**3.2. Vectors**

Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) are insects of medical and veterinary importance since they are involved in the transmission of diverse pathogens [24]. The blood-feeding females are usually considered as the only natural vectors of protozoan *Leishmania* species. Among the phlebotomine sand flies recorded in the New World, 56 species, all belonging to the genus *Lutzomyia*, are involved in the transmission of the *Leish‐ mania* spp. [25].

To determine the transmission of leishmaniasis in south-eastern Mexico, vectors were captured near La Libertad, municipality of Escárcega, Campeche (<200 m) and in a medium-sized subperennial forest located 8 km southeast of this village. Near La Libertad, nine sand flies species were collected using CDC light traps, a Shannon trap and a mouth aspirator; the most abundant species were *Lutzomyia deleoni* (61.3%) and *Lu. cruciata* (13.7%). The highest number of sand flies was obtained with CDC traps (46.5%) followed by direct searching in natural shelters (37.0%) and in the Shannon trap (16.5%). The highest peak of abundance of *Lu. cruciata* was obtained in March. The population peak of *Lu. cruciata* was related to the low temperature (21– 22°C), high levels of humidity (≥80%) and low rainfall. The anthropophilic species like *Lu.cruciata, Lu. ovallesi, Lu. panamensis, Lu. shannoni* had the lowest densities around the village. In contrast, at 8 km southeast of La Libertad in the forest, 16 species were caught using CDC light traps and a Shannon trap. The most abundant species were *Lu. olmeca* (21.7%), *Lu. cruciata* (19.2%), and *Lu. ovallesi* (14.1%). *Lu. olmeca* had the highest leishmania infection rate of all sand flies collected in the forest. In both study areas, more females than males were captured. The low densities of anthropophilic sand flies species captured near the village and the abundance of *Lu. olmeca* females (zoophilic species) suggested the sylvatic transmission of *Leish‐ mania* in the Yucatan peninsula [26, 27].

Further entomological studies were carried out at 150 km, east of La Libertad in the village of La Guadalupe and the nearby village of Dos Naciones, municipality of Calakmul, Campeche. Using Shannon traps, Disney traps and CDC light traps, 15 sand fly species (*Brumptomyia* and *Lutzomyia*) were caught. In both study locations, the number of sand fly species caught was very similar but the predominant species differed. In the Dos Naciones village, *Lu. panamensis* (1682), *Lu. ovallesi* (504), *Lu. cruciata* (332), and *Lu. olmeca* (329) had the highest abundance; while in La Guadalupe *Lu. cruciata* (754) and *Lu. olmeca* (244) were most abundant. In both locations, the numbers of sand flies attracted to Shannon traps peaked between 18.00 and 22.00 hours. Given the abundance *of Lu. olmeca* in the collections made with Shannon and Disney traps (it was the only species caught in the latter), this species was confirmed as the primary vector of *L. (L.) mexicana* in Calakmul county. Dos Naciones and La Guadalupe differ in terms of vegetation structure, with much more severe and extensive deforestation around La Guadalupe than around Dos Naciones. Destruction of habitats by humans has led to the decrease in sand fly diversity and abundance. However, some species of medical importance have adapted to deforested habitats becoming closely associated with human settlements. Moreover, there is evidence that the deforestation could increase domestic transmission of *Leishmania* parasites [28].

Although the abundance of sand fly vectors was determined in three foci, the species of *Leishmania* in vectors had yet to be determined. Using the PCR method, kDNA of the parasite was amplified from sand flies collected in the forest. Because only two species of *Leishmania* have been reported in the Yucatan Peninsula, specific primer for *Leishmania* genus, namely 13 A (GTGGGGGAGGGGCGTTCT) and 13 B (ATTTTACACCAACCCCCAGTT), B-4 (TCGTACTCCCCGACATGCCTC) for the subgenus *Viannia*, and M1.1 (CCAGTTTC-GACCGCCGGAGC) for the subgenus *Leishmania* were used. Both *Lu. olmeca* and *Lu. cruciata* were found infected by *L. (L.) mexicana* [23].

#### **3.3. Reservoirs**

disease in a given area, is of the same species as that found in suspected mammalian reservoirs

Based on both the clinical and epidemiological features of the disease, as well as on the biological characteristics of the parasite in laboratory animals [19–21], *L. (L.) mexicana* Biagi, 1953 emend. Garnham, 1962, was considered the main agent of LCL in the Yucatan Peninsula. Nevertheless, its characterization at the genus and sub-genus level was not done in those

Therefore, from January 1990 to July 1992, 153 patients with LCL determined by both clinical diagnosis and parasite visualization (smear, biopsy and/or isolation-culture) were studied. All of them were infected in the state of Campeche. Parasite isolation by needle aspirates taken from the edge of the lesions was positive in 49%. Isolates were characterized by isoenzyme markers (glucose phosphate isomerase, mannose phospate isomerase, nucleoside hydrolase, phosphoglucomutase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase). Seventy (93.3%) were identified as *L. (L.) mexicana* Biagi, 1953 emend. Garham, 1962 and 5 (6.7%) as *Leishmania (Viannia) braziliensis* Viannia 1911 emend. Matta, 1916 [22]. Later on, a study to identify *Leishmania* parasites isolated from humans and wild rodents from the state of Campeche, using IFA with Mabs was carried out. The main purpose was to determine if the parasites of both types of hosts were of the same biotype. All the isolates obtained from wild rodents reacted with monoclonal antibodies M7 and M8 and were thus identified as *L. (L.) mexicana*. No differences in reactivity patterns were found among the

Finally, to assure that *L. (L.) mexicana* is the main agent causing LCL in the state of Campeche, a study leading to the identification by PCR of *L. (L.) mexicana* in the potential vectors *Lutzomyia*

Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) are insects of medical and veterinary importance since they are involved in the transmission of diverse pathogens [24]. The blood-feeding females are usually considered as the only natural vectors of protozoan *Leishmania* species. Among the phlebotomine sand flies recorded in the New World, 56 species, all belonging to the genus *Lutzomyia*, are involved in the transmission of the *Leish‐*

To determine the transmission of leishmaniasis in south-eastern Mexico, vectors were captured near La Libertad, municipality of Escárcega, Campeche (<200 m) and in a medium-sized subperennial forest located 8 km southeast of this village. Near La Libertad, nine sand flies species were collected using CDC light traps, a Shannon trap and a mouth aspirator; the most abundant species were *Lutzomyia deleoni* (61.3%) and *Lu. cruciata* (13.7%). The highest number of sand flies was obtained with CDC traps (46.5%) followed by direct searching in natural shelters (37.0%) and in the Shannon trap (16.5%). The highest peak of abundance of *Lu. cruciata* was obtained in March. The population peak of *Lu. cruciata* was related to the low temperature (21– 22°C), high levels of humidity (≥80%) and low rainfall. The anthropophilic species like

different strains of *L. (L.) mexicana* from humans and wild rodents [22].

*olmeca* and *Lutzomyia cruciata* was performed [23].

and insect vectors, is very important [18].

142 The Epidemiology and Ecology of Leishmaniasis

studies.

**3.2. Vectors**

*mania* spp. [25].

Leishmaniases is a complex of zoonotic diseases, which are infections transmitted from animals to humans. Identifying a reservoir of such zoonosis requires extensive ecological, entomological, mammalian, parasitological and epidemiological studies. The World Health Organization enumerated five criteria to incriminate a primary reservoir [17]. Thus, a step-by-step method is needed to investigate a primary reservoir of a zoonosis.

The first step is to identify animals that harbour the parasite. The first attempt to find leishmanial hosts close to the Yucatan Peninsula was carried out in Belize in the early 1960s [29–31]. However, the south of Belize has to be considered as a different endemic area of the disease since this area is not part of the Biotic Province of the Peninsula of Yucatán [32]. In the Yucatan Peninsula, *L. (L.) mexicana* was identified by PCR in the base of tails of two heteromid rodents *Heteromys desmarestianus* Desmarest, 1817 and *Heteromys gaumeri* J.A. Allen and Chapman, 1897; and five cricetid rodents *Ototylomys phyllotis* Merriam, 1901, *Peromyscus yucatanicus*J.A. Allen and Chapman, 1897, *Oryzomys melanotis* Thomas 1893, *Sigmodon hispidus* Say and Ord, 1825, *Reithrodontomys gracilis* J.A. Allen and Chapman, 1897; and one marsupial *Marmosa mexicana* Merriam, 1897 [18, 33, 34]. Thus, many small terrestrial mammals of the Peninsula seemed to be able to harbour the parasite. However, the sand‐fly vector is absent from the driest and unstable habitats such as corn‐ and bean‐fields [27]. Due to *Sigmodon*'s preference for those disturbed areas, this species is not a potential reservoir of *L. (L.) mexicana* in the Yucatan Peninsula. The endemic area of *L. (L.) mexicana* is limited to the humid medium‐ size forest (height < 12 m), which characterizes southern Campeche and Quintana Roo states and the low‐stature forest (height < 8 m) from eastern and southern Yucatan state and northern Campeche. This area occupies 19,839 km2 [16].

The second step to identify a primary reservoir is that the species, which is relatively abundant in the focus to provide a food source for sand flies. In southern Campeche, *H. gaumeri* was the most abundant rodent in four of the five studied foci (total 46%), followed by *O. phyllotis* (19%) and *P. yucatanicus* (13%). Hence, the least abundant species such as *H. desmarestianus, Oryzomys* and *Reithrodontomys* as well as the marsupial *Marmosa* cannot be primary reser‐ voirs of *L. (L.) mexicana* in the Yucatan Peninsula [34]. Because of the geographic isolation of the Yucatan Peninsula, two species of rodents exist only in the Yucatán Peninsula, the Gaumer's spiny pocket‐mouse, *H. gaumeri* and the Yucatán deer‐mouse, *P. yucatanicus*; two of the three potential reservoirs of *L. (L.) mexicana* in the area [32].

In the Yucatan Peninsula, a well‐defined transmission season has been demonstrated, which is limited to the coolest months of the year, from November to March [35]. Thus, an important step to demonstrate a primary reservoir in an area of seasonal transmission is that the individuals of the reservoir species survive the infection and keep the parasite until the next transmission season, which is more than 7 months. In the field, *S. hispidus* and *O. melanotis* do not live for more than 6 months while *Heteromys* spp., *O. phyllotis* and *P. yucatanicus* can live for more than 2 years [34]. However, in order to survive such a long time, the course of infection has to be relatively non‐pathogenic. In other words, the immune system of the reservoir must react to the presence of the parasite in such a way that while preventing it from doing any irrevocable damage, the parasite is not eliminated. The infection by *L. (L.) mexicana* in small rodents is mild and painless lesions situated at a non‐vital part of the animal, the tail, and thus do not weaken their survival in the wild [33]. Subclinical infections, detected by PCR in the absence of visible lesion, are common [34]. Moreover, the existence of cryptic parasites in *P. yucatanicus* during the warmest months and the presence of a low‐temperature trigger of clinical infection placed the Yucatán deer‐mice in the best position as a primary reservoir of *L. (L.) mexicana* [36]. This type of infection either asymptomatic or with a mild pathology results from an ancient parasitic association in a well‐balanced host‐parasite relationship [37]. The next step to implicate a primary reservoir is that the proportion of animals infected is high enough (20%) to infect the vector during the transmission season (OMS 1984). In the state of Campeche, the seasonal prevalence of infection of *H. gaumeri* range from 88% (*n* = 32) to 29% (*n* = 7), *O. phyllotis* from 100% (*n* = 33) to 27% (*n* = 17), and *P. yucatanicus* from 18% (*n* = 17) to 50% (*n* = 14). Those alarming prevalence in the municipality of Calakmul, Campeche, indicate the need to study each focus individually in order to assess their consequences on human health [34].

The last step to identify the primary reservoir is that the species of parasite is identical in all hosts (reservoir, vector and human) thus, the geographic and temporal distributions of humans and the transient micro-habitat of reservoir and vector need to overlap. Leishmaniasis existed first among wild animals and sand flies and the forest was not very populated by humans. However, with the human displacements due to overpopulation of some areas, new settlements appear constantly deep in the forest. A deforested ring around village limits the contact with the reservoir species and vectors. However, due to the Mayan slash-and-burn agriculture human enters deep into the forest exposing themselves to the bites of infected sand flies. Moreover, subsistence hunting takes place during the night mainly when the agricultural season is over [38]. Moreover, with the ecological protection of large forests, such as the Calakmul Biosphere Reserve in the State of Campeche, the incidence of wild zoonotic diseases might increase.

In conclusion, *O. phyllotis* and the two endemic rodents *H. gaumeri* and *P. yucatanicus* have been incriminated as *L. (L.) mexicana* reservoirs based on their geographic and temporal distributions and the overlap of reservoir, vector and human habitats. All three rodent species are long-lived and the course of the infection is relatively non-pathogenic. Human encroachment into wild areas increases their contact with the infected vector.

#### **3.4. Seasonal transmission**

[29–31]. However, the south of Belize has to be considered as a different endemic area of the disease since this area is not part of the Biotic Province of the Peninsula of Yucatán [32]. In the Yucatan Peninsula, *L. (L.) mexicana* was identified by PCR in the base of tails of two heteromid rodents *Heteromys desmarestianus* Desmarest, 1817 and *Heteromys gaumeri* J.A. Allen and Chapman, 1897; and five cricetid rodents *Ototylomys phyllotis* Merriam, 1901, *Peromyscus yucatanicus*J.A. Allen and Chapman, 1897, *Oryzomys melanotis* Thomas 1893, *Sigmodon hispidus* Say and Ord, 1825, *Reithrodontomys gracilis* J.A. Allen and Chapman, 1897; and one marsupial *Marmosa mexicana* Merriam, 1897 [18, 33, 34]. Thus, many small terrestrial mammals of the Peninsula seemed to be able to harbour the parasite. However, the sand‐fly vector is absent from the driest and unstable habitats such as corn‐ and bean‐fields [27]. Due to *Sigmodon*'s preference for those disturbed areas, this species is not a potential reservoir of *L. (L.) mexicana* in the Yucatan Peninsula. The endemic area of *L. (L.) mexicana* is limited to the humid medium‐ size forest (height < 12 m), which characterizes southern Campeche and Quintana Roo states and the low‐stature forest (height < 8 m) from eastern and southern Yucatan state and northern

[16].

The second step to identify a primary reservoir is that the species, which is relatively abundant in the focus to provide a food source for sand flies. In southern Campeche, *H. gaumeri* was the most abundant rodent in four of the five studied foci (total 46%), followed by *O. phyllotis* (19%) and *P. yucatanicus* (13%). Hence, the least abundant species such as *H. desmarestianus, Oryzomys* and *Reithrodontomys* as well as the marsupial *Marmosa* cannot be primary reser‐ voirs of *L. (L.) mexicana* in the Yucatan Peninsula [34]. Because of the geographic isolation of the Yucatan Peninsula, two species of rodents exist only in the Yucatán Peninsula, the Gaumer's spiny pocket‐mouse, *H. gaumeri* and the Yucatán deer‐mouse, *P. yucatanicus*; two of the three

In the Yucatan Peninsula, a well‐defined transmission season has been demonstrated, which is limited to the coolest months of the year, from November to March [35]. Thus, an important step to demonstrate a primary reservoir in an area of seasonal transmission is that the individuals of the reservoir species survive the infection and keep the parasite until the next transmission season, which is more than 7 months. In the field, *S. hispidus* and *O. melanotis* do not live for more than 6 months while *Heteromys* spp., *O. phyllotis* and *P. yucatanicus* can live for more than 2 years [34]. However, in order to survive such a long time, the course of infection has to be relatively non‐pathogenic. In other words, the immune system of the reservoir must react to the presence of the parasite in such a way that while preventing it from doing any irrevocable damage, the parasite is not eliminated. The infection by *L. (L.) mexicana* in small rodents is mild and painless lesions situated at a non‐vital part of the animal, the tail, and thus do not weaken their survival in the wild [33]. Subclinical infections, detected by PCR in the absence of visible lesion, are common [34]. Moreover, the existence of cryptic parasites in *P. yucatanicus* during the warmest months and the presence of a low‐temperature trigger of clinical infection placed the Yucatán deer‐mice in the best position as a primary reservoir of *L. (L.) mexicana* [36]. This type of infection either asymptomatic or with a mild pathology results from an ancient parasitic association in a well‐balanced host‐parasite relationship [37].

Campeche. This area occupies 19,839 km2

144 The Epidemiology and Ecology of Leishmaniasis

potential reservoirs of *L. (L.) mexicana* in the area [32].

To know the timing of the transmission cycle in each focus of leishmaniasis is very important because high-risk seasons might be restricted. Thus, intervention measures such as prevention through medical education could be conducted before the high-risk period. Moreover, epidemiological and ecological studies could be limited to that season and consequently the cost of research could be diminished.

Based on this rationale, all the results of previous research were analysed focusing on the timing of transmission of *L. (L.) mexicana* in the state of Campeche, Peninsula of Yucatan, Mexico [35]. The study included the timing of incidence of LCL in humans during 1993–1994, as well as the rate and time of infection in rodents and sand flies between February 1993 and March 1995. Rodents and sand flies were found infected between November and March, when men carried out their field activities and were exposed.

In summary, the median-size humid forests of the Yucatan Peninsula have ideal ecological conditions for *L. (L.) mexicana* transmission, particularly in the winter season when high humidity and low temperature, support the growth of sand fly population and trigger the appearance of lesion in the reservoir. This transmission cycle occurs when men enter the forest for agriculture, hunting and gum collecting. Although the number of human cases of LCL reported each year has peaked from March to July, if the incubation period is considered, there is a strong correlation with the abundance, rates and timing of infection of both reservoirs and vectors (**Figure 2**). Based on these results, for the first time in the world, a seasonal transmission (from November to March) of LCL caused by *L. (L.) mexicana* in the sylvatic region of the state of Campeche was determined.

**Figure 2.** Monthly percentage of patients and rodents infected by *Leishmania (Leishmania) mexicana*, and relative abundance of *Lutzomyia cruciata* in a forest at 8 km from the village of La Libertad, Campeche, Mexico.
