Skin sampling collection
The database repository of the Veterinary Pathology Service of the Veterinary Teaching Hospital (OVUD) of the Department of Veterinary Medicine of Perugia (central Italy) was searched to identify histopathology records of skin biopsies from dogs living in CanL endemic areas, namely those dogs recently estimated to be at medium–high risk (prevalence rates: 10–20%) for CanL [20].
The skin FFPE blocks of the dogs were included in the study if: (i) the histopathology previously performed by a board-certified (CB) veterinary pathologist confirmed features consistent with LHD and/or GD compatible with Leishmania infection; and (ii) an adequate amount of FFPE tissue (> 0.5 cm2) was stored in the biorepository at the time of the investigation. The present study, therefore, included 30 dogs whose samples has been collected between 2005 and 2020.
Histopathology and immunohistochemistry
Slides containing 4-µm-thick sections were prepared from the skin FFPE blocks and stained with H&E for histopathologic examination. H&E-stained slides were checked for lesions in the dermis as follows: inflammation (presence/absence); severity (mild, moderate, severe); pattern (perivascular, interstitial, band-like, nodular, diffuse); depth (superficial, mid dermis, deep); and relative percentage of inflammatory cells (macrophages, lymphocytes, plasma cells, neutrophils, multinucleated giant cells, eosinophils and mast cells). H&E staining was used to characterize amastigote forms of Leishmania according to their size, shape and location inside macrophages, and to estimate the parasite load. The number of microscopic fields that were positive for Leishmania amastigotes was counted, and the percentage of positive fields was calculated based on the average number of positive fields per skin sample. The grading system used for H&E-stained samples was: (i) negative (−, no amastigotes found); (ii) suspect [+/−, < 5% positive microscopic fields, at 400× magnification, field number (FN) = 22 mm], or samples where the detection of parasites was inconclusive or difficult and where differentiation from phagocytosed cell debris was necessary); (iii) positive [+, with mild parasite load (5–25% positive microscopic fields); ++, moderate parasite load (25–50% positive microscopic fields); +++, intense parasite load (> 50% positive microscopic fields)]. A minimum of 10 high-power fields at 400× magnification were evaluated.
For IHC, a protocol described by Tafuri et al. [13] was used, with slight modifications. Briefly, immunohistochemical labeling was performed on 4-μm-thick serial sections mounted on poly-L-lysine-coated slides. After deparaffination in xylene and rehydration in graded alcohols, antigen retrieval was performed in a microwave by immersion of slides in a pre-heated citrate solution (pH 6.0). The slides were then washed with Tris-buffered saline (TBS) buffer and incubated in 3% H2O2 for 10 min. After protein blocking (ab93677; Abcam, Cambridge, UK), the slides were incubated overnight in a humidified chamber with the serum of a dog naturally infected with Leishmania infantum (titre 1:320), at a 1:2000 dilution, which was applied as the primary antibody. As a secondary antibody, a horseradish peroxidase-conjugated goat polyclonal anti-dog antibody (ab112835; Abcam) was applied at 1:200 dilution and incubated for 1 h at room temperature. After the secondary antibody, immunolabeling was revealed with DAB (3,3′-diaminobenzidine; ab64238; Abcam), and Mayer’s hematoxylin was applied as a counterstain. A known positive skin sample from a sick dog due to CanL was used as a positive control. Negative controls were incubated with TBS, omitting the primary antibody. In addition, skin sections collected at necropsy from PCR-negative dogs from a non-endemic country were used as negative controls. The skin tissue parasite loads were semi-quantitatively analyzed by the presence of immunolabeled amastigote forms of Leishmania associated with the chronic inflammatory reaction in the dermis at 400× magnification; the grading system was the same as that described for the morphological (H&E) examination.
Molecular methods
Five 5-mm-thick sections of each FFPE skin tissue block were cut and handled with a new disposable razor blade and new gloves to prevent cross-contamination with Leishmania DNA. After each block was cut, the microtome blade, tweezers and entire cutting area were carefully cleaned with a 0.1 M solution of sodium hypochlorite to break down any potential contamination.
The sections were deparaffinized at room temperature by two consecutive immersion washes, 30 min each wash, in 1 ml of xylene and then rinsed twice, 5 min each rinse, with 1 ml of 100% ethanol. The samples were centrifuged at 10,000 g for 5 min, and the liquid was decanted between each change. Total genomic DNA was extracted using the ExgeneTM Clinic SV Mini Kit (GeneAll, Seoul, Korea), according to the manufacturer’s protocol. The extracted DNA was used in a cPCR protocol to amplify a final 120-bp fragment of conserved region of the Leishmania kinetoplastic (k) DNA minicircle, according to Francino et al. [21]. The Leishmania-specific primers used were NP13A (forward: 5′- AACTTTTCTGGTCCTCCGGG -3′) and NP13B (reverse: 5′- CCCCCAGTTTCCCGCCC -3′). Reaction mixtures were prepared in a total reaction volume of 50 μl that contained 25.0 μl of EconoTaq PLUS GREEN 2× Master mix (Lucigen Corporation, Middleton, WI, USA), 1 μM of sense primer, 1 μM of reverse primer and 1 μl of extracted DNA, ranging from 50 to 100 ng for each reaction. The conditions for the cPCR amplification were: initial denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s and extension at 72 °C for 1 min, for 50 cycles; and a final extension at 72 °C for 5 min.
The reaction was carried out in a StepOnePlus™ instrument (Applied Biosystems, Thermo Fischer Scientific, Foster City, CA, USA). Each reaction included a negative (sterile water) and a positive control (DNA extracted from L. infantum-cultured promastigotes).
Samples (each sample: 15 μl) of the amplification products were electrophoresed in a 1.2% agar gel for 30 min at 100 V in TBE buffer (89 mM Tris borate, 2.0 mM EDTA, pH 8.3) with 5 ul of EuroSafe Nucleic Acid Stain (EuroClone S.p.A., Pero, Italy) and 10 ul of SharpMassTM 100 ready-to load DNA ladder (100 bp) (EuroClone S.p.A.) to determine the PCR fragment size. The gel was visualized under UV transillumination.
The amplicons obtained from cPCR were directly sequenced in both directions using a 16-capillary ABI PRISM 3130 × l Genetic Analyzer, assembled and edited with SeqScape software v 2.5 (all Applied Biosystems). The assembled sequences were compared to Leishmania spp. sequences available in GenBank using the Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/; accessed on 05 Feb 2022).
Detection and quantification of L. infantum DNA was performed using a real-time PCR commercial kit (TIB Molbiol, Genova, Italy), as described by Solano-Gallego et al. [22]. The kit was based on a couple of primers and a fluorescent resonance energy transfer (FRET) probe specific for L. infantum kinetoplast DNA minicircles. Real-time PCR was carried out using LightCycler FastStart DNA MasterPLUS Hybridization Probes (Roche, Mannheim, Germany) in a LightCycler II instrument (Roche); the composition of the reaction mix and thermal cycling conditions were according to the manufacturer’s protocol. A no-template control (water) and a negative control were included in the qPCR run in order to exclude the risk of contamination. Parasite load was quantified using the absolute quantification method. Serial tenfold dilutions of recombinant plasmid containing the target DNA with a known copy number (ranging from 1 × 108 to 1 × 100 copies/μl) were used to generate the qPCR standard curve. Results were expressed as target DNA copies per microliter (copies/μl) of extract.
A grading system based on the amount of target DNA copies per microliter of extract was used. Samples were arbitrarily classified as negative (−, no amplification), low (+, 1–100 copies/μl), moderate (++, 101–10,000 copies/μl) or intense (+++, > 10,000 copies/μl).
Statistical analysis
The frequencies of positive results obtained from all the skin samples through the diagnostic tests (e.g. H&E staining, IHC, cPCR and qPCR) with 95% confidence intervals (CI) were calculated and compared using the Chi-square test (χ2) test. An accepted level of significance was set at p < 0.05. In addition, the degree of agreement between the evaluated tests was determined by the Kappa coefficient (κ) value and interpreted as follows: κ = 0.01–0.20, scant agreement; κ = 0.21–0.4, fair agreement; κ = 0.41–0.60, moderate agreement; κ = 0.61–0.80, good agreement; and κ ≥ 0.80, almost total agreement.
A preliminary statistical descriptive analysis of the dependent variables was performed taking into consideration the severity, the depth and the pattern of dermal inflammation. The position indices (median and mode) were calculated in order to find the “central trend” of the variables, as well as to determine the value corresponding to the maximum observed absolute frequency. The associations between the histologic variables and grading of the parasitic load detected by H&E staining, IHC and qPCR were assessed using the χ2 test. To verify any correlations between variables, we used the Pearson test (Gaussian distribution data) and the Spearman test (non-normal data). Associations between histological variables and the presence of the parasite were assessed with the Mann–Whitney U-test. The calculations were carried out using the SPSS computer program for epidemiologist V.11.30 (IBM Corp., Armonk, NY, USA).