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Canine leishmaniosis (CanL) is an emerging zoonosis caused by Leishmania infantum and transmitted in southern Europe by phlebotomine sand flies of the subgenus Phlebotomus (Larroussius). Endemic foci of CanL have been recorded in northern continental Italy since early 1990s and attributed to the northward expansion of vector populations due to climatic changes in association with travelling/relocated infected dogs from the southern Mediterranean littoral. In this study, further spread of endemic Leishmania foci was monitored during 2018-2019 in five regions (Aosta Valley, Piedmont, Lombardy, Veneto and Friuli-Venezia Giulia), with focus to territories where investigations were not performed, or they have been inconclusive. Clinical cases of CanL identified by local veterinary practitioners and confirmed by reference diagnosis centers were regarded as autochthonous if their origin from, or travel to, areas endemic for CanL were excluded in the previous ≥2 years. Around these index cases, i) serosurveys for L infantum were carried out where indicated, ii) sampling from potential autochthonous cases in healthy or clinically-suspected resident dogs was intensified by collaborating veterinary practitioners, and iii) suitable sites were investigated for the presence of competent phlebotomine vectors. Fifty-seven municipalities whose enzootic status of CanL was unreported before 2018, were identified as endemic. The stability of 27 foci recorded over the past decade, was also confirmed. Competent phlebotomine vectors, mainly Phlebotomus perniciosus, were collected for the first time in 23 municipalities. The newly recorded endemic municipalities appear to be distributed over a west-to-east decreasing gradient: 30 in Piedmont, 21 in Lombardy, 4 in Veneto and 2 in Friuli-Venezia Giulia. As regards Veneto, it should be noted that a relatively restricted territory was investigated as several municipalities of the region had already been surveyed and detected as endemic for CanL in the past. Cold climate conditions of the easternmost region of Friuli-Venezia Giulia bordering non-endemic territories of Slovenia, are probably less favorable to L infantum transmission., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

We compared results of a serum immunofluorescence assay (IFA) and lymph node quantitative PCR (qPCR) in dogs classified as exposed, infected, or sick because of leishmaniasis. We also determined how IFA or qPCR results changed in response to treatment and reflected different clinical and clinicopathologic improvement of dogs. We included 108 dogs in our retrospective study: 12 exposed, 25 infected, and 71 sick, as classified according to Canine Leishmaniasis Working Group standards. Between-group comparison showed higher IFA values ( p < 0.01) for sick dogs; qPCR values were higher for sick than infected dogs ( p < 0.01). A novel clinical and clinicopathologic score was created and applied to 50 sick dogs. Using this score, 41 were reclassified as partially recovered (PR) within 3 mo, and 37 as totally recovered (TR) 3-6 mo after presentation. Statistically significant differences in IFA values were found between the sick and TR dogs ( p < 0.01), but not between sick and PR dogs ( p = 0.98). During follow-up, qPCR revealed a progressive decrease in parasite load, with a statistically significant difference in sick versus PR ( p < 0.01), sick versus TR ( p < 0.01), and PR versus TR ( p < 0.01) dogs. A decrease of 1 point in the clinical score corresponded to 1.3 Leishmania/μL qPCR decrease ( p < 0.01) and decrease of 1:42 in IFA ( p < 0.01). Our findings confirm that the clinical status of dogs affected by leishmaniasis is closely related to parasite load and antibody level, both before and after treatment.

This paper reports the findings of a study on the presence of various species of filarial nematodes in dogs in Liguria, north-west Italy, a region traditionally considered free from the disease. Between 2009 and 2012 blood samples were taken from 365 dogs in rural areas in Liguria. The blood samples were then submitted to Knott's test, histochemical staining, polymerase chain reaction (PCR) and the enzyme-linked immunosorbent assay (ELISA) for Dirofilaria immitis antigens. Overall, 35 of the 365 dogs were positive using Knott's test for microfilariae (prevalence 9.6%; 95% confidence interval (CI): 6.6-12.6%). Acanthocheilonema reconditum was the most prevalent species (8.0%), while Dirofilaria repens (1.4%) and Dirofilaria immitis (0.6%) were less common. One co-infection by D. repens and A. reconditum was observed. All morphological identifications were confirmed by histochemical staining and PCR. In addition, a retrospective analysis of data on D. immitis antigens in 11,363 samples of canine sera was carried out. Sera were collected and analysed for D. immitis antigens by the Istituto Zooprofilattico Sperimentale (IZS) of Piedmont, Liguria and Aosta Valley (Imperia section) between 2004 and 2013 during annual tests for leishmaniasis on autochthonous dogs throughout Liguria. Serological data from IZS showed an overall seroprevalence of 0.65% (95% CI: 0.50-0.80%) for D. immitis throughout the region. The present study updates the epidemiological map of canine filarial infections in Italy and suggests the need for surveillance and prophylaxis in Liguria.

In this study, we compared a rapid immunochromatographic test (Speed Leish K; BVT Groupe Virbac, La Seyne sur Mer, France) with an indirect immunofluorescence assay (IFAT) and Western blotting (WB) for the detection of Leishmania infantum antibodies in dogs. A total of 250 serum samples were collected from 125 L. infantum-positive and 125 L. infantum-negative dogs. Among the positive samples, 81 were strongly positive at low IFAT dilutions, while 44 were low-reactivity sera (IFAT titers, 1:40 to 1:80). The sensitivity and specificity of the Speed Leish K were 96.3% and 100%, respectively, compared with those of the IFAT. When IFAT low-reactivity sera (titers, 1:40 or 1:80) were tested with the Speed Leish K, using WB results as a reference, the sensitivities were 93.75% for sera with a 1:80 titer and 73.33% for sera with a 1:40 titer, and the specificity was 100%. The Speed Leish K is easy to use and performs well, so it can be considered a quick and reliable tool for the diagnosis of L. infantum infection in dogs.

In this study, a recombinant chimeric antigen (CA) ELISA was validated as a single test for both human and dog leishmaniasis. Serum panels included 327 human and 339 canine IFAT-positive and 1113 human and 1078 canine IFAT-negative samples. CA-ELISA was carried out using the same serum dilution, and labelled protein A as secondary reagent. Test performances were calculated using ROC analysis. For the human panel, the test showed diagnostic accuracy (DA) 0.974, specificity (Sp) 97.12%, sensitivity (Se) 91.44%, and concordance (K) 0.88. The dog panel showed DA 0.998, Sp 99.54%, Se 98.54%, and K 0.98. The proposed method is the best recombinant antigen-based ELISA, and can be used as IFAT substitute for mass screening.

In this study we compared a commercial enzyme linked immunosorbent assay (ELISA) rapid test (Snap CLATK Canine Leishmania Antibody Test Kit, IDEXX-Snap) with indirect immunofluorescence assay (IFA) and Western blot (WB) for the detection of Leishmania infantum antibodies in dogs. In total sera from 234 dogs were collected: 59 positives and 51 doubtful sera (IFA 1:40-1:80) from an L. infantum endemic area and 124 negative sera from a non-endemic area were tested. To evaluate the Snap CLATK's performances on whole blood, blood in EDTA and sera from 37 dogs were tested in parallel with Snap CLATK. Snap CLATK sensitivity and specificity compared to IFA were 91.1% and 99.2%, while compared to WB were 93.4% and 98.3%, respectively. When IFA doubtful sera (titers of 1:40 or 1:80) were tested Snap CLATK, using WB as reference, sensitivity and specificity were 90.9% and 100%, respectively. Moreover, a complete concordance was observed when Snap CLATK rapid assay was carried out on whole blood or sera from 37 dogs. The Snap CLATK has demonstrated simplicity and performance and can be considered a quick and reliable alternative for the diagnosis of L. infantum infection in dogs.

We report the results of a survey to determine the prevalence of canine leishmaniasis and the presence of sand flies in northwestern Italy, where autochthonous foci of canine leishmaniasis have not been reported. Active foci of canine leishmaniasis were identified, which suggests that the disease is now also endemic in continental climate areas.

Canine infections with Leishmania infantum are important as a cause of serious disease in the dog and as a reservoir for human visceral leishmaniasis (VL). Accurate diagnosis of canine infections is essential to the veterinary community and for VL surveillance programs. A standardized ELISA using a purified recombinant antigen (rK39) specific to VL was compared to the immunofluorescent antibody test (IFAT) as the standard. The ELISA was developed, optimized and evaluated using sera from 6368 dogs. The standardized ELISA and IFAT results were highly concordant. The timing and pattern of ELISA and IFAT seroconversion in dogs followed prospectively after natural infections were very similar. Antibodies reacting with rK39 were more common in asymptomatic canine infections than reported for subclinical human VL. The rK39 ELISA is a relatively simple and rapid assay for assessing the infection status of dogs, and is an alternative to IFAT, especially when screening large numbers of samples.

An epidemiological survey was carried out on the distribution of canine leishmaniasis in Western Liguria (Northern Italy). Blood sera from 21146 dogs collected from 1990 to 1996 were subjected to the indirect immunofluorescence test (IFAT). From 1990 to 1994 (16690 dogs), only monthly seroprevalences were calculated (min 14%-max 60.5%). Dog sera sampled from October 1994 to May 1996 (4456 dogs) were divided into two subgroups (I: October 1994-May 1995; II: October 1995-May 1996). Seroprevalence was 30.3% and 22.1% in these two groups, respectively. Data analysis by log-linear models revealed that serological positivity to Leishmania sp. was significantly associated with a dog's outdoor lifestyle, with a rural environment and with the male sex. Of the 218 dogs which were tested twice seroconversion rates were also higher in outdoor, rural and male dogs while negativization rates were lower in the same groups. Basic reproductive numbers (R0) were 1.53 (subgroup I) and 1.28 (subgroup II).