transmission or other arthropod-born transfer has not been ruled out (74). In contrast, sand flies are thought to transmit the disease to people from wild mammals. The risk of direct transmission from infected dogs to humans is unknown (75).

In Iraq, (76) suggested that Ph. sergenti might be the vector, while in the USA (77) has suggested that both Ph. papatasi and Ph. alexandri might be the vector.
2.8. Clinical classification of leishmaniasis :
There are six species of Leishmania recognized to cause disease in humans, they are very similar morphologically but produce strikingly different pathological responses (78) (Table,2.2).

(Table,2-2): Leishmania Species Pathogenic for Humans, their Vectors, Host Range and Disease manifestations (78).
Species Host range Main vector Disease manifestations
L. tropica Humans Phlebotomus
sergenti Cutaneous leishmaniasis (urban, dry Oriental sore)
L. donovani Dogs, savannah

rodents, humans Phlebotomus
argentipes, Lutzomyia longipalpis Visceral leishmaniasis

(kala-azar), PKDL
L. major Desert and savannah
rodents; Rhombomys, Psammomys, Arvicanthis Phlebotomus

papatasi Cutaneous leishmaniasis,
(rural, wet Oriental sore)
L. braziliensis

Complex Sloth, dog Lutzomyia
umbratilis
and many others Cutaneous leishmaniasis,
Mucocutaneous Leishmaniasis
L. aethiopica Rock hyrax Phlebotomus
longipes Cutaneous leishmaniasis,
Diffuse cutaneous leishmaniasis

L. Mexicana Complex

Forest rodents Lutzomyia
flaviscutellata,
Lutzomyia olmeca

Cutaneous leishmaniasis,
Diffuse cutaneous leishmaniasis

a- Skin ulcer b- DCL c- MCL

d- VL e –PKDL
Figure (2-4): Clinical classification of leishmaniasis (Medical Books Online; 79, 58)

2.8.1 Cutaneous Leishmaniasis :
People can carry some species of Leishmania asymptomatically for
long periods, without becoming ill. In humans, the reported incubation period for cutaneous leishmaniasis can be as short as 1-2 weeks or as long as several months when it is caused by New World species. (80).
Cutaneous Leishmaniasis manifests itself starting from small erythematous papules through nodules and to ulcerative lesions. Unusual clinical manifestations are sporotrichoid patterns, i.e., subcutaneous nodules developing along lymphatics, and hyperkeratosis, i.e., thick adherent scale, as well as leishmaniasis recidivans, also known as lupoid leishmaniasis (81). In the Middle East, it was very difficult and even impossible to discern by the clinical picture whether cases were caused by L. major or L. tropica (82).
( 83) revealed that the Cutaneous leishmaniasis (CL) is a worldwide public health and asocial problem in many developing countries. It can affect the skin and mucous membranes and is caused by different Leishmania species widely spread in the New and Old World (the Eastern Hemisphere). Old World cutaneous leishmaniasis (OWCL) is present in many endemic areas in North Africa, Mediterranean, Middle East, Indian subcontinent and Central Asia. The species responsible for OWCL are mainly L. major and L. tropica. L. infantum and L. donovani can also cause localized cutaneous leishmaniasis but are observed less frequently in the Mediterranean areas. Diffuse CL is uncommon and is caused by L. aethiopica in Africa, with regard to transmission; OWCL is divided into two main groups:
Anthroponotic cutaneous leishmaniasis (ACL), when the parasite is transmitted from person to person and is generally characterized by large and/or multiple cutaneous lesions with a variable tendency to self-care. It is an urban disease and is geographically distributed in the Middle East, the Indian Subcontinent, and Western Asia. ACL is mostly caused by L. tropica. Lesions may persist for 6 to 15 months before healing with significant scarring. Cutaneous leishmaniasis has been also categorised into four different clinical forms (84).
Zoonotic cutaneous leishmaniasis (ZCL), when the parasite is transmitted from a range of animals to humans. It is seen in rural areas and is geographically distributed in the Middle East, Northwestern China and North Africa. ZCL is mostly caused by L. major and often heals spontaneously in about two to four months if not contaminated, although in some cases it may persist for as long as five years (83).

2.8.1.1. Localized :

In the localized form the parasite is confined to the skin. After an incubation period of 1- 12 weeks a papule or bump develops at the site of the insect bite. The papule grows and turns into an ulcer. A typical lesion of the localized form of CL is a painless papule or ulcer covered with an adherent crust of dried exudates. Most people with CL have one or two lesions varying in size from 0.5 to 3cm in diameter, usually occur on exposed parts of the body such as the face, arms or legs. There is, however, considerable variation: people may have as many as 200 simple skin lesions; some lesions grow but do not ulcerate (nodules), and some Leishmania species also infect the lymphatic system producing lesions along the lymphatic channels (nodular lymphangitis). Secondary bacterial infection is common, causing pain and serious disability. Most lesions heal spontaneously over months or years, leaving permanent scarring with skin thinning. Scarring of leishmaniasis is typical with a de-pigmented centre and a pigmented border (85).Figure 2.4.a.
2.8.1.2. Diffuse leishmaniasis :
Diffuse cutaneous leishmaniasis (DCL) produces disseminated and chronic skin lesions resembling those of lepromatous leprosy. It is difficult to treat(86), Diffuse leishmaniasis affects only the skin but with generalized skin lesions. It is seen mainly in Africa transmitted by L. aethiopica (83) Figure 2.4.b.
2.8.1.3. Recidivans :
In Leishmaniasis recidivance (LR) which is a relapsing form of oriental sore. Its frequently is about 1% of all L. tropica lesions. The lesions continue to spread over a period of many years with partial healing (44).
2.8.1.4. Mucosal leishmaniasis :
In mucosal leishmaniasis, the parasite may spread to the mucous membranes, especially those of the nose, mouth and throat, and cause extensive damage and disfiguration (Figure 2-4.c). It is seen mainly in South America but it can also be caused by species from Old World countries including L. tropica, L. major and L. infantum. Leishmaniasis is a seasonal disease in temperate regions. Infections are acquired in the warmer months when sand flies are active (6).
Cutaneous leishmaniasis is rarely fatal. This form of leishmaniasis often heals spontaneously, although some lesions may persist for long periods or leave scars. The mucocutaneous form caused by L. braziliensi L. panamensis or L. guyanensis rarely heals spontaneously and is disfiguring. nasopharynx can be fatal (87).

2.8.2 Visceral leishmaniasis:
Visceral leishmaniasis, or Kala-azar, is associated with prolonged fever, splenomegaly, hepatomegaly, substantial weight loss, progressive anaemia, pancytopenia, and hypergammaglobulinemia. It can be impaired by serious bacterial infections and is usually fatal if left untreated (Figure 2.4.d). A serious late sequel to Kala-azar is post-Kala Azar dermal leishmaniasis (PKDL) which appears within months or years of the cure of VL (88 ).
(89) said that the Post kala-azar a dermal leishmaniasis is a form of diffuse cutaneous leishmaniasis and a sequel of visceral leishmaniasis that may appear in affected individuals up to 20 years after the being partially treated, untreated or in those considered adequately treated (Figure 2.4.e).

2.8.3 Mucocutaneous leishmaniasis :
Mucocutaneous leishmaniasis (MCL) is a severe form of CL, as it produces disfiguring lesions and mutilations of the face, nose and throat (85). They commonly appear in the mouth and nose where they erode underlying tissue and cartilage (90). (85) revealed that if the lesions spread to the roof of the mouth and the larynx, they may prevent speech. Beside other systemic symptoms include fever, weight loss, and anaemia. There is always a substantial danger of bacteria infecting the already open sores (Figure 2.4.c).

2.9. Immunology of leishmaniasis :
(91) showed that the clinical and experimental evidence indicates that vector, parasite, and host factors all influence the evolution and outcome of leishmanial infection. The failure of the vertebrate host to control the infection is apparently related to two major factors:
The ability of some strains of Leishmania spp. to resist the microbicidal effect of activated macrophages, and The breakdown of host-protective cellular immune responses.
As obligate intracellular parasites, these microorganisms have evolved complex strategies for evading host defence mechanisms that occur before the complement-mediated killing process, during the toxic effect of oxygen metabolites produced during the macrophage-induced respiratory burst, and after the non-oxidative killing effect of lysosomal hydrolysis or nitric oxide from L-arginine entry into host cells (92).
The immune responses to Leishmania spp infection are enormously complex and while they may accelerate healing, some responses exacerbate the disease depending on the particular circumstances (93). Immune response outcomes are contingent on the genetic variation in the mammalian host, genetic variation in the parasites between species, and chance factors such as the inoculum size, location, and a number of infective bites received (94,14).
Studies of several membrane surface enzymes and other macromolecules synthesized by leishmanial parasites have provided new insights about their functions in the establishment of infection, virulence determinants which are responsible for evasion of such defences are often developmentally regulated, allowing these organisms to survive in the immunologically hostile environment of the host (95).

2.10. Genomic variation of Leishmania :
The genomes of Leishmania spp. vary from 29 Mb in L. amazonensis; (96) to 33 Mb in L. major, L. tropica and L. braziliensis (97 ) and are organised into a variable number of chromosomes (i.e., 34 in L.amazonensisand , L.mexicana , 35 in L. brasiliensis , 36 in L.major, L.donovani and L.tropica) (98).
Despite the striking variability in pathogenicity and tissue tropism of different Leishmania species, their genomes are remarkably similar, displaying a high degree of conservation in gene content and architecture. The genomes of Leishmania spp. are characterised by a high gene density, the presence of long arrays of polycistronic gene clusters and almost complete absence of introns (99). However, careful examination of protein-coding genes in Leishmania allowed the identification of a relatively small number of species-specific genes, the majority of which encode predicted proteins of unknown function
( 100). Several transcriptomic studies have investigated the Leishmania-induced regulation of gene expression in infected tissues with the aim to link such responses to disease outcome (101,102).
(103) revealed that the Leishmania spp. And other trypanosomatids have unique genomic organization traits compared with eukaryotes, such as genes without introns, polycistrons and small chromosomes with high gene densities. Moreover, these ?agellates possess a single mitochondrion called the kinetoplast, which contains a large network of kinetoplast DNA (kDNA).

2.10.1 Chromosomal DNA :
2.10.1.1. Ribosomal DNA (rDNA):
The ribosomal RNA (rRNA) genes are located mostly on chromosome 27, usually as multiple copies of tandem head-to-tail repeats of approximately 12.5 Kb (104). Among the different components of these genes, the most variable ITS regions are ideal for species-typing (105,106). Like most eukaryotes, large ribosomal subunit (LSU) is composed of 28S, 5.8S and 5S rRNAs, whereas the small subunit (SSU) contains 18S rRNA (107 ).

2.10.1.1.1. Ribosomal RNA transcription units :
The 18S rRNA is a structural RNA of the ribosomal SSU. The high conservation of this gene and its ?anking regions make it suitable for reconstructing phylogenetic relationships.
The 5.8S rRNA is a non-coding component of the ribosomal LSU and is part of the 45S precursor that also contains the 18S and 28S rRNAs. The 28S rRNA acts as a ribozyme, catalysing peptide bond formation. Leishmania species are atypical in its composition, as they contain two large (24Sa and 24Sb) and four small rRNA molecules (108).

2.10.1.1.2. Internal transcribed spacer :
The internal transcribed spacer (ITS) refers to the non-coding spacer DNA located between the SSU and LSU rRNAs. The ITS1 region ranges from 50 to 350 bp and is located between the 18S rRNA and 5.8S rRNA genes (109). It has sufficiently high conservation to be a Leishmania PCR target but its polymorphism allow species typing, such as differentiating L. aethiopica, L. tropica, L. major, L. turanica and the L. donovani complex, L. mexicana, L. amazonensis, L. guyanensis, L. braziliensis (105,109, 110, 111, 112). The ITS2 region is of 50 to 650 bp-long and is located between the 5.8S rRNA and LSU rRNA genes. Ampli?cation of ITS2 with generic PCR primers revealed substantial differences between the Old and New World Leishmania spp. but also between species complexes and species of these subgenera (106,109).