POLYMERASE CHAIN REACTION IN DETECTING Leishmania sp IN SYMPTOMATIC AND ASYMPTOMATIC SEROPOSITIVE DOGS

In human and canine renal histological studies of visceral leishmaniasis (VL), the etiological agent is rarely found in situ. The objective of this study was to evaluate PCR in identifying the etiological agent in spleen, liver, lymph node, and kidneys of VL-seropositive dogs. Twenty-five symptomatic (case group) and 15 asymptomatic (control group) VL-seropositive dogs of different breeds, sexes, and ages from Teresina, Piauí State, Brazil, were used. Serologic diagnosis was made by enzyme-linked immunosorbent assay and indirect immunofluorescence test. Animals were subjected to euthanasia and necropsy. Renal fragments were immersed in buffered formaldehyde solution. Spleen, liver, lymph node, and kidney samples were collected and frozen at -70oC until DNA extraction. After dehydration and diaphanization, renal fragments were infiltrated and embedded in paraffin, cut at 3 μm, and stained with hematoxylin-eosin (HE). DNA amplification used an automatic thermocycler with specific Leishmania primers. All case-group dogs and 2 controls showed positive results in spleen, liver, or lymph node PCRs. There was a significant difference by Fisher exact test. In symptomatic seropositive dogs, renal histopathological evaluation showed one animal (4%) with amastigote forms of Leishmania in inflammatory infiltrate, and kidney PCRs detected Leishmania DNA in eight animals (32%). The conclusion was that PCR is more precise than the conventional histopathology in detecting the Leishmania parasite in kidney.


INTRODUCTION
In Brazil, visceral leishmaniasis (Kala-azar) is caused by the protozoan parasite Leishmania (Leishmania) chagasi of the Kinetoplastida order and Trypanosomatidae family (16).It is an anthropozoonosis transmitted to man or other susceptible animals by Lutzomyia longipalpis (5).
Leishmania chagasi promastigotes can be found in the digestive tract of invertebrate hosts, and amastigotes in the macrophage cytoplasm of vertebrate hosts (16).
Diagnosis of canine visceral leishmaniasis is complex for two main reasons: the clinical signs are diverse and similar to other diseases; and the immunological appearance is nonspecific.Today, there is no diagnostic test that can offer 100% specificity and sensitivity (12).
Several types of diagnostic test are used, including clinical examination, and parasitological, immunological and molecular methods.
The clinical manifestations of leishmaniasis depend on the complex interactions between the virulence characteristics of the infecting Leishmania species and the host immune response (13).
Polymerase chain reaction (PCR) allows amplification (in vitro replication) of specific DNA segments from specifically annealed oligonucleotides (primers) on the edges of the target region.The reaction depends on thermostable polymerase DNA and occurs over several cycles, each typically consisting of denaturation, annealing, and extension (15).
Leishmania are members of the Kinetoplastide order, they have a special structure in the mitochondria containing extranuclear DNA, called kinetoplast (kDNA).
Leishmania genus has a region of approximately 200 base pairs kept in the different species, and the remaining sequence varies between species and subspecies (1).
The kDNA forms a complex network of circulating molecules, the minicircles and maxicircles, corresponding to approximately 15% to 35% of the total cell DNA (2).
With the technological advances in molecular biology, it is possible to identify Leishmania in cases of subclinical diseases and in those with few parasites, helping treatment follow-up and a more precise evaluation of its prevalence in endemic areas (15).
Leishmania DNA has been detected in different clinical samples from cases of visceral and cutaneous leishmaniasis in man and canine (6,7,9,10,14,17).In research with L. donovani in humans, the parasite was detected in 21 of 22 patients by PCR using bone marrow and blood samples (6).
In general, the etiological agent is not commonly identified in renal histological studies (11).We found no reports in literature using PCR to detect Leishmania in the kidney.
Previous studies have not identified the parasite in renal histological sections.The scope of this study was to evaluate parasite presence by PCR in the kidneys of dogs with visceral leishmaniasis (VL) and in the spleen, liver, and lymph node of symptomatic and asymptomatic VL-seropositive dogs.Renal histology, and spleen, liver, lymph node and kidney PCR were evaluated in both groups.

Clinical samples
Animals were subjected to euthanasia, and renal parenchyma fragments were collected for histopathology.Spleen, liver, lymph node, and kidney samples were collected and frozen at -70ºC until DNA extraction.

Renal histology
After dehydration and diaphanization, renal fragments were infiltrated and embedded in paraffin, cut at 3 µm, placed on slides, stained with hematoxylin-eosin (HE), and examined by light microscopy.

Polymerase Chain Reaction (PCR)
DNA extraction from spleen, liver, lymph node, and kidney (cortical and medullar region) samples was performed with approximately 25 mg of tissue.The QIAamp DNA Mini Kit was used.Amplification was performed using an automatic thermocycler (Applied Biosystems Each assay contained a negative control, in which no DNA was added to the reaction mixture, and a positive control, in which parasite DNA (obtained from Leishmania donovani culture) was included as a template in the PCR.
The mixture was incubated at 94ºC for 3 min and 30 s; followed by 35 cycles of 30 s at 93ºC, 1 min at 60ºC, 1 min at 72ºC.At the end of the PCR cycles, all tubes were incubated for 10 min at 72ºC and then at 4ºC.
After thermocycling, the reactions were separated by electrophoresis (80V for 50 min) in 1.5% agarose gel with Tris/Borate/EDTA (TBE) buffer.Gel was stained with ethidium bromide and photographed under ultraviolet illumination.

Statistics
PCR results were compared by the Fisher exact test using Statistical Analysis System (SAS).The other data were analyzed by frequency of occurrence.

RESULTS
All case-group animals (symptomatic seropositive, n=25) were PCR positive in spleen, liver, or lymph node.Only two control-group animals (asymptomatic seropositive, n=15) were PCR positive in the same organs.The Fisher exact test showed significant difference at 1% probability between both groups (Table 1).
Eight case-group and no control-group animals were PCR positive in renal tissues.
There was a significant difference at 5% probability between both groups by the Fisher exact test (Table 1).
Renal histopathological evaluation of symptomatic animals (case) showed that 4% (1/25) had Leishmania in the inflammatory infiltrate, and PCR evaluation detected Leishmania DNA in the kidneys of 32% (8/25) dogs.Interstitial inflammatory mononuclear cells with macrophages were found in this infiltrate, including amastigote forms of Leishmania sp in their cytoplasm (Figure 1).The electrophoresis analysis indicated the amplified products of Leishmania in the kidney (Figure 2).

DISCUSSION
The animals in this study were naturally infected and at different stages of the disease.In all symptomatic VL-seropositive dogs (25/25), Leishmania sp was detected in mononuclear phagocytic system organs (spleen, liver, or lymph node) by PCR.In asymptomatic seropositive dogs, 13.33% (2/15) were PCR positive in the same organs.There was a significant difference between both groups by the Fisher exact test.This suggests that dogs with disease symptoms should present a higher parasite load than those asymptomatic.A PCR study on blood and bone marrow aspirate found Leishmania parasites in 18% (4/22) of clinically symptomatic dogs and in 7.6% (40/523) of asymptomatic dogs (14).In research with L. donovani in humans, the parasite was detected in 21 of 22 patients by PCR using bone marrow and blood samples (6).With technical advances in molecular biology, it is possible to identify Leishmania in cases of subclinical diseases and in those with few parasites, improving the precision of prevalence evaluation in endemic areas (15).
Renal histopathological evaluation showed that one animal (4%) had amastigote forms of Leishmania in inflammatory infiltrate, and PCR detected Leishmania (32%) in renal fragments of symptomatic VL-seropositive dogs.This confirmed that PCR was a more precise test for detecting the parasite in this organ.Several authors have reported parasite absence in renal histopathological evaluation (3,4,8,11).None of the consulted literature shows this protozoan in renal tissue by PCR.
In conclusion, there were more positive results by PCR in symptomatic seropositive dogs.The VL etiological agent could be involved in the histopathology of glomerular lesions, and PCR is more precise than conventional histopathology in detecting the Leishmania parasite.
Seventy-three dogs of different breeds, ages and sexes, caught by the Zoonosis Control Center in Teresina, Piauí State, between January and March 2002, were evaluated.They were all subjected to enzyme-linked immunosorbent assay and indirect immunofluorescence test.Forty VL-seropositive dogs were randomly distributed into two groups.The case group consisted of 25 VL-seropositive dogs with clinical manifestation of the disease.The control group consisted of 15 VL-seropositive dogs without any clinical signs of the disease at physical examination.

Figure 1 .
Figure 1.Histopathological aspects of the kidney in symptomatic VL-seropositive dogs.The infiltrate showed interstitial inflammatory mononuclear cells with macrophages including amastigote forms of Leishmania sp in their cytoplasm.HE staining.Bar: 5 µm.