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. 2011 Jun;105(4):319–323. doi: 10.1179/136485911X12987676649827

The PCR-based detection of Leishmania major in Mus musculus and other rodents caught in southern Iran: a guide to sample selection

M Parhizkari *, M H Motazedian †, Q Asqari *, D Mehrabani *
PMCID: PMC4090797  PMID: 21871168

In recent years, rural cutaneous leishmaniasis (CL) has become one of the most challenging public-health problems in several countries. Rodents play an important role as the ‘reservoir’ hosts of the parasite (Leishmania major) that causes the vast majority of the CL found in rural Iran. The present study deals with the role of rodents in the epidemiology of CL in Fars province, in the south of Iran, where the incidence of the disease has doubled over the last decade. Samples, of skin, foot pads, ears, livers and spleens, were collected from 89 rodents (three Meriones persicus, one Me. libycus, 64 Tatera indica and 21 Mus musculus), that had been caught in an endemic area, killed, and fixed in formalin [5% (w/v) formaldehyde]. When each sample was checked for L. major DNA by PCR, at least one sample from each of three (100%) of the Me. persicus, 35 (54·7%) of the T. indica and nine (42·9%) of the Mu. musculus was found to contain L. major DNA. Only 60% of the ear samples, 56% of the footpad samples, 38% of the liver samples and 38% of the spleen samples from the PCR-positive rodents were, however, found PCR-positive. Four of the rodents (two T. indica and two Mu. musculus) were each found to have PCR-positive ear and footpad samples but PCR-negative liver and spleen samples. Only four of the rodents (all T. indica) had PCR-positive spleen and/or liver samples but PCR-negative ear and footpad samples. It is therefore recommended that, for the PCR-based detection of L. major in Iranian rodents, priority is given to the collection and investigation of both ear and footpad samples.

Zoonotic cutaneous leishmaniasis (CL) caused by Leishmania major is now endemic in 14 of the 29 provinces of Iran, with many rural foci in the north, east and south of the country (Yaghoobi–Ershadi et al., 2005). Several species of rodents have been identified as ‘reservoir’ hosts for the causative parasite in Iran, including Meriones libycus, Me. hurrianae, Nosokia indica, Rhombomys opimus, Tatera indica and Rattus norvegicus. (Seyedi–Rashti and Nadim, 1967; Nadim and Faghih, 1968; Seyedi–Rashti and Salehzadeh, 1990; Javadian et al., 1998; Motazedian et al., 2010). Unfortunately, because L. major may cause no or only minor skin lesions in such hosts, it is not always easy to detect the infections in wild rodents (WHO, 1990; Moemenbellah–Fard et al., 2003). Most infected rodents have been identified by the collection of skin scrapings from ears or feet and the culture of these samples (Edrissian et al., 1982). PCR has, however, already been found useful for the identification of possible reservoir hosts, without the need for culture and parasite isolation (Alexander et al., 1998; Oliveira et al., 2005; Brandão–Filho and Shaw, 2006).

The main aim of the present study was to use a PCR-based assay and ear, footpad, liver and spleen samples to explore the prevalences of leishmanial infection among rodents that had been trapped in various parts of Fars province, in southern Iran.

ANIMALS AND METHODS

Study Areas

Fars province lies in southern Iran, at 27′2″–31′42″N, 50′42″–55′36″E, and covers about 1,300,000 km2 or 8·1% of Iran. Its mean altitude is 1540 m above sea level and its total population is about 4 million. Rodents were trapped in various parts of the province, including Shiraz, Kazerun, Firooz Abad, Fassa, Sarvestan, Kherem, Zarghan, Marvdasht, Sepidan, Arsanjan, Estahban, Kohmere Sorkhi, Nobandgan, Kavar, Khafr, Lar, Abade, Bavanat, Khoram Bid, Niriz and Moshkan.

Sampling

Rodents were caught alive, in wire traps, between April 2004 and April 2005 (Mehrabani et al., 2007). The traps were set inside and outside homes, in agricultural plantations close to houses and in both rural and urban settings. The trapped animals were identified by staff of Shiraz University’s Department of Biology, using the relevant taxonomic criteria (Eisenberg and Redford, 1999), before being humanely killed (using methods approved by the Iranian Veterinary Organization), checked for skin lesions and then stored in formalin [5% (w/v) formaldehyde] until they could be processed further.

The Fars Environmental Protection Organization gave a permit for the trapping.

DNA Extraction and PCR

Small samples (each of about 30–40 mm3) of both ears, both rear footpads, the liver and the spleen were collected from each fixed animal. Each sample was homogenized in a 1·5-ml microcentrifuge tube, using a glass rod, and then suspended in 200 μl lysis buffer [50 mm Tris-HCI (pH 7·5), 1 mm EDTA, 1% (v/v) Tween 20 and 15 μl of a solution that contained 19 mg proteinase K/ml]. Each sample was then incubated at 37°C for 48 h, homogenized again with a clean glass rod, and then incubated at 100°C for 15 min. After each resultant lysate was allowed to cool, the DNA in the lysate was recovered by phenol: chloroform: isoamyl-alcohol extraction (Pourmohammadi et al., 2008). Precipitated DNA was resuspended in 50 μl double-distilled water, and stored at −20°C until it could be tested for leishmanial kinetoplast DNA in a PCR-based assay (Pourmohammadi et al., 2008).

Data Analysis

For the rodent species collected in sufficient numbers (i.e. T. indica), χ2 tests, conducted using the SPSS software package (SPSS Inc, Chicago, IL), were used to see which single type of sample (i.e. footpad, ear, liver or spleen), when investigated by PCR, gave a good indication of whether a rodent would be found PCR-positive (by the examination of any sample). A P-value of <0·05 was considered indicative of a statistically significant agreement.

RESULTS

Overall, 89 rodents (three Me. persicus, 64 T. indica, 21 Mus musculus, and one Me. libycus) were trapped and checked for leishmanial infection. Although no lesions were seen, 47 of the rodents — three (100%) of the Me. persicus, 35 (54·7%) of the T. indica, nine (42·9%) of the Mu. musculus and none of the Me. lybicus — were found PCR-positive for leishmanial (all L. major) DNA.

With T. indica, the types of samples most frequently found PCR-positive were the ears (45·3%) and liver (29·7%) (Table 1). With Mu. musculus, the footpads (38·5%) and ears (30·7%) were the samples most frequently found PCR-positive (with all of the liver and spleen samples found PCR-negative; Table 1). In contrast, all of the samples from the three Me. persicus gave a positive result in the PCR (Table 1).

Table 1. Results of the PCR-based detection of Leishmania major DNA in samples from rodents caught in southern Iran.

Meriones persicus Tatera indica Mus musculus Meriones libycus
No. of rodents trapped 3 64 21 1
no. and (%) with PCR-positive:
    Right rear foot 3 (100) 10 (15·6) 3 (14·3) 0 (0)
    Left rear foot 3 (100) 6 (9·4) 2 (9·5) 0 (0)
    Right ear 3 (100) 16 (25·0) 1 (4·8) 0 (0)
    Left ear 3 (100) 13 (20·3) 3 (14·3) 0 (0)
    Liver 2 (67) 19 (29·7) 0 (0·0) 0 (0)
    Spleen 1 (33) 18 (28·1) 0 (0·0) 0 (0)
    Any sample 3 (100) 35 (54·7) 9 (42·9) 0 (0)
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With T. indica, the PCR-based examination of either just footpad samples (P<0·002) or just ear samples (P<0·001) gave a good indication of whether the rodent would be found PCR-positive (i.e. by the examination of all samples). The PCR-positive T. indica were trapped in the Shiraz focus and rural areas in Kavar, Firooz Abad, Fassa, Sarvestan, Khrameh, Sepidan, Kohmereh Sorkhi and Nobandegan. All the T. indica trapped in Khafr and Abadeh were found PCR-negative. The nine PCR-positive Mu. musculus came from Kavar, Sepidan, Estahban, Moshkan and Niriz.

Four of the rodents (two T. indica and two Mu. musculus) were each found to have PCR-positive ear and footpad samples but PCR-negative liver and spleen samples (Table 2). Only four of the rodents (all T. indica) had PCR-positive spleen and/or liver samples but PCR-negative ear and footpad samples (Table 2).

Table 2. The combinations of organs/tissues, from rodents caught in southern Iran, found PCR-positive for Leishmania major DNA.

Meriones persicus Tatera indica Mus musculus Meriones libycus
No. of rodents trapped 3 64 21 1
no. and (%) with PCR-positive:
    Foot/feet only 0 (0) 3 (3·1) 4 (19·0) 0 (0)
    Ear(s) only 0 (0) 4 (6·3) 3 (14·3) 0 (0)
    Liver only 0 (0) 2 (3·1) 0 (0) 0 (0)
    Spleen only 0 (0) 4 (6·3) 0 (0) 0 (0)
    Foot/feet and ear(s) only 0 (0) 2 (3·1) 2 (9·5) 0 (0)
    Foot/feet and liver only 0 (0) 0 (0) 0 (0) 0 (0)
    Foot/feet and spleen only 0 (0) 0 (0) 0 (0) 0 (0)
    Ear(s) and liver only 0 (0) 4 (6·3) 0 (0) 0 (0)
    Ear(s) and spleen only 0 (0) 1 (1·6) 0 (0) 0 (0)
    Spleen and liver only 0 (0) 4 (6·3) 0 (0) 0 (0)
    Ear(s), foot/feet and liver only 2 (67) 2 (3·1) 0 (0) 0 (0)
    Ear(s), spleen and liver only 0 (0) 6 (9·4) 0 (0) 0 (0)
    Ear(s), foot/feet and spleen only 1 (33) 3 (4·7) 0 (0) 0 (0)
    Ear(s), foot/feet, spleen and liver 0 (0) 1 (4·8) 0 (0) 0 (0)
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DISCUSSION

In the detection of leishmanial infection in rodents, the investigation of biopsy/necropsy samples by PCR is more sensitive than the investigation of similar samples as smears or by culture (Nicolas et al., 2002; Oliveira et al., 2005; Parvizi et al., 2008). Even though the samples investigated in the present study had been formalin-fixed, the overall prevalence of L. major detected (52·8%) was markedly higher than the value (16·2%) estimated, using microscopy and culture, for rodents caught elsewhere in southern Iran (Motazedian et al., 2006; Mehrabani et al., 2007). The results of rodent trapping in Fars (present study) indicate that T. indica is the most common rodent in the province, and the results of the subsequent PCR indicated that, at the time of the trapping, about one in every two (54·7%) T. indica in the province was infected with L. major. In earlier studies, T. indica infected with L. major have been detected in southern or south–eastern Iran (Javadian et al., 1998; Mohebali et al., 2004; Mehrabani et al., 2007) as well as the east and west of the country (Rassi et al., 2001).

Meriones libycus is considered to be the main ‘reservoir’ host in the foci of zoonotic CL in northern and central Iran (Yaghoobi–Ershadi et al., 2004; Pourmohammadi et al., 2008). In the present study, this species and Me. persicus appeared to be rare in Fars province, although all three specimens of Me. persicus that were caught were found PCR-positive for L. major DNA, perhaps indicating that, despite their relative rarity, Meriones spp. are also important reservoir hosts for ZCL in southern Iran (Rassi et al., 2006).

The present report appears to be the first to describe natural infection of Iranian Mu. musculus with L. major. Such infection may be epidemiologically significant because Mu. musculus is so widespread in and around housing, especially in urban areas, and probably occurs in every Iranian city and village. Laboratory mice bred from wild Mu. musculus are important experimental hosts of L. major (Mauricio et al., 2001). After Aebischer et al. (1993) infected C57BL/6 mice with L. major, they were able to find the parasites in the lymph glands, livers, spleens and bone marrow of the rodents. Nicolas et al. (2002) infected BALB/C mice with L. major and, 6–12 months later, found necropsy samples (of ears, lymph glands, livers, spleens, bone marrow, blood, foot pads and tail skin) to be PCR-positive for the parasite’s DNA. As Phlebotomus papatasi can be infected by feeding on laboratory mice experimentally infected with L. major (Kimblin et al., 2008), naturally infected mice may play an important role as reservoirs for human infection. The presence of Mu. musculus infected with L. major may explain why, in recent years, ZCL (which is usually considered a rural disease) has been increasingly detected in some urban areas of Iran (Razmjou et al., 2009). In the present study, none of the livers and spleens of the Mu. musculus caught was found PCR-positive, perhaps indicating that L. major does not spread as much in Mu. musculus of the wild type as it tends to do in laboratory mice.

Taken together, the present results indicate that, for the PCR-based detection (or, presumably, the culture-based isolation) of L. major in Iranian rodents, priority is given to the collection and investigation of both ear and footpad samples.

Acknowledgments

The authors thank the Office of the Vice-Chancellors for Research of Shiraz University of Medical Sciences, for financial support. They are also grateful to Dr M. Lerch, of the Seattle Biomedical Research Institute, for her advice and editing of the English manuscript.

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