Interleukin-27 Regulates Adaptative Immune Responses Associated With Control of Parasite Replication in Canine Leishmaniasis.

Interleukin 27 (IL-27) is a cytokine that regulates susceptibility to Leishmania infantum infection in humans and experimental models. This cytokine has not yet been described in canine leishmaniasis (CanL). Therefore, we investigated whether IL-27 has a regulatory role in CanL. The EBI3 and p28 subunits of IL-27 were measured in splenic leukocytes culture supernatant from dogs with CanL and compared to control dogs. We also correlated EBI3 and p28 levels with IL-21, anti-L. infantum antibodies and parasite loads. We performed functional assays followed by IL-27 blockade and measured parasite loads, production of cytokines in splenic leukocytes culture supernatant, and the expression of PD-1, CTLA-4, phospho-Stat-1/3, T-bet, GATA3 and nitric oxide production (NO). Both IL-27 subunits increased in the supernatant of dogs with CanL compared to control dogs. EBI3 and p28 levels showed a moderate positive correlation with IL-21 (r = 0.67, p < 0.0001 and r = 0.45, p < 0.012, respectively), and the EBI3 subunit was positively associated with anti-L. infantum IgG antibodies (r = 0.38, p < 0.040) and parasite load (r = 0.47, p < 0.009). IL-27 and IL-21 participate of immune responses in CanL. IL-27 may be associated with the failure of immunity to control parasite replication via upregulation of the expression of PD-1, CTLA-4, T-bet and NO in splenic leukocytes from dogs with CanL. These findings suggest that the pathways regulated by IL-27 are involved in CanL pathogenesis in the host, and may be targets for new therapies.

Presence of Leishmania sp. amastigotes in the reproductive tract of dogs with visceral leishmaniasis.

BACKGROUND: Although canine visceral leishmaniasis (CVL) transmission primarily occurs through the bite of phlebotomine sand flies infected with Leishmania infantum, alternative routes may exist. METHODS: Thirty-four dogs diagnosed with CVL were sampled for parasitological investigation in tissues from the reproductive tract. RESULTS: Amastigotes of Leishmania sp. were present in 79% (27/34) of the reproductive system samples, with distinct infection rates depending on the tissue. CONCLUSIONS: Our data confirms that alternative routes, such as horizontal and vertical transmissions, should be considered in the epidemiological chain of CVL.

Fatal case of imported visceral leishmaniasis in a dog caused by Leishmania infantum in French Guiana.

Here we described a case of fatal canine visceral leishmaniasis (VL) in French Guiana, a non-endemic VL Amazonian area. The dog was a 2-year-old pug imported from Brazil to French Guiana. Initially seen for a pruriginous lesion on the muzzle which healed after treatment, the dog was in a deteriorated condition and had sublingual, foreleg and eye ulcers, one month later. A visceral leishmaniasis was suspected by the veterinarian. The dog was hospitalized awaiting results, which revealed the presence of L. infantum. However, the dog succumbed suddenly before the results were returned. Few imported and scarce autochthonous canine VL cases have been previously reported in French Guiana, raising the need for local epidemiological surveillance, considering the possibility of unusual transmission routes of the parasite.

[Epidemiological characteristics of local visceral leishmaniasis in Beijing Municipality from 2021 to 2023].

OBJECTIVE: To investigate the epidemiological characteristics of local visceral leishmaniasis in Beijing Municipality from 2021 to 2023, so as to provide insights into formulation of the visceral leishmaniasis control strategy. METHODS: Epidemiological data of visceral leishmaniasis cases reported in Beijing Municipality from 2021 to 2023 were collected from the National Health Informatization Disease Prevention and Control Information System, and the epidemiological characteristics of local visceral leishmaniasis cases were analyzed using a descriptive epidemiological method. In November 2021 and 2023, 50 to 100 permanent residents were voluntarily selected within a 100 meter radius of sites where patients lived or acquired Leishmania infections, and venous blood was sampled for anti-Leishmania antibody testing. Venous blood was sampled from dogs for anti-Leishmania antibody testing in natural villages where patients lived or acquired Leishmania infections, or in districts where Leishmania infected dogs were reported. In addition, sandflies were captured with CO2 mosquito traps and fine mesh nets in natural villages where patients lived or acquired Leishmania infections from May to September, 2021 and 2023, for sandfly species identification. RESULTS: A total of 4 local visceral leishmaniasis cases were reported in Beijing Municipality from 2021 to 2023, with ages of 2 to 77 years, and acquiring Leishmania infections in Mentougou District (2 cases), Changping District (1 case), and Yanqing District (1 case). The anti-Leishmania antibody testing was all negative in 73 human blood samples and the sero-prevalence of anti-Leishmania antibody was 25.00% in 36 venous blood samples from domestic dogs in 2021, with a total of 4 520 Phlebotomus chinensis captured. The sero-prevalence of anti-Leishmania antibody was 0.51% in 198 human blood samples and 13.58% in 243 venous blood samples from domestic dogs in 2023, with 16.10%, 25.00%, 17.78% and 3.13% sero-prevalence in dogs sampled from Mentougou District, Changping District, Yanqing District and Haidian District, respectively (P = 0.011), while a total of 1 712 Ph. chinensis were captured, including 1 421 female sandflies (86.54%). CONCLUSIONS: The prevalence of local visceral leishmaniasis was low in Beijing Municipality from 2021 to 2023; however, there is a risk of further spread in the epidemic foci. Intensified visceral leishmaniasis surveillance and control is recommended.

[Prevalence of canine Leishmania infections in Xin'an County, Luoyang City in 2020].

OBJECTIVE: To investigate the prevalence of canine Leishmania infections in villages endemic for visceral leishmaniasis in Xin'an County, Luoyang City, so as to provide insights into visceral leishmaniasis prevention and control. METHODS: All dogs were captured from Huzhanggou Village, Xin'an County, Luoyang City in August 2020, where local cases with visceral leishmaniasis lived. The basic characteristics of dogs were collected, and venous blood was collected via the ear or neck veins of dogs. The serum anti-Leishmania antibody was detected using the rk39 immunochromatographic test and Leishmania nucleic acid was detected using PCR assay, and the prevalence of Leishmania infection was estimated in dogs. RESULTS: A total of 133 domestic dogs were captured from Huzhanggou Village, with a median age of 18.0 (28.5) months. The sero-prevalence of anti-Leishmania antibody was 24.81% (33/133) and the prevalence of a positive PCR assay was 14.29% (19/133) in dogs. The median ages of Leishmania-infected and uninfected dogs were 24.0 (36.0) months and 12.0 (18.0) months, respectively (U = 872.000, P = 0.000), and the prevalence of Leishmania infection was 55.56% (5/9) in "mangy dogs" and 24.19% (30/124) in asymptomatic dogs (χ2 = 2.793, P = 0.095). CONCLUSIONS: There are a large number of asymptomatic dogs with Leishmania infections in Xin'an County, Luoyang City, with a high transmission risk of visceral leishmaniasis. Timely prevention and control measures are required to control the spread of visceral leishmaniasis.

Development and preliminary study of the rLiNTPDase2 rapid test: A lateral flow immunochromatographic assay for Canine Visceral Leishmaniasis.

Canine Visceral Leishmaniasis (CVL) is the most fatal form of Leishmania infection in dogs and is caused by L. infantum in the Americas. This parasite follows a zoonotic life cycle, raising concerns within domestic households, where dogs act as the primary reservoir of the parasite. Accurately detecting infected dogs is vital for effective epidemiological control in both canine and human populations. However, existing diagnostic methods in Brazil have limitations, particularly in detecting asymptomatic and oligosymptomatic dogs, leading to ineffective disease control. To address this challenge, we evaluated a novel recombinant antigen from L. infantum, the rLiNTPDase2. Previous studies have confirmed its high performance via ELISA, leading us to assess its suitability for a Lateral Flow Immunochromatographic Assay (LFIA), which is ideal for point-of-care testing. Standardization of the assay involved testing two nitrocellulose membranes (HF135 and HF120, Millipore), three blocking protocols, and five sample dilutions (1:10, 1:20, 1:40, 1:80, and 1:160). Following the chosen conditions (HF120 membrane, 1-minute blocking protocol, and 1:80 sample dilution), we validated our assay with a sample size of 78 dogs, comprising 32 negatives and 46 positives, including symptomatic (n=23), oligosymptomatic (n=17), and asymptomatic (n=6) cases. The results revealed a sensitivity of 86.9 %, specificity of 62.5 %, and accuracy of 76.9 %, which is consistent with ELISA performance for the same samples. Compared to DPP-LVC, our assay demonstrated promising results in detecting asymptomatic and oligosymptomatic cases. This study underscores the suitability of the rLiNTPDase2 antigen for the LFIA format, suggesting its potential as a novel point-of-care diagnostic test for CVL.

Strategic use of 4 % deltamethrin impregnated-collar in seropositive dogs reduces the incidence of seroreactivity to visceral leishmaniasis in dogs from endemic areas.

The effectiveness of a visceral leishmaniasis (VL) control strategy based on the application of 4 % deltamethrin impregnated collars (DIC) exclusively in seropositive dogs was assessed between 2018 and 2019, through a prospective study. The effectiveness of DIC-collaring was evaluated by comparing the incidence rate of anti-leishmanial antibodies among dogs from two endemic districts in Brazil. In one of the areas, the conventional control measure which is based on the non-compulsory euthanasia of LV seropositive dogs, was practiced by the official healthy service as a regular procedure, whereas strategic collaring, conceived in this study, was carried out in the other. Results of serological tests applied to serum samples collected from all domiciled dogs were evaluated in three consecutive times, spaced by around 200 days. Incidence rates of VL seroreactivity were compared between districts in the same period of time as well as within the same district, in consecutive periods. Based on the results, the risk of infection in the population under conventional control measure was up to four times higher than the risk of infection where DIC-collaring was used. The strategic use of collar proposed here emerged as a promising measure for VL control in dogs from endemic areas. Strategic collaring does not rely on the euthanasia of infected animals, an extremely controversial procedure, and instead of being used in all dogs, as collaring is normally recommended; only seropositive dogs are intervened. Strategic use of DIC has the potential to drastically reduce costs, if compared to mass collaring canine population.

Feline leishmaniosis in the Mediterranean Basin: a multicenter study.

BACKGROUND: Cats are now recognized as competent hosts for Leishmania infantum and a blood source for sand fly vectors. Although canine leishmaniosis (CanL) is endemic in Mediterranean Basin countries, large-scale epidemiological studies are lacking for feline leishmaniosis (FeL). This study aimed to assess the prevalence of L. infantum infections, associated risk factors, clinical signs, and clinicopathological abnormalities in domestic cat populations from six Mediterranean Basin countries. METHODS: From 2019 to 2022, blood and serum samples of cats (n = 2067) living in Italy (n = 300), Greece (n = 297), Portugal (n = 295), France (n = 231), Israel (n = 313), and Spain (n = 631) were collected along with animal data (i.e., age, sex, breed, housing conditions, and geographical origin), clinical signs, and laboratory blood test parameters. Cats were grouped according to their age as kittens (up to 1 year), young (older than 1 and younger than 7 years), mature (between 7 and 10 years), and senior (older than 10 years). Serum samples were tested for L. infantum by immunofluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA), and blood samples of seropositive cats were tested for L. infantum kinetoplast deoxyribonucleic acid (kDNA). Viral infection by feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) was molecularly addressed in all cats enrolled. Statistical analysis was performed to evaluate the association between the risk of L. infantum infection and independent variables, and among co-infection of L. infantum with FIV and/or FeLV, clinical signs, and clinicopathological abnormalities. RESULTS: Overall, 17.3% (358/2067) of cats scored positive for L. infantum by serological tests. Specifically, 24.7% were from Portugal, 23.2% from Greece, 16.6% from Israel, 15% from Spain, 13.3% from France, and 12.6% from Italy. Leishmania infantum DNA was detected in 15 seropositive animals. Housing condition and FIV infection proved to be risk factors for FeL. Leishmania seropositivity was significantly associated with weight loss, lymphadenomegaly, gingivostomatitis, and oral ulcers, as well as with reduced albumin and albumin/globulin ratio, increased total globulins and total proteins, leukocytosis, and thrombocytosis. CONCLUSIONS: This study provides, for the first time, a large-scale epidemiological survey on FeL and its clinical presentation, revealing that L. infantum circulates among domestic cats, especially shelter/free-roaming and FIV-infected animals, living in CanL endemic countries of the Mediterranean Basin.

Unraveling molecular mechanistic disparities in pathogenic visceral Leishmania resistance between reptiles and mammals through comparative transcriptomic analyses.

Leishmaniasis is one of the most important neglected tropical parasitic diseases, manifesting various clinical forms depending on the parasite species and the genetic background of the host. In order to elucidate the underlying mechanisms of reptilian defense against pathogenic Leishmania species and to delineate the global gene expression profile alterations during host-pathogen interaction, we established experimental animal and cell models using both heterothermic lizards (Phrynocephalus przewalskii) and homothermic mammals (BALB/c mice) infected with pathogenic Leishmania infantum (high virulence HCZ strain) and Leishmania donovani (low virulence 801 strain). Overall, the lizards didn't show any obvious clinical symptoms or immune responses in vivo. Using RNA-seq methodology, differentially expressed genes identified in the HCZ and 801-comparison groups of P. przewalskii were primarily associated with arginine biosynthesis, the MAPK signaling pathway and the PI3K-Akt signaling pathway. In contrast, higher parasite loads, exacerbated hepatic inflammatory lesions and enhanced immune responses were observed in BALB/c mice, with DEGs predominantly associated with immunological diseases, innate and adaptive immune responses. By integrating transcriptional data from reptile and mammalian hosts, we elucidated the pivotal role of amino acid metabolism and lipid metabolism in parasite control. In contrast to findings from animal experiments, Leishmania parasites effectively infected peritoneal macrophages of lizards in vitro, demonstrating a high infection rate. Furthermore, we used RT-qPCR to detect changes in cytokine expression in macrophages and found that Th1-type cytokines were significantly upregulated in lizards, facilitating the clearance of the HCZ strain 24 hours post-infection. Conversely, cytokine expression was generally suppressed in BALB/c mice, allowing immune evasion by the parasites.

Relapses in canine leishmaniosis: risk factors identified through mixed-effects logistic regression.

BACKGROUND: Canine leishmaniosis (CanL), caused by Leishmania infantum, is an important vector-borne parasitic disease in dogs with implications for human health. Despite advancements, managing CanL remains challenging due to its complexity, especially in chronic, relapsing cases. Mathematical modeling has emerged as a powerful tool in various medical fields, but its application in understanding CanL relapses remains unexplored. METHODS: This retrospective study aimed to investigate risk factors associated with disease relapse in a cohort of dogs naturally infected with L. infantum. Data from 291 repeated measures of 54 dogs meeting the inclusion criteria were included. Two logistic mixed-effects models were created to identify clinicopathological variables associated with an increased risk of clinical relapses requiring a leishmanicidal treatment in CanL. A backward elimination approach was employed, starting with a full model comprising all potential predictors. Variables were iteratively eliminated on the basis of their impact on the model, considering both statistical significance and model complexity. All analyses were conducted using R software, primarily employing the lme4 package, and applying a significance level of 5% (P < 0.05). RESULTS: This study identified clinicopathological variables associated with an increased risk of relapses requiring a leishmanicidal treatment. Model 1 revealed that for each 0.1 increase in the albumin/globulin ratio (A/G) ratio, the odds of requiring treatment decreased by 45%. Conversely, for each unit increase in the total clinical score (CS), the odds of requiring treatment increase by 22-30%. Indirect immunofluorescence antibody test (IFAT) was not a significant risk factor in model 1. Model 2, incorporating individual albumin and globulins values, showed that dogs with high IFAT titers, hyper beta-globulinemia, hypoalbuminemia, anemia, and high CS were at increased risk of relapse. Both models demonstrated a good fit and explained a substantial amount of variability in treatment decisions. CONCLUSIONS: Dogs exhibiting higher CS, dysproteinemia, anemia, and high IFAT titers are at increased risk of requiring leishmanicidal treatment upon clinical relapse in CanL. Regular monitoring and assessment of risk factors prove essential for early detection of relapses and effective intervention in CanL cases. The contrasting findings between the two models highlight the complexity of aspects influencing treatment decisions in this disease and the importance of tailored management strategies to improve outcomes for affected dogs.

First Molecular Evidence of Leishmania Infantum in Domestic Cats and Associated Risk Factors from the Black Sea Region of Türkiye.

PURPOSE: The objectives of the present study are to determine the molecular prevalence of Leishmania spp. in the owned domestic cats in the Black Sea Region of Türkiye and analyze the associated risk factors in FeL. METHODS: Conjunctival swabs (CS), blood, demographic, and clinical data were collected from 150 owned cats brought to the Veterinary Teaching Hospital during 2020-2022. Leishmania kinetoplast DNA (kDNA) from CS was screened by TaqMan Real-Time PCR (qPCR) with the genus-specific primers and a probe. RESULTS: All qPCR positive products were also amplified and sequenced to identify Leishmania species by ITS1 primers. Molecular prevalence of L. infantum found as 12.6% (19/150) in the observed cats in the Black Sea Region of Türkiye. There was a significant difference (p < 0.05) between neutered and intact cats with regarding to L. infantum positivity. Intact cats found to be 0.368 times more prone to be L. infantum-positive (L+). Dermatological lesions were found the most common (26.3%) problems in the L + cats. The median leucocyte count was the only parameter that was found statistically (p < 0.05) lower in the L + group (6.60) than the negative group (L-) (8.96), when comparing the WBC, NEU/LYM, MONO/LYM, EOS/LYM and PLT/LYM values. CONCLUSION: This study presented the molecular occurrence of FeL in the Black Sea Region of Türkiye for the first time indicating that the carrier status of the cats makes them alternative reservoirs for possible zoonotic transmission of L. infantum in this zone.

Canine leishmaniosis global prevalence over the last three decades: a meta-analysis and systematic review.

Leishmaniosis by Leishmania infantum is a zoonotic vector-borne disease transmitted to humans and dogs by the bite of female sand-flies. The domestic dog is the main reservoir and infected dogs may show or not clinical symptoms. The prevalence of infection in dogs varies according to the population studied, the geographic area, and the diagnostics employed. This study aims to estimate the global prevalence, subgrouping per continent, country, diagnostic test and selected risk factors. Cross-sectional studies (n=150; from 1990 to 2020) estimating the prevalence of the infection by Leishmania infantum were extracted from four electronic databases. The pooled global prevalence obtained by random-effects meta-analysis was 15.2 % (95 %CI 13.6-16.9), mostly in rural (19.5 %) and owned dogs (16.5 %). Prevalence varied if the diagnosis was made by western blot (WB, 32.9 %), cellular immunity tests (27.5 %), ELISA (17 %), PCR (16.9 %), IFAT (15.9 %), rapid tests and direct agglutination test (DAT, 11.5 %), cytology/immunohistochemistry (13.1 %), culture (8.6 %). A small studies bias (P<0.005) in the overall prevalence meta-analysis, due to the impact of small-size studies on the overall results was found. Moreover, a continent-related bias was found regarding rapid test, DAT (P=0.021), and WB (P<0.001), as these assays are mainly used in South American studies. A study period bias (P=0.033) and a publication year bias (P=0.002) were detected for PCR, as the test was not employed before the year 2000. In conclusion, a high prevalence of canine leishmaniosis worldwide and high heterogeneity among studies were found.

Discovery of new antigens for serodiagnosis of visceral leishmaniasis in humans and dogs adds valuable tools to our arsenal.

Surveillance and sustained control of visceral leishmaniasis (VL) require reliable serodiagnostic tools. rK39, the gold standard antigen for VL diagnosis, is limited by its documented poor sensitivity in certain endemic regions, such as East Africa, and by the longevity of its antibodies, making it difficult to distinguish active from cured infections. In a recent publication in mBio, Roberts et al. (A. J. Roberts, H.B. Ong, S. Clare, C. Brandt, et al., mBio 15:e00859-24, 2024, https://doi.org/10.1128/mbio.00859-24) identified new immunogenic Leishmania candidates in dogs and humans. In dogs, combined antigens LdBPK_290790.1 + LdBPK_362700.1 (D4 +D46) distinguished symptomatic from asymptomatic infections. For humans, LdBPK_323600.1 (D36) antigen produced short-lived antibodies and performed well in patient cohorts from Bangladesh and Ethiopia, but not Kenya. This study adds promising new candidates to our serodiagnostic toolbox but highlights the need for more antigen discovery studies that may have to be focused on regional performance.

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.

Prevalence of Leishmania infection in 205 cats from a referral hospital population in Brazil (2021-2022).

Leishmaniases are a group of neglected diseases of significant public health concern, with Brazil being the primary focus of this disease in the Americas. The municipality of Sobral, in the state of Ceará, is a historical focus of visceral leishmaniasis in both humans and dogs, but data on Leishmania spp. infections in cats are limited. Between April 2021 and February 2022, 205 cats from a referral hospital population were sampled and tested for Leishmania spp. by real-time PCR. Eight cats (3.9%; 95% CI: 1.7-7.5%) tested positive. Among these, three (37.5%) displayed clinical signs compatible with feline leishmaniosis. Non-domiciled cats showed significantly higher positivity compared to domiciled ones (Fisher's exact test, P = 0.0124). Considering their potential role as reservoirs of L. infantum, it is crucial to conduct further studies to understand the Leishmania spp. circulating among cats in Sobral and to implement measures for reducing their exposure to phlebotomine sand fly vectors in this important focus of leishmaniases.

The erythrocyte sedimentation rate and other markers of inflammation in cats tested for Leishmania infantum and feline immunodeficiency virus antibodies.

BACKGROUND: In endemic areas, Leishmania infantum and feline immunodeficiency virus (FIV) co-infection occurs in cats, and may favour a progressive course of feline leishmaniosis. Abnormalities in serum protein fractions have been reported, but inflammation markers have scarcely been studied. Erythrocyte sediment rate (ESR) is a marker of inflammation that is poorly used in veterinary medicine, but it has been evaluated in EDTA blood using a recently introduced automatic device. We studied ESR and a pool of feline markers of inflammation (MoI) in cats L. infantum (Li+) and/or FIV antibody-positive (Li+FIV+/FIV+) with the aims (a) to evaluate ESR as MoI in cats with the infectious and clinical conditions considered and (b) to provide data about a pool of MoI never investigated in the feline infections studied and in other cat diseases before. METHODS: This prospective controlled study included 35 study group cats (Li+, n = 20; FIV +, n = 8; Li+FIV+, n = 7) and ten healthy antibody-negative control cats. Clinical findings at physical examination and selected clinical pathological abnormalities related to inflammation were statistically analysed in relation to the infectious status and ESR values. RESULTS: ESR values were higher in Li+, FIV+, and Li+FIV+ cats compared with control cats, and 40% of the study group cats had ESR values above the reference interval (RI). ESR positively correlated with some positive MoI and negatively with some negative MoI studied. Additionally, a higher prevalence of ESR values above the RI has been detected in cats with hypoalbuminemia or hypergammaglobulinemia and higher ESR values were measured in cats with serum protein electrophoresis (SPE) fraction abnormalities. Correlations were also found with erythrocytes, hemoglobin, hematocrit and some erythrocyte indices. FIV+ and Li+FIV+ cats had a higher prevalence of increased ESR values, and almost all had SPE abnormalities and more severe clinical presentations compared with Li+ cats. CONCLUSIONS: Abnormal levels of MoI were found in almost all parameters studied, particularly in FIV+ and Li+FIV+ cats. Also, ESR can be used as a marker of inflammation in cats with L. infantum and/or FIV infection.

Canine visceral leishmaniasis: Seroprevalence and georeferencing in the state of Santa Catarina, Brazil.

Canine visceral leishmaniasis (CVL) has long been considered an endemic disease in the northern and northeastern regions of Brazil, while the southern region remains non-endemic. However, in recent years, several cases of CVL have been reported in southern states. The objective of this work was to determine the seroprevalence of CVL in dogs in the state of Santa Catarina, Brazil, through immunochromatographic tests (DPP®) and ELISA (Enzyme-Linked Immunosorbent Assay) and its correlation with environmental characteristics through georeferencing. Blood samples from dogs (n = 1227) were collected in six mesoregions of the state and evaluated by the rapid test (DPP®). Positive samples were sent to Lacen (Central Public Health Laboratory) in Santa Catarina to be tested using ELISA. Information obtained from the epidemiological questionnaire was subjected to statistical analysis (Chi-square and Student's t-test; P < 0.05) to verify the correlation between serology and the analyzed variables. The locations (GPS) of the samples were used for georeferencing and creating heatmaps (Kernel Method). Four animals that died from CVL were necropsied and organ samples were collected for molecular analysis (PCR), immunohistochemistry, and histopathology (HE). Of the 1227 dogs analyzed, 22 (1.8%) were reactive in the DPP® and of these, 7 (0.6%) were also positive in the ELISA. A correlation (P < 0.01) was observed between positive serology and region, environment, access to the street, and clinical signs. The positive cases were concentrated in the eastern region of the state, in low-altitude areas with average rainfall and higher average temperatures, and in more populated areas close to forest fragments. PCR, HE, and immunohistochemistry, along with serology, have proven to be efficient for characterizing positive cases.

Leishmania infantum detection in Nyssomyia neivai and dogs in Southern Brazil.

BACKGROUND: The sand fly Nyssomyia neivai is one of the most abundant species in Southern Brazil. It is frequently found in areas that are foci of visceral leishmaniasis in the state of Santa Catarina, caused by Leishmania infantum. In this region, the main vector of L. infantum, Lutzomyia longipalpis, has not been detected. In the absence of L. longipalpis, this study aimed to identify the sand fly fauna and diagnose any potential Leishmania spp. infection in sand flies and in dogs in a region of Southern Brazil that experienced a recent canine visceral leishmaniasis outbreak. METHODS: This report includes a survey of the sand fly fauna at the Zoonosis Control Center of the Municipality of Tubarão (Santa Catarina, Brazil). Molecular tests were conducted to investigate Leishmania spp. natural infection in sand flies using polymerase chain reaction (PCR). In positive females, in addition to morphological identification, molecular analysis through DNA barcoding was performed to determine the sand fly species. Additionally, the dogs were tested for the presence of Leishmania spp. using a non-invasive technique for the collection of biological material, to be assessed by PCR. RESULTS: A total of 3419 sand flies, belonging to five genera, were collected. Nyssomyia neivai was the most abundant species (85.8%), followed by Migonemyia migonei (13.3%), Pintomyia fischeri (0.8%), Evandromyia edwardsi (< 0.1%), and species of the genus Brumptomyia. (0.1%). Out of the 509 non-engorged females analyzed by PCR, two (0.4%) carried L. infantum DNA. The naturally infected females were identified as Ny. neivai, in both morphological and molecular analysis. In addition, two out of 47 conjunctival swabs from dogs tested positive for L. infantum, yielding an infection rate of 4.2%. CONCLUSIONS: These results confirm the presence of Ny. neivai naturally infected with L. infantum in an area where dogs were also infected by the parasite, suggesting its potential role as a vector in Southern Brazil.

Ehrlichia spp. infection worsens cardiac damage in dogs with canine visceral leishmaniasis.

In endemic areas for canine visceral leishmaniasis (CVL), the occurrence of coinfection with other pathogens, such as Ehrlichia spp., has been associated with worsening of the clinical condition. The study aimed to evaluate the occurrence of histological changes in the myocardia of dogs naturally infected with Leishmania chagasi with or without coinfection with Ehrlichia spp.. We evaluated paraffin-embedded myocardial sections from 31 dogs, affected by either L. chagasi alone or coinfected with L. chagasi and Ehrlichia spp., to compare the extent and degree of cardiac damage. The blocks were divided into two groups. G1 (dogs infected only by L. chagasi) and G2 (dogs coinfected with L. chagasi and Ehrlichia spp.). The right atrium free wall, right ventricle free wall, left ventricle, and interventricular septum of all groups were evaluated. Cardiac alterations were observed in 41.93% (52/124) of the fragments evaluated and inflammatory infiltrate was the most common pattern found. The G2 group showed a higher incidence of myocarditis, with 61.53% (32/52), compared to the G1 group, in which 20 out of 72 cases (27.7%) exhibited histopathological changes (p <0.05). These findings confirmed that coinfection can potentiate cardiac damage in dogs.