Enhanced apoptotic index in hepatocytes, Kupffer cells, and inflammatory infiltrate showed positive correlation with hepatic lesion intensity, parasite load, and clinical status in naturally Leishmania-infected dogs.

It is unknown if Leishmania amastigote infections affect hepatocytes and Kupffer cell apoptosis, and the role played by apoptosis in liver lesions in leishmaniasis is still unclear. Clinically affected and subclinically infected dogs with leishmaniosis and uninfected controls were assessed. Parasite load, biochemical markers for evaluation of liver damage, morphometry (area, perimeter, number of inflammatory focus, major and minor diameters), apoptosis in hepatic tissue (hepatocytes, Kupffer cells, and inflammatory infiltrates) and cellularity in inflammatory foci were quantified. The parasite load in clinically affected dogs proved to be higher than in the other groups. All morphometric parameters (area, perimeter, number of inflammatory focus, major and minor diameters) from clinically affected were higher than the values found in the subclinically infected and uninfected control dogs. Only clinically affected dogs presented high levels of ALT, FA, GGT and cholesterol in serum. Strong positive correlation was observed between biochemical markers for evaluation of liver damage (ALT, FA, GGT and cholesterol) and hepatic apoptosis (hepatocytes, Kupffer cells, and inflammation). Clinically affected dogs showed a more intense hepatic lesion. Hepatocytes showed a higher rate of apoptosis in Leishmania-infected dogs than in uninfected control dogs. The Kupffer cell apoptotic index and apoptosis within the inflammatory infiltrates were higher in clinically affected dogs. The apoptotic index evaluated in hepatocytes, Kupffer cells, and inflammatory infiltrates showed a positive correlation with the intensity of the hepatic lesion, parasite load, and clinical status. Apoptotic cells also showed positive immunostaining for TUNEL, Bcl2, and Bax. Our data showed that hepatic apoptosis was related to the severity of liver damage, the progression of infection, and the parasite load in leishmaniasis. Apoptotic regulated cell recruitment modulated the inflammatory response and favored the survival and dissemination of parasites, depending on the clinical status of the Leishmania-infected dogs.

Characterization and Proteomic Analysis of Plasma EVs Recovered from Healthy and Diseased Dogs with Canine Leishmaniosis.

Dogs are highly valued companions and work animals that are susceptible to many life-threatening conditions such as canine leishmaniosis (CanL). Plasma-derived extracellular vesicles (EVs), exploited extensively in biomarker discovery, constitute a mostly untapped resource in veterinary sciences. Thus, the definition of proteins associated with plasma EVs recovered from healthy and diseased dogs with a relevant pathogen would be important for biomarker development. For this, we recovered, using size-exclusion chromatography (SEC), EVs from 19 healthy and 20 CanL dogs' plasma and performed proteomic analysis by LC-MS/MS to define their core proteomic composition and search for CanL-associated alterations. EVs-specific markers were identified in all preparations and also non-EVs proteins. Some EVs markers such as CD82 were specific to the healthy animals, while others, such as the Integrin beta 3 were identified in most samples. The EVs-enriched preparations allowed the identification of 529 canine proteins that were identified in both groups, while 465 and 154 were only identified in healthy or CanL samples, respectively. A GO enrichment analysis revealed few CanL-specific terms. Leishmania spp. protein identifications were also found, although with only one unique peptide. Ultimately, CanL-associated proteins of interest were identified and a core proteome was revealed that will be available for intra- and inter-species comparisons.

Antibodies elicited by the CaniLeish® vaccine: long-term clinical follow-up study of dogs in Spain.

The prevention of canine leishmaniosis in healthy dogs requires a multimodal approach combining repellents with an effective vaccine. A vaccine that modulates the cell-mediated immune response against the protozoan has been available in Europe since 2012 (CaniLeish®, Virbac, France). The aim of the present study was to monitor dogs vaccinated with CaniLeish® to examine the kinetics of the antibody response and the safety and tolerance of CaniLeish®. Dogs vaccinated with CaniLeish® were monitored for 12 months. In follow-up visits at baseline (primovaccination or annual booster) (Visit 1, V1), and 1 (V2), 4 (V3), 8 (V4) and 12 (V5) months later, we examined antibody response kinetics using two serology techniques (IFAT and Speed Leish K™). Tolerance to CaniLeish® and its safety were also monitored. Anti-L. infantum IgG antibodies were determined in 242 dogs (125 dogs after primovaccination (Group P) and 117 dogs after booster vaccination (Group B). In addition, 46, 22 and 19 dogs were followed for 2, 3 and 4 years, respectively. At baseline, 100% of dogs in Group P returned negative IFAT and Speed Leish K™ test results while 9.4% (11/117) in Group B tested IFAT positive though Speed Leish K™ negative. In subsequent visits, seropositivity was detected by IFAT in 31.2% (Group P) and 41% (Group B) of the dogs in V2; 16.8% (Group P) and 10.2% (Group B) in V3; 6.4% (Group P) and 8.5% (Group B) in V4; and 3.2% (Group P) and 5.9% (Group B) in V5. All dogs tested Speed Leish K™ negative except two, in which it was later confirmed by molecular testing that they were not infected. Adverse events that could be associated with the vaccine were detected in 20 out of 314 dogs (6.4%). The good clinical status of all dogs was confirmed in an exhaustive clinical exam and haemato-biochemical profile. The Canileish® vaccine was well-tolerated with exceptions that did not appear to be related to age, sex, race or size of vaccinated dogs. Anti-L. infantum antibodies were detected by IFAT in 31.9-40.3% of the dogs 1 month after vaccination, and these antibodies could still be detected in 3.2% of the dogs 1 year later. This means that veterinarians need to use other tools (eg. PCR) to correctly diagnose seropositive dogs.

Commercially approved vaccines for canine leishmaniosis: a review of available data on their safety and efficacy.

Canine leishmaniosis is an important vector-borne zoonosis caused mainly by Leishmania infantum. Diagnosis and treatment of affected individuals can be particularly complex, hindering infection control in endemic areas. Methods to prevent canine leishmaniosis include the use of topical insecticides, prophylactic immunotherapy and vaccination. Four vaccines against canine leishmaniosis have been licensed since 2004, two in Brazil (Leishmune®, the production and marketing licence of which was withdrawn in 2014, and Leish-Tec®) and two in Europe (CaniLeish® and LetiFend®). After several years of marketing, doubts remain regarding vaccine efficacy and effectiveness, potential infectiousness of vaccinated and infected animals or the interference of vaccine-induced antibodies in L. infantum serological diagnosis. This review summarises the scientific evidence for each of the vaccines commercially approved for canine leishmaniosis, while discussing possible weaknesses of these studies. Furthermore, it raises the need to address important questions related to vaccination impact in Leishmania-endemic countries and the importance of post-marketing pharmacological surveillance.

La leishmaniose canine est une importante zoonose à transmission vectorielle causée principalement par Leishmania infantum. Le diagnostic et le traitement des personnes atteintes peuvent être particulièrement complexes, entravant la lutte contre l'infection dans les zones d'endémie. Les méthodes de prévention de la leishmaniose canine comprennent l'utilisation d'insecticides topiques, l'immunothérapie prophylactique et la vaccination. Quatre vaccins contre la leishmaniose canine ont été homologués depuis 2004, deux au Brésil (Leishmune®, dont la licence de production et de commercialisation a été retirée en 2014 et Leish-Tec®) et deux en Europe (CaniLeish® et LetiFend®). Après plusieurs années de commercialisation, des doutes subsistent quant à l'efficacité et à l'effet du vaccin, au potentiel infectieux des animaux vaccinés et infectés ou à l'interférence des anticorps induits par le vaccin dans le diagnostic sérologique de L. infantum. Cette revue résume les données scientifiques de chacun des vaccins commercialement approuvés pour la leishmaniose canine, tout en discutant des possibles faiblesses de ces études. En outre, il soulève la nécessité de répondre à des questions importantes liées à l'impact de la vaccination dans les pays où la Leishmania est endémique et à l'importance de la surveillance pharmacologique post-marketing.

Canine neutrophils cooperate with macrophages in the early stages of Leishmania infantum in vitro infection.

Leishmania infantum is the aetiological agent of human visceral leishmaniasis and canine leishmaniasis, both systemic and potentially fatal diseases. Polymorphonuclear neutrophils (PMN) are the first cells to phagocyte this parasite at the inoculation site, but macrophages (MØ) are the definitive host cells, ensuring parasite replication. The interaction between dog MØ, PMN and L infantum promastigotes was in vitro investigated. It was observed that promastigotes establish contact with blood monocyte-derived MØ mainly by the tip of the flagellum. These cells, that efficiently bind and internalize parasites, underwent major morphological changes, produced nitric oxide (NO) and released histone H1 in order to inactivate the parasite. Transfer of intracellular parasites from PMN to MØ was confirmed by flow cytometry, using L infantum expressing a green fluorescent protein. The interaction of MØ with L infantum-infected PMN lead to NO production and release of extracellular traps, which may contribute to parasite containment and inactivation. This study highlights for the first time the diversity of cellular and molecular events triggered by the interaction between canine PMN and MØ, which can promote a reduction of parasite burden in the early phase of L infantum infection.

Use of acute phase proteins for the clinical assessment and management of canine leishmaniosis: general recommendations.

BACKGROUND: Dogs with canine leishmaniosis (CanL) due to Leishmania infantum can show a wide spectrum of clinical and clinicopathological findings at the time of diagnosis. The aim of this paper is to describe the possible application of acute phase proteins (APPs) for the characterization and management of this disease, based on previously published information on the utility of APPs in CanL and the experience of the authors in using APPs as analytes in the profiling of canine diseases. MAIN BODY: Dogs diagnosed with L. infantum infection by serology, polymerase chain reaction, cytological or histopathological identification, can be divided into three groups based on their clinical condition at physical examination and their APPs concentrations: Group 1: dogs with no clinical signs on physical examination and APPs in reference range; Group 2: dogs with changes in APPs but no clinical signs on physical examination; Group 3: dogs with clinical signs and changes in APPs. This report describes the main characteristics of each group as well as its association with the clinical classification schemes of CanL. CONCLUSION: APPs concentration can be a useful clinical tool to characterize and manage CanL.

Serological and entomological survey of canine leishmaniasis in Lampedusa island, Italy.

BACKGROUND: During last decade Lampedusa island (Italy) has been interested by a deep social change caused by the massive arrival of migrants from north Africa. The goal of this study was to evaluate current CanL burden and risk factors for Visceral Leishmaniosis (VL) on Lampedusa, actually based on very few data obtained in a previous study performed fifteen years ago. Two hundred and forty-two dogs were enrolled for the detection of Leishmania infantum infection by serology. In addition, an entomological investigation was performed to confirm the presence of Leishmania-vectors. RESULTS: Seroprevalence was of 54.13%. 223 sand flies specimens were collected. Among them, 4 species were identified: Phlebotomus perniciosus, P. papatasi, P. neglectus, Sergentomia minuta, with P. perniciosus the most abundant (67.7%; p < 0.01). CONCLUSION: The high proportion of seropositive dogs together with the presence of the most competent vector for L. infantum, P. perniciosus, demonstrate that L. infantum abundantly circulates in the island and may constitute a risk for people, particularly for hosted migrants.

Canine Leishmania infantum infection: an imported case in UK after staying in the Canary Islands.

Leishmaniosis is reported in the Iberian Peninsula and the Balearic Islands, but the Canary Islands are deemed free. In the present communication, we report a clinical leishmaniosis due to Leishmania infantum in a dog that was presumptively infected during its stay on Tenerife. The result of Leishmania serology (whole-cell based ELISA with L. infantum antigen) was high positive (test score of 82.2 at a cut-off value of 12.0). This result was further confirmed with quantitative real-time polymerase chain reaction (qPCR) for Leishmania spp. on a blood sample. A medium load of parasites was detected (48 parasites/ml blood). L. infantum was identified by RFLP analysis of the ITS-1 PCR product. Confirmation that leishmaniosis is endemic to the Canary Islands would further require study on local dogs with no travel history as well as reassessment on frequency and distribution of Phlebotomus spp. as well as Leishmania spp. detection in the sand fly vector. However, this case strongly suggests that L. infantum is present on the Canary Islands. Although transmission seems to be still exceptional, preventive measures in dogs travelling to the Canaries should be considered.

The use of Escherichia coli total antigens as a complementary approach to address seropositivity to Leishmania antigens in canine leishmaniosis.

Canine leishmaniosis (CanL) is a major veterinary concern and a public health issue. Serological data are essential for disease management. Several antigens used in serological assays have specificity related problems preventing relevant seropositivity values establishment. Herein we report significant seropositivity level disparity in a study cohort with 384 dogs from eight countries, for antigens traditionally used in CanL - soluble promastigote Leishmania antigens (SPLA) and K39 recombinant protein (rK39): 43·8 and 2·9% for SPLA and rK39, respectively. To better understand the reasons for this disparity, CanL-associated serological response was characterized using, for complement serological evaluation, a ubiquitous antigen - soluble Escherichia coli antigens (SECAs). Using cohorts of CanL dogs and dogs without clinical evidences of CanL from non-endemic regions of Portugal, the serological response of CanL animals followed specific trend of seropositivity rK39 > SPLA > SECA absent in non-diseased animals. Using receiver operating characteristic curve analysis, these characteristic trends were converted in ratios, SPLA/SECA, rK39/SECA and rK39/SPLA, that presented high predictive for discriminating the CanL cohort that was potentiated when applied in a scoring system involving positivity to four out of five predictors (rK39, SPLA, SPLA/SECA, rK39/SECA and rK39/SPLA). In fact, this approach discriminated CanL with similar sensitivity/specificity as reference antigens, diminishing seropositivity in European cohort to 1·8%. Ultimately, non-related antigens like SECA and seropositivity ratios between antigens enable different perspectives into serological data focusing on the search of characteristic serological signatures and not simple absolute serology values contributing to comprehensive serological status characterization.

Insights on adaptive and innate immunity in canine leishmaniosis.

Canine leishmaniosis (CanL) is caused by the parasite Leishmania infantum and is a systemic disease, which can present with variable clinical signs, and clinicopathological abnormalities. Clinical manifestations can range from subclinical infection to very severe systemic disease. Leishmaniosis is categorized as a neglected tropical disease and the complex immune responses associated with Leishmania species makes therapeutic treatments and vaccine development challenging for both dogs and humans. In this review, we summarize innate and adaptive immune responses associated with L. infantum infection in dogs, and we discuss the problems associated with the disease as well as potential solutions and the future direction of required research to help control the parasite.

Detection of intracellular IFN-γ and IL-4 cytokines in CD4+ and CD8+ T cells in the peripheral blood of dogs naturally infected with Leishmania infantum.

Canine leishmaniosis (CanL) is a systemic zoonotic disease the clinical manifestations of which can range from self-healing cutaneous lesions to disseminated visceral disease. Effective activation of cellular immunity is the cornerstone of resistance against Leishmania infantum in infected dogs. The aim of this cross-sectional, controlled study was the intracellular detection of interleukin 4 (IL-4) and interferon-γ (IFN-γ) in CD4+ and CD8+ lymphocytes in the peripheral blood of 40 dogs naturally infected with L. infantum by applying flow cytometry. The percentage of CD4+IL-4+ and CD8+IL-4+ lymphocytes (with or without immunostimulation) was low in the clinically healthy and subclinically infected dogs in contrast to clinically affected ones. In the same groups of dogs, the percentage of CD4+IFN-γ+ and CD8+IFN-γ+ T cells in their resting phase and following specific immunostimulation with Leishmania soluble antigen (LSA) was also low. CD4+IL-4+ and CD8+IL-4+ T cell percentage was higher in sick compared to clinically healthy and subclinically infected dogs, after immunostimulation. The corresponding figure of CD8+IL-4+ cells in sick dogs after LSA immunostimulation was also increased thus underlining the important role these cells may play in humoral immunity and perhaps the progression of CanL.

Erythrocyte sedimentation rate in heartworm naturally infected dogs "with or without" Leishmania infantum seropositivity: an observational prospective study.

Canine heartworm disease by Dirofilaria immitis and canine leishmaniosis by Leishmania infantum (CanL) are both vector-borne diseases with frequently overlapping endemicity and able to trigger the acute phase response, being characterized by variations in acute phase proteins (APP). Recently, erythrocyte sedimentation rate (ESR), an indicator of inflammation, has gained attention in veterinary medicine, proving useful in several conditions that include CanL active forms in dogs. This study aims to evaluate ESR in heartworm-infected dogs, compare levels with heartworm-infected and L. infantum seropositive dogs as well as clinically healthy dogs, and assess correlations with other laboratory parameters. From October 2022 to January 2023, a prospective observational study was conducted enrolling heartworm-infected (Dirofilaria group) and heartworm-infected L. infantum seropositive (Dirofilaria/Leishmania group) animals subgrouped according to the CanL clinical form (Dirofilaria/Leishmania active and non-active groups). A group of clinically healthy dogs (control group) was also included. For each dog enrolled physical examination and laboratory tests (complete blood count, biochemical panel including APP, serum protein electrophoresis) were performed. Dirofilaria and Dirofilaria/Leishmania groups presented a significantly higher ESR level compared to healthy dogs. Dirofilaria/Leishmania active group had the highest ESR level among the groups considered. Dirofilaria/Leishmania non-active group had an ESR similar to the Dirofilaria group, but significantly higher and lower compared to the control and the Dirofilaria/Leishmania active group, respectively. A significant positive correlation between ESR and C-Reactive Protein has been found in all groups except for the Dirofilaria/Leishmania non-active group. In Dirofilaria/Leishmania active group a strong positive correlation between ESR and gamma globulins percentage as well as a strong negative correlation between ESR and albumin, albumin/globulins ratio were found. Overall, the ESR was confirmed to be an inflammation marker as well as a helpful disease index, being notably increased in heartworm-infected dogs affected by an active form of CanL.

Urinary neutrophil gelatinase-associated lipocalin as early biomarker for renal disease in dogs with leishmaniosis.

Canine leishmaniosis (CanL), caused by Leishmania sp., presents a wide array of symptoms; renal dysfunction is frequently observed in these dogs and is associated with a poor prognosis and increased mortality. The traditional biomarkers namely urea and creatinine can detect renal damage but only in advanced stages of the disease. However, it has been shown that the symmetric dimethylarginine assay (SDMA) or the protein/creatinine ratio (UPC) and are early biomarkers of renal dysfunction. Their elevation occurs earlier than that of creatinine, but other novel biomarkers such as neutrophil gelatinase-associated lipocalin (NGAL) are currently under investigation. Our objective was to determine whether the urine NGAL-creatinine ratio (uNGAL/c) can provide very early diagnosis of kidney disease in CanL. In total, 68 dogs were included in the study: 15 healthy dogs and 53 dogs with CanL who were classified according to International Renal Interest Society (IRIS) classification: IRIS 1 (N= 34), IRIS 2 (N= 9) and IRIS 3/4 (N= 10). IRIS 1 was subdivided according to proteinuria in IRIS 1NP (13 dogs with UPC < 0.2), IRIS 1BL (8 dogs with UPC = 0.2-0.5) and IRIS 1 P (13 dogs with UPC > 0.5). Blood samples were collected for complete hematological and biochemistry analysis including plasma NGAL. Urinalysis included specific gravity, UPC, CysC and NGAL expressed as a ratio with creatinine. The mean concentrations of pCysC and SDMA in CanL, show a statistically significant increase from IRIS 1NP, not being statistically significant for pCysC in the IRIS 1BL group. The UPC show a statistically significant increase from IRIS 1NP. In all groups with CanL for uCysC/c and uNGAL/c was observed a statistically significant increase. The uNGAL/c in the group proteinuric animals, presents a positive correlation with all renal biomarkers studied. In the group of non-proteinuric animals, the uNGAL/c presents a positive correlation with SDMA and UPC. The uNGAL/c can be considered a reliable indicator of renal disease in dogs diagnosed with CanL who are non-azotemic and non-proteinuric.

Efficacy of a combination of nucleotides and lactoferrin in maintaining stable or improving the clinical picture and laboratory findings of leishmaniotic dogs: A randomized controlled study.

This prospective, randomized, controlled, therapeutic study aimed to evaluate the efficacy of a product containing nucleotides and lactoferrin in maintaining or improving the clinical picture and laboratory findings of canine leishmaniosis (CanL). The safety and tolerance of this combination were also assessed. Forty Leishmania infantum-seropositive dogs, not requiring leishmanicidal and/or leishmaniostatic treatment, were enrolled in the study and randomized into treatment (TG) and placebo (CG) groups. Products A (containing nucleotides and lactoferrin) and B (placebo) were blindly administered to TG and CG, respectively, as palatable tablets at a rate of 1 tablet per 10 kg of weight once every 24 h for 6 months. Following inclusion (T0), dogs were followed up after 3 (T90) and 6 (T180) months. At each time point, for all animals enrolled physical examination and laboratory tests (complete blood count, biochemical panel including C-reactive protein [CRP] and ferritin, and serum protein electrophoresis) were performed. The immunofluorescence antibody test to detect antibodies for L. infantum (T0, T180), Ehrlichia canis (T0, T90, and T180), and Anaplasma phagocytophilum (T0, T90, and T180) was executed. A CanL-dedicated clinical score, using a validated scale from 0 (i.e., absence of clinical signs) to 19, was assigned. Four dogs (n=2 in TG, n=2 in CG) did not complete the study. No statistically significant differences in CanL clinical score were observed between CG and TG at T0, T90 and T180. Both TG and CG showed significant variations in anti-L. infantum antibody titres (p=0.0001 and p=0.004, respectively). In TG, antibody titres decreased in 77.8 %, increased in 5.5 %, and remained stable in 16.7 % of dogs, while in CG, decreased in 27.8 %, increased in 50 %, and remained stable in 22.2 % of dogs. During the study, CRP and ferritin remained stable in TG and significantly increased in CG. At T180, 9 out of 18 dogs (50 %) enrolled in the CG, and 1 out of 18 (5.6 %) enrolled in the TG, developed an active form of leishmaniosis. No side effects were reported in any patient included. In conclusion, a 6-month oral administration of a supplement containing nucleotides and lactoferrin was effective in maintaining a stable clinical score, improving antibody titres and potentially reducing the progression from non-active to active forms in L. infatum seropositive dogs. Furthermore, the product was well-tolerated, easy to administer, and free of side effects.

Trends and Gaps in the Scientific Literature about the Effects of Nutritional Supplements on Canine Leishmaniosis.

In canine leishmaniosis (CanL), complex interactions between the parasites and the immunological background of the host influence the clinical presentation and evolution of infection and disease. Therefore, the potential use of nutraceuticals as immunomodulatory agents becomes of considerable interest. Some biological principles, mainly derived from plants and referred to as plant-derived nutraceuticals, are considered as supplementation for Leishmania spp. infection. This study provides a systematic review regarding the use of nutraceuticals as a treatment using a text mining (TM) and topic analysis (TA) approach to identify dominant topics of nutritional supplements in leishmaniosis-based research, summarize the temporal trend in topics, interpret the evolution within the last century and highlight any possible research gaps. Scopus® database was screened to select 18 records. Findings revealed an increasing trend in research records since 1994. TM identified terms with the highest weighted frequency and TA highlighted the main research areas, namely "Nutraceutical supports and their anti-inflammatory/antioxidant properties", "AHCC and nucleotides in CanL", "Vit. D3 and Leishmaniosis", "Functional food effects and Leishmaniosis" and "Extract effects and Leishmaniosis". Despite the existing academic interest, there are only a few studies on this issue so far, which reveals a gap in the literature that should be filled.

Corrigendum: Clinical validation of circulating immune complexes for use as a diagnostic marker of canine leishmaniosis.

[This corrects the article DOI: 10.3389/fvets.2024.1368929.].

Clinical validation of circulating immune complexes for use as a diagnostic marker of canine leishmaniosis.

Introduction: Canine leishmaniosis (CanL) is a systemic disease that affects dogs. When multiplication of the parasite cannot be controlled, dogs consistently show high levels of antigen and IgG antibodies, which lead to the formation of circulating immune complexes (CIC). Timely intervention to reduce the parasite load and CIC levels is crucial for preventing irreversible organ damage. However, a diagnostic test to quantify CIC levels is currently lacking. Methods: In this real-world study, we aimed to examine the performance of a new ELISA to measure CIC levels in dogs naturally infected with Leishmania infantum. Thirty-four dogs were treated according to their clinical condition and followed for 360 days. Before (day 0) and after treatment (days 30, 90, 180, 270, and 360), all dogs underwent a physical examination, and blood samples were obtained for CBC, biochemical profile, serum protein electrophoresis and IFAT. Serum PEG-precipitated CIC were determined by ELISA. Results: Our results indicate higher CIC levels in dogs in advanced disease stages showing higher antibody titres (p < 0.0001, r = 0.735), anemia (p < 0.0001), dysproteinemia (p < 0.0001), and proteinuria (p = 0.004). Importantly, dogs responding well to treatment exhibited declining CIC levels (p < 0.0001), while in poor responders and those experiencing relapses, CIC were consistently elevated. CIC emerged as a robust discriminator of relapse, with an area under the curve (AUC) of 0.808. The optimal cut-off to accurately identify relapse was an optical density of 1.539. Discussion: Our findings suggest that declining CIC levels should be expected in dogs showing a favorable treatment response. Conversely, in dogs displaying a poor response and recurrent clinical relapses, CIC levels will be high, emphasizing the need for vigilant monitoring. These findings suggest that CIC could serve as a valuable biomarker for disease progression, treatment efficacy, and relapse detection in CanL. Our study contributes to enhancing diagnostic approaches for CanL and underscores the potential of CIC as a complementary tool in veterinary practice. As we move forward, larger studies will be essential to confirm these findings and establish definitive cut-offs for clinical application.

Performance of a point-of-care test for the detection of anti-Leishmania infantum antibodies is associated with immunofluorescent antibody titer and clinical stage of leishmaniosis in dogs from endemic regions.

Canine leishmaniosis (CanL) is caused by the protozoal parasite Leishmania infantum, which is transmitted by sand flies in warm climates across the world. Because dogs are considered a primary domestic reservoir for the parasite that causes leishmaniosis in humans, it is important from a One Health perspective that CanL be properly managed. In endemic regions, CanL is a common differential diagnosis in sick dogs because the clinical signs and clinicopathological disorders of the disease are non-specific, variable, and may overlap those of other common conditions. Diagnosis is based on the presence of compatible clinical signs, laboratory abnormalities, and confirmation by serological and parasitological evidence of infection. Here, we describe the performance of a point-of-care (POC) immunoassay that uses recombinant antigens to detect canine anti- L. infantum antibodies in a convenience sample set from a diagnostic laboratory, a group of canine patients with clinical staging, and in apparently healthy dogs from endemic areas. An immunofluorescence antibody test (IFAT) was used as the semiquantitative reference method. In the convenience sample set with high IFAT titers (≥ 1:800), the POC immunoassay demonstrated perfect agreement with IFAT (100%; 90/90). Using samples from dogs staged as either LeishVet Stage 2 or 3 or LeishVet Stage 1, positive agreement of the POC immunoassay with the IFAT was 98.8% (82/83) and 83.8% (31/37), respectively. The negative agreement with IFAT was 98.9% (272/275) in apparently healthy dogs from endemic areas of Greece and Italy. Since the performance of the POC immunoassay was associated with IFAT titer and clinical stage of CanL, the test may help veterinarians when determining if CanL is likely responsible for a patient's clinical picture or when evaluating an apparently healthy patient prior to vaccination.

Twenty-year evolution of Leishmania infantum infection in dogs in Valdeorras (Galicia, Northwestern Spain): implication of climatic factors and preventive measures.

BACKGROUND: Abiotic factors play a significant role in the evolution of Leishmania infantum infection due to its vectorial nature. This study aims to assess the evolution in the detection of new L. infantum infection cases in Valdeorras (Ourense, Northwestern Spain) over a 20-year period and how different climatic variables and preventive measures may have affected it. METHODS: Indirect immunofluorescence antibody tests (IFAT) were performed on serum samples collected from dogs attending the 'Servicios Veterinarios de Sil' veterinary clinic (Valdeorras, Northwestern Spain) between May 2003 and April 2023 to detect L. infantum exposure. The percentage of new cases of L. infantum infection was calculated from May of one year to April of the following year. Climatic conditions in the region, global sales of ectoparasiticides and the number of vaccines against L. infantum delivered in the veterinary clinic from 2003 to 2022 were recorded. Statistical analyses were conducted to determine the associations between these factors and the percentage of new cases of L. infantum infection. RESULTS: A total of 2909 dogs were assessed, and 3785 IFAT tests were performed between May 2003 and April 2023. The mean percentage of new seropositive cases over the 20-year period studied was 21.65 ± 10.8%, with a decline from the beginning to the end of the period studied. The percentage was significantly higher between May 2003 and April 2008 compared with the other periods (May 2008 to April 2013, May 2013 to April 2018 and May 2018 to April 2023). There was a positive correlation between the percentage of new cases of L. infantum infection and the maximum relative humidity in winter. Conversely, there was a negative correlation between the percentage of new cases and sales of ectoparasiticides and vaccination against L. infantum. CONCLUSIONS: This study is one of the longest evaluations of the evolution of L. infantum infection in a fixed location and its association with external factors including climatic conditions and preventive measures. The results confirm that Valdeorras is a high-risk area for L. infantum infection. The use of ectoparasiticides and vaccines against L. infantum has been shown to play a significant role in preventing L. infantum infection, highlighting the crucial role of veterinarians in the fight against this disease.

Urinary Tamm-Horsfall protein excretion is a potential early biomarker of renal distal tubular damage in canine leishmaniosis.

This study evaluates distal tubular damage in early stages of renal disease in dogs with naturally acquired leishmaniosis. Pherograms of urinary proteins separated in vertical electrophoresis system (SDS-PAGE) were evaluated. Peptide fingerprint and fragmentation (MALDI-TOF TOF) identified bands located at 100 and 60 kDa as Tamm-Horsfall protein (THP) and albumin, respectively. The variables examined were: urine protein to creatinine ratio (UPC), total number of bands, quantification of THP urinary excretion through the optical density (OD %) of bands located at 100 kDa, blood creatinine, and urine specific gravity (USG). Positive correlation was found between UPC and the number of bands (ρ = 0.75849, P = <0.0001). Negative correlation was identified between UPC and OD % of 100 kDa bands (ρ = -0.85332, P = <0.0001), and the number of bands and OD % of 100 kDa bands (ρ = -0.74479, P = <0.0001). The area under the ROC curve was 0.991 (95 % CI, 0.976-1). The optimal cut-off UPC that better discriminated between urines with high or low OD% of THP was 0.46 with 92.6 % sensitivity and 96.2 % specificity. Our findings indicate that non azotemic dogs with borderline proteinuria might excrete low amount of THP, which could suggest tubular damage in early stages of chronic kidney disease.