Molecular diagnosis of canine visceral leishmaniasis: Identification of Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR

Acta Tropica 111 (2009) 289–294

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Molecular diagnosis of canine visceral leishmaniasis: Identification of Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR Patrícia Flávia Quaresma a , Silvane Maria Fonseca Murta b , Eduardo de Castro Ferreira a , Ana Cristina Vianna Mariano da Rocha-Lima a, Ana Amélia Prates Xavier a, Célia Maria Ferreira Gontijo a,∗ a Laboratório de Leishmanioses, Centro de Pesquisas René Rachou, Fundac¸ão Oswaldo Cruz Avenida Augusto de Lima, 1715 Barro Preto 30190-002 Belo Horizonte, Minas Gerais, Brazil b Laboratório de Parasitologia Celular e Molecular Centro de Pesquisas René Rachou, Fundac¸ão Oswaldo Cruz Avenida Augusto de Lima, 1715 Barro Preto 30190-002 Belo Horizonte, Minas Gerais, Brazil

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Article history: Received 25 July 2008 Received in revised form 17 April 2009 Accepted 17 May 2009 Available online 23 May 2009 Keywords: Canine visceral leishmaniasis Leishmania infantum chagasi Molecular diagnosis Real-time PCR PCR-RFLP

a b s t r a c t The efficacies of polymerase chain reaction (PCR) procedures for the diagnosis of canine visceral leishmaniasis (CVL), and of PCR-restriction fragment length polymorphism (RFLP) analysis for the identification of Leishmania species, have been assessed. Quantitative real-time PCR employing a SYBR Green dyebased system was standardised for the quantification of Leishmania kDNA minicircles. Skin, peripheral blood and bone marrow samples collected from 217 dogs, asymptomatic or symptomatic for CVL, were analysed. The PCR method, which was based on the amplification of a 120 bp kDNA fragment conserved across Leishmania species, was able to detect the presence in clinical samples of protozoan parasite DNA in amounts as low as 0.1 fg. Bone marrow and skin samples proved to be more suitable than peripheral blood for the detection of Leishmania by PCR and presented positive indices of 84.9% and 80.2%, respectively. PCR-RFLP analysis indicated that 192 of the PCR-positive dogs were infected with Leishmania infantum chagasi, whilst L. braziliensis was identified in two other animals. Quantitative PCR revealed that bone marrow samples from dogs presenting positive conventional tests contained a higher number of copies of Leishmania kDNA than peripheral blood, although no significant differences were detected between symptomatic and asymptomatic dogs in terms of parasite load. This study demonstrates that PCR can be used for the detection of Leishmania in clinical samples derived from naturally infected dogs, and that PCR-RFLP represents a rapid and sensitive tool for the identification of Leishmania species. Additionally, qPCR is effective in quantifying Leishmania DNA load in clinical samples. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Zoonotic visceral leishmaniasis (VL), the etiological agent of which is Leishmania infantum chagasi (Shaw, 2006), is widely distributed within the Americas. In Brazil, the growing number of registered urban cases has received special attention as new foci have arisen (Gontijo and Melo, 2004). Dogs constitute the main host reservoir of L. infantum chagasi and, hence, play an important role in the domestic transmission cycle of VL. The progression of canine visceral leishmaniasis (CVL) during Leishmania infection is, however, very variable amongst infected dogs in that some animals may never develop the disease whilst others suffer severe symptoms that can lead to early death (Alvar et al., 2004). On the other hand, cutaneous parasitism is commonly present in both asymptomatic and symptomatic

∗ Corresponding author. Tel.: +55 31 3349 7755; fax: +55 31 3349 7795. E-mail address: gontijo@cpqrr.fiocruz.br (C.M.F. Gontijo). 0001-706X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2009.05.008

dogs and is crucial to the transmission dynamics of the disease by acting as a source of infection for phlebotomine sand flies (Silva et al., 2001). CVL is routinely diagnosed using the indirect immunofluorescent assay test (IFAT), although asymptomatic dogs may yield false negative results (Almeida et al., 2005) and cross reaction with other parasites and other Leishmania species can occur (Schulz et al., 2003; Ferreira et al., 2007). Considering the limitations of conventional methods, alternative diagnostic tests are needed for CVL. In this context, polymerase chain reaction (PCR) has been shown to provide a rapid and sensitive technique for parasite detection (Leontides et al., 2002; Schonian et al., 2003; Cortes et al., 2004). With the ready availability of molecular tools, many researchers have started to use PCR-based methods for CVL diagnosis in different settings (Lachaud et al., 2001, 2002; Gomes et al., 2007). Epidemiological surveillance, however, requires not only parasite detection but also species identification. Knowledge of the species of Leishmania involved is extremely important with respect to the epidemiology of leishmaniasis, particularly in endemic areas with

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simultaneous occurrence of visceral and cutaneous forms of the disease. Several studies have reported that PCR-restriction fragment length polymorphism (RFLP) is efficient for the identification of Leishmania species (Volpini et al., 2004; Andrade et al., 2006; Ferroglio et al., 2006; Rotureau et al., 2006). Additionally, application of PCR-RFLP requires only small amounts of DNA and allows differentiation between the New World species L. (Leishmania) amazonensis, L. (Viannia) braziliensis and L. (L.) infantum chagasi (Andrade et al., 2006). In order to obtain information concerning disease progression and the role of dogs as a source of infection for the vector of CVL, it is also important to determine the parasitic density in different tissue samples. Quantitative real-time PCR (qPCR) has been applied to a wide range of biological sources of bacteria, mycobacteria, viruses, fungi and parasites, including Leishmania (Espy et al., 2006; De Monbrison et al., 2007; De Paiva Cavalcanti et al., 2008). The aim of the present study was to diagnose CVL by employing PCR for parasite detection in various tissue samples from dogs presenting different clinical manifestations, PCR-RFLP for the identification of Leishmania species, and qPCR for the quantification of Leishmania DNA. 2. Materials and methods 2.1. Clinical samples The procedures used for the collection of clinical specimens were those approved by the Ethics Committee for Animal Experimentation (CEUA-FIOCRUZ, P 0119-02) and adopted by the Brazilian College of Animal Experimentation (COBEA). The study involved a total of 217 dogs (Canis familiaris) living in an area of Belo Horizonte, Minas Gerais, Brazil, that is endemic for VL. Clinical specimens corresponding to peripheral blood, bone marrow aspirates, and skin fragments obtained from the inner ears of the dogs, were used as samples for both conventional and molecular diagnoses. 2.2. Conventional diagnosis Parasitological examinations involved the analysis of Giemsastained slides containing bone marrow smears or imprints of skin fragments for the detection of amastigote forms, and bone marrow culture in Novy-MacNeal-Nicolle-liver infusion tryptose (NNN-LIT) medium supplemented with 20% foetal bovine serum. Serological examinations included IFAT assays carried out using the Canine Leishmaniasis IFAT Kit (Bio-Manguinhos/FIOCRUZ, Rio de Janeiro, RJ, Brazil) and enzyme-linked immunosorbent assays (ELISA) performed with the Canine Leishmaniasis EIE Kit (BioManguinhos/FIOCRUZ), both kits being applied according to the manufacturer’s instructions. Direct agglutination tests (DAT) were performed as described previously (Oskam et al., 1996). 2.3. Molecular diagnosis 2.3.1. Extraction of DNA DNA was extracted from promastigotes of L. (L.) infantum chagasi (MHOM/BR/74/PP75) using the phenol-chloroform method (Sambrook et al., 1989) in order to obtain a PCR-positive control. Isolation of DNA from clinical samples was performed with the aid of commercial kits (GE Healthcare, São Paulo, SP, Brazil) used according to the manufacturer’s instructions. The weights of skin fragments (10 mg each) and the volume of blood and bone barrow aspirates (300 ␮l) used for DNA extraction were standardised. In order to assess the effectiveness of extraction of DNA from clinical samples, amplification of the constitutive canine ␤-globin

gene was performed using the primers: 5 CAA CTT CAT CCA CGT TCA CC 3 and 5 ACA CAA CTG TGT TCA CTA GC 3 (Greer et al., 1991). Samples that were positive for the expected amplicon from ␤-globin were validated, whereas those that were negative for this amplicon were excluded from the study. 2.3.2. PCR of Leishmania DNA DNA extracted from clinical samples was submitted to PCR using primers for Leishmania that had been designed to amplify a 120 bp fragment of the conserved region of kDNA minicircles (Degrave et al., 1994). The sequences of the primers employed were 5 (C/G)(C/G)(G/C) CC(C/A) CTA T(T/A)T TAC ACC AAC CCC 3 and 5 GGG GAG GGG CGT TCT GCG AA 3 . Reaction mixtures were prepared in a final volume of 25 ␮l containing 2 ␮l of DNA template and 1× buffer solution (1.0 mM Tris–HCl; 5.0 mM KCl; 1.5 mM MgCl2 ; pH 8.0), 200 ␮M dNTPs, 10 pmol of each primer and 1.25 U of Taq DNA polymerase (GE Healthcare). The amplification conditions were as follows: 94 ◦ C for 4 min, followed by 30 cycles of denaturation at 94 ◦ C for 30 s, annealing at 60 ◦ C for 30 s and extension at 72 ◦ C for 30 s, with a final extension step at 72 ◦ C for 10 min. Amplicons were analysed on silver-stained 6% polyacrylamide gels. 2.3.3. Identification of Leishmania species PCR-RFLP was performed, as described previously (Volpini et al., 2004; Andrade et al., 2006), on the 120 bp fragments amplified from kDNA minicircles present in clinical samples derived from dogs presenting positive PCR tests. The non-purified PCR products (5.0 ␮l) were digested with 1 U of the restriction enzyme Hae III (Promega, Madison, WI, USA), and the restriction fragments obtained were compared with the molecular profiles of the WHO reference strains L. (L.) amazonensis (IFLA/BR/67/PH8), L. (Viannia) braziliensis (MHOM/BR/75/M2903) and L. (L.) infantum chagasi (MHOM/BR/74/PP75). 2.3.4. Quantification of Leishmania kDNA An ABI 5700 real-time PCR instrument (Applied Biosystems, Foster City, CA, USA) was used to perform qPCR on DNA isolated from blood and bone marrow samples from 35 dogs presenting positive PCR assays. Reaction mixtures were prepared in a final volume of 25 ␮l containing 5 ␮l of DNA template and 1× SYBR Green buffer, 3.0 mM MgCl2 , 250 ␮M dNTP, 10 pmol of each primer (as detailed above for conventional PCR), 1 U AmpliTaq Gold DNA polymerase (Applied Biosystems) and DNAse-and RNAse-free distilled water (Gibco, Invitrogen Corp., Carlsbad, CA, USA). The amplification conditions were as follows: 94 ◦ C for 10 min, followed by 40 cycles of denaturation at 94 ◦ C for 15 s, and annealing and extension at 60 ◦ C for 1 min. Standard curves were prepared for each assay using known quantities of TOPO pCR 2.1 plasmids (Invitrogen Corp.) containing a cloned canine gene (␤globin; 118 bp) and the 120 bp kDNA from the parasite. Serial dilutions (10×) of recombinant plasmids containing 104 to 107 plasmid copies, representing a concentration range of 1–1000 parasites since a kDNA network presents about 104 minicircles, were employed in the construction of the calibration curves. Concentrations of kDNA that were not within the linear range were estimated using Sequence Detection System Gene-Amp 5700 data analysis software (Applied Biosystems) that allows assessment of the dissociation curve and the fluorescence intensity of the samples. The fluorescence intensity of each sample, which is proportional to the amount of DNA present, was expressed in terms of the PCR threshold cycle (CT ) defined as the number of PCR cycles required for the fluorescence signal to exceed the detection threshold (background noise). The canine housekeeping gene (-globin was used to normalise the concentration of input DNA.

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Table 1 Positive results from serological and parasitological conventional examinations and positivity of PCR for Leishmania detection in dogs from Belo Horizonte, Minas Gerais, Brazil, distributed according to clinical status. Clinical status of dogs assayed

Serological assaysb

Parasitological assaysb

PCR assaysa Skin

Peripheral bloodb

Bone marrowb

PCR-positive in at least one clinical samplec

b

Symptomatic

107/144 (74.3%)

83/144 (57.6%)

111/135 (82.2%)

102/131 (77.8%)

113/128 (88.3%)

143/144 (98.6%)

Asymptomatic

40/73 (54.8%)

19/73 (26.0%)

51/67 (76.1%)

47/67 (70.1%)

50/64 (78.1%)

69/73 (94.5%)

Total

147/217 (67.7%)

102/217 (47.0%)

162/202 (80.2%)

149/198 (75.3%)

163/192 (84.9%)

212/217 (97.7%)

a b c

Samples tested by PCR – positive for constitutive canine ␤ globin gene. Values represent the number of positive tests/total number of dogs tested using the assay, with the percentage of positive results obtained shown in parenthesis. Values relate to dogs that tested positive for the presence of Leishmania DNA in at least one of the three clinical samples assayed.

2.4. Statistical analysis Statistical analysis was performed with the aid of MINITAB version 13. Differences in the frequencies of positive results for each test, comparative analyses of PCR results amongst different clinical samples, and comparisons between clinical groups were performed using the ␹2 -test and Bonferroni multiple comparisons methodology. Results of qPCR concerning the amounts of DNA present were analysed using Student’s t-test, the paired t-test, ANOVA and Tukey multiple comparison tests. The confidence interval employed in all statistical analyses was 95%. 3. Results 3.1. Allocation of animal to groups Based on clinical evaluation, animals were allocated to either the asymptomatic group (presenting no clinical signs of VL; n = 73) or the symptomatic group (presenting at least one symptom of VL including loss of weight, lymphadenopathy, dry exfoliative dermatitis, ulcers, periorbital alopecia, diffuse alopecia, ocular signs or onychogryphosis; n = 144). On the basis of the results of parasitological and serological tests, dogs were allocated into either the negative conventional examination (NCE) group (n = 70) or the positive conventional examination (PCE) group (with at least one positive conventional test; n = 147). The NCE group, comprising negative parasitological and negative serological (NPNS) examination animals, included 33 asymptomatic and 37 symptomatic dogs (n = 70). The PCE group was further categorised into two subgroups, namely, negative parasitological and positive serological (NPPS) examination animals comprising 21 asymptomatic and 24 symptomatic dogs (n = 45), and positive parasitological and positive serological (PPPS) examination animals comprising 19 asymptomatic and 83 symptomatic dogs (n = 102).

tested, no significant differences were detected between the proportions of positive PCR results obtained for asymptomatic and symptomatic dogs with any of the tissue sample types studied. This was also the case when PCR-positive results obtained with at least one of the clinical sample types were considered (Table 1). In contrast, the proportions of positive serological and parasitological tests for symptomatic dogs were significantly (P < 0.05) greater than for asymptomatic animals. Comparison of the efficiency of the PCR assay with those of conventional serological and parasitological tests in the detection of CVL-positive dogs revealed that PCR was the most accurate diagnostic procedure and that conventional parasitological tests exhibited the lowest sensitivity (P = 0.000). When PCR-positive results obtained with at least one of the clinical sample types were considered, no significant differences were detected between NCE dogs and those of the PCE group (Table 2). However, a significantly (P = 0.004) larger proportion of PCR-positive results were obtained for PPPS dogs compared with NPNS and NPPS animals. 3.3. Identification of Leishmania species by PCR-RFLP PCR-RFLP with Hae III digestion permitted identification of the infecting species of Leishmania in 194 of the 212 PCR-positive dogs. The majority (n = 192; 98.9%) of these animals were infected with L. infantum chagasi (Fig. 1), while only two dogs were infected with L. Table 2 Positivity of PCR for Leishmania detection in clinical samples obtained from dogs previously classified according to the results of conventional diagnostic procedures. NCE groupa

PCE groupb

NPNS dogs (n = 70)c

NPPS dogs (n = 45)c

PPPS dogs (n = 102)c

Skine

51/66 (77.3%)

29/41 (70.7%)

82/95 (86.3%)

Bloode

40/63 (63.5%)

23/41 (56.1%)

86/94 (91.5%)

Bone Marrowe

45/60 (75.0%)

23/35 (65.7%)

95/97 (97.9%)

PCR-positive in at least one clinical sampled

67/70 (95.6%)

40/45 (88.9%)

102/102 (100%)

Clinical sample

3.2. Assessment of PCR performance Table 1 shows the results of the serological, parasitological and PCR tests conducted on clinical samples derived from 217 dogs and distributed according to clinical status. DNA from 15, 19 and 25 samples, respectively, of skin, blood and bone marrow was excluded from further PCR procedures since the constitutive canine ␤-globin gene could not be amplified in these extracts. Comparative statistical analyses revealed no significant differences between the proportion of positive PCR results obtained using either skin or bone marrow samples, although percentages of positive tests obtained with bone marrow were significantly (P = 0.017) higher than with blood samples. Thus, amongst the tissues studied, bone marrow and skin appeared to be more appropriate for the detection of Leishmania by PCR. With respect to the clinical status of the animals

a Animals with negative conventional examinations (NCE) comprising dogs with negative parasitological and serological (NPNS) tests. b Animals with at least one positive conventional examination (PCE) comprising dogs with negative parasitological and positive serological (NPPS) tests and dogs with positive parasitological and positive serological (PPPS) tests. c Values represent the number of positive tests/total number of samples tested using the assay, with the percentage of positive results obtained shown in parenthesis. d Values relate to dogs that tested positive for the presence of Leishmania DNA in at least one of the three clinical samples assayed. e Samples tested by PCR – positive for constitutive canine ␤ globin gene.

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Fig. 1. Restriction fragment length polymorphisms of 120 bp kDNA amplicons from Leishmania obtained with restriction enzyme Hae III and analysed on silver-stained 10% polyacrylamide gel. MM: 50 bp molecular size marker; lanes: La – L. amazonensis (IFLA/BR/67/PH8), Lb – L. braziliensis (MHOM/BR/75/M2903), Lc – L. infantum chagasi (MHOM/BR/74/PP75); 01 to 04 – amplified DNA from skin samples.

braziliensis. Remarkably, Leishmania co-infection was not detected in any animal. 3.4. Quantification of Leishmania kDNA Initially, a standard curve was obtained using 10× serial dilutions of a recombinant plasmid containing fragments from both Leishmania kDNA and the canine ␤-globin gene. The CT values obtained were linear over a wide range of plasmid concentrations (104 to 107 plasmid copies) for both genes with correlation coefficients (r2 ) of 0.9938 for Leishmania kDNA and of 0.9806 for ␤-globin DNA. Plots of the temperature-dependent dissociation of the SYBRGreen fluorescent marker from kDNA and ␤-globin PCR amplicons showed fluorescence as a single peak, indicating that a single PCR product was generated in each assay. The melting temperatures (Tm ) were 56.0 ◦ C for kDNA and 53.0 ◦ C for ␤-globin. Examination of the number of copies of Leishmania kDNA in blood compared with bone marrow samples indicated no differences in DNA levels for the 35 dogs assessed by qPCR. Nevertheless, analysis of the number of copies of kDNA in animals from the PCE group revealed a significantly (P = 0.014) higher parasite concentration in bone marrow compared with blood (Fig. 2A). No differences in parasite concentration were, however, detected between the bone marrow and the blood samples in animals from the NCE group (data not shown). Comparison of the Leishmania DNA load between the PCE and NCE groups showed that animals of the former group displayed significantly (P = 0.044) higher parasite densities in bone marrow samples (Fig. 2B). No such differences were observed, however, in blood samples (data not shown). The examination groups NPNS, NPPS and PPPS were analysed separately in order to assess whether positive diagnostic tests could be correlated with parasite density, and a significant (P = 0.004) difference was detected in the number of DNA copies in bone marrow samples between PPPS dogs and NPPS/NPNS animals (Fig. 2C). Clinical samples from 21 asymptomatic and 14 symptomatic dogs were submitted to qPCR, but no significant differences (P = 0.933 for bone marrow; P = 0.537 for peripheral blood) were observed in the number of copies of Leishmania kDNA between the clinical groups (data not shown). 4. Discussion In the present study, conventional PCR exhibited a reliable performance with high positive indices as demonstrated by the results obtained for different tissue samples from a large number of animals. The molecular assay was able to detect Leishmania kDNA in concentrations as low as 0.1 fg DNA/␮l. Since it is known that one

Fig. 2. Quantification of Leishmania DNA by qPCR showing: (A) peripheral blood and bone marrow samples from PCE dogs: a significant difference in parasite load between these tissues was observed in PCE dogs (P = 0.014); (B) bone marrow samples from NCE and PCE dogs: the parasite load was significantly higher in the bone marrow samples from PCE animals (P = 0.044); and (C) bone marrow samples from NPNS, NPPS and PPPS animals: the number of copies of Leishmania kDNA was significantly higher in bone marrow samples from PPPS dogs compared with NPPS and NPNS animals (P = 0.004).

parasite contains approximately 300 fg DNA (Vergel et al., 2005), PCR assays carried out under the conditions stated herein would allow the detection of DNA representing 1/3000 of one parasite. Amongst the different types of clinical samples employed in the present study, bone marrow and skin were considered to be the most appropriate for the detection of Leishmania DNA by PCR. The main drawback of using bone marrow samples is, however, the difficulty of collecting material, especially in large-scale epidemiological surveys. Skin is clearly a reliable source of Leishmania DNA in dogs, and the biopsy of a small skin fragment is a simple and safe procedure. Additionally, Manna et al. (2004) have reported that canine skin, regardless of the presence of cutaneous lesions, allows more efficient detection of Leishmania DNA by PCR than other tissues.

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Analysis of the performance of PCR with samples from both asymptomatic and symptomatic dogs has shown it to be a reliable method for Leishmania DNA detection irrespective of the clinical status of the animal. In contrast, serological and parasitological methods gave higher positive indices for samples from symptomatic dogs than from asymptomatic animals. PCR is, therefore, a more effective method for the diagnosis of CVL because it does not possess the limitations associated with conventional diagnostic methods, especially in situations where infection is not apparent. The applicability of PCR for CVL diagnosis was further supported by its performance with samples from animals presenting both positive and negative results according to conventional tests. Results from the present study demonstrate that PCR is able to detect CVL infection even when conventional tests fail to diagnose the disease. As expected, a higher percentage of PCR-positive tests were obtained for PPPS dogs than for NPNS or NPPS animals, further highlighting the sensitivity of PCR. The lower proportion of positive results obtained by serological (67.7%) and parasitological (47.0%) methods compared with those obtained by PCR may reflect underestimations of the numbers of CVL-infected animals in VL-endemic areas. Indeed, the major concern in the control of the canine disease is the presence of asymptomatic dogs that appear to be negative for CVL on the basis of serological surveys. Recent determinations, using PCR-based approaches, of the prevalence of CVL infection in endemic areas have shown that the percentage of infected animals is higher than those that develop the disease (Sideris et al., 1999; Solano-Gallego et al., 2001). Hence, the use of PCR together with other diagnostic methods would help to determine the extent of sub-clinical infection in endemic areas and would provide a more accurate estimate of the number of asymptomatic dogs. PCR-RFLP has been shown to be a practical, safe and rapid method for the identification of parasite species in CVL-infected animals. It was not possible to identify the species of Leishmania in only 18 (8.5%) of the 212 PCR-positive dogs tested, and this was possibly due to a low yield of 120 bp amplicon (insufficient for Hae III digestion assays) or to inhibition of the PCR occasioned by excess host DNA relative to the amount of parasite DNA. The vast majority of dogs examined in the present study were infected with L. infantum chagasi, with only two animals (1.0%) showing infection with L. braziliensis. The area from which the dogs under study were selected is endemic for both cutaneous and visceral leishmaniasis, although most recorded human and canine cases are the latter. Andrade et al. (2006) employed PCR-RFLP to identify Leishmania species in canine samples from the same municipality and showed that, with the exception of one animal infected with L. braziliensis, all animals harboured L. infantum chagasi. In both the present study, and that cited above, no cross-infection was detected. Campaigns to control VL in Brazil involve the euthanasia of seropositive dogs, although the infecting species of Leishmania is not revealed by the serological methods employed. Use of PCR-RFLP would avoid the unnecessary killing of dogs with L. braziliensis infection and, hence, reduce the costs of running such campaigns in developing countries. A standardised SYBR Green dye-based real-time PCR system was effective for the quantification of L. infantum chagasi kDNA and, mainly because of the target employed, exhibited very high sensitivity. Such sensitivity was partly due to the high copy number (around 10,000 copies per parasite; Nicolas et al., 2002) of kDNA minicircles. In the present study, the number of DNA copies varied widely but extremely high values were rarely obtained. Ferrer (2002) has assumed that under natural conditions, phlebotomine sand flies transmit a low number of promastigotes (100–1000 parasite), but that these are sufficient to initiate the disease. However, host immune competence and other intrinsic factors influence the level of parasitic infection more profoundly than the vector load (Travi et al., 2001).

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Quantification of Leishmania kDNA in PCE animals revealed that, in comparison with blood samples, bone marrow presented higher parasitic loads, suggesting a correlation between parasitic load and increased sensitivity of conventional diagnostic tests with this tissue. This may reflect the fact that parasites in bone marrow are more established and have had sufficient time to stimulate the immune system. In blood samples, no correlation between parasitic load and the sensitivity of diagnostic tests could be detected, and it appears that the blood serves as a medium for parasite transport instead of acting as a reservoir tissue. Manna et al. (2006) reported that parasite quantification in blood samples, especially those derived from asymptomatic dogs, appeared not to be significant in determining the course of infection. Higher numbers of copies of Leishmania kDNA were detected in bone marrow samples derived from dogs presenting positive parasitological results in comparison with animals showing negative results in one or all conventional VL tests. It should be noted, however, that the conventional parasitological methods used in the present study were in vitro culture of bone marrow aspirates and optical microscopy of bone marrow smears, and this may account for the direct correlation observed between positive parasitological tests and high loads of Leishmania DNA. In despite of this, the quantity of Leishmania DNA found in bone marrow was higher than that in blood samples, in accordance with a recent report relating to natural infections (Francino et al., 2006). Contrary to observations from earlier studies (Reis et al., 2006; Rodríguez-Cortés et al., 2007), no significant differences were detected between the quantities of Leishmania kDNA in asymptomatic and symptomatic dogs (data not shown). This finding was possibly the result of either the sample size employed or the number of tissue samples analysed (blood and bone marrow). Such limitations excluded the prospect of making a full comparison of differences amongst the various infected organs in the host. The positive correlation observed between symptomatic animals and a high parasitic load is not consensual. CVL can begin either as an anergic condition with generalised dissemination, high parasite numbers and no or few clinical signs, or as a hyper-reactive form in which the animal displays severe clinical symptoms of the disease despite no parasites being detected (Quinnell et al., 2003; Alvar et al., 2004; Poot et al., 2005). Further investigations using diverse tissue samples and a larger number of animals exhibiting different clinical forms of the disease are required to clarify such issues. The results of the present study suggest that PCR-based methodologies for CVL diagnosis are reliable, in that they are highly sensitive and reproducible and involve a relatively short procedure time. They may represent useful tools in epidemiological surveillance within CVL endemic areas where animals display different clinical forms of the disease. In addition, quantification of kDNA using the SYBR Green dye-based real-time PCR is an important step in validating the qPCR technique. The described method involves lower costs compared with other molecular techniques for the quantification of parasites, and has been shown to be a valuable tool in parasite burden studies and also in detecting ongoing VL infections. Acknowledgements The authors wish to thank Fundac¸ão Oswaldo Cruz (FIOCRUZ/PAPES III) and Fundac¸ão de Amparo à Pesquisa de Minas Gerais (FAPEMIG) for financial support for this project. References Almeida, M.A.O., Jesus, E.E.V., Sousa-Atta, M.L.B., Alves, L.C., Berne, M.E.A., Atta, A.M., 2005. Clinical and serological aspects of visceral leishmaniasis in Northeast Brazilian dogs infected with Leishmania chagasi. Vet. Parasitol. 127, 227–232.

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