anti-Leishmania Antibodies Research Articles

Epidemiological investigation on a visceral leishmaniasis case in Zhengzhou City of Henan Province

To perform an epidemiological investigation on a case with visceral leishmaniasis in Zhengzhou City, Henan Province, and to identify the source of infection, so as to illustrate the transmission chain and assess the risk of local leishmaniasis transmission. The medical data were collected from a case with visceral leishmaniasis in Zhengzhou City, and the patient's bone marrow smears were detected by microscopy. Serum anti-Leishmania antibody test and PCR assay were performed among high-risk residents and all dogs in the village where the patient lived. Sandflies were captured using light traps and artificial traps, and the captured female Phlebotomus chinensis was subjected to PCR assay. The internal transcribed spacer 1 (ITS1) gene was amplified with a nested PCR assay using the genomic DNA extracted from visceral leishmaniasis patients, positive dogs and sandflies, and the sequences were aligned with those download from NCBI. In addition, a phylogenetic tree was created based on the ITS1 gene. The visceral leishmaniasis patient had recurrent irregular fever, reduced complete blood counts, low hemoglobin, and a large number of Leishmania amastigotes in bone marrow smears, and was therefore diagnosed as visceral leishmaniasis. Both rk39 rapid diagnostic test and PCR assay tested negative among 324 residents living neighboring the patient's residence, while 21.39% (43/201) dogs were positive for rk39 rapid diagnostic test and 13.93% (28/201) positive for PCR assay. There were 17 female Ph. chinensis tested positive for Leishmania (0.82%) by PCR assay, and the ITS gene sequences from visceral leishmaniasis patients, positive dogs and sandflies shared a 100% homology with L. infantum. The Leishmania species was therefore characterized as L. infantum. L. infantum infection occurs in visceral leishmaniasis patients, dogs and sandflies in Zhengzhou City, indicating a complete transmission chain and a high transmission risk of visceral leishmaniasis by L. infantum. Intensified control measures are required to prevent local transmission of leishmaniasis in Zhengzhou City.

FATORES DE RISCO E SOROPREVALÊNCIA DE ASSINTOMÁTICOS DA LEISHMANIOSE VISCERAL HUMANA EM ÁREA DE TRANSMISSÃO INTENSA EM PETROLINA/PE, BRASIL

OBJECTIVES: To identify the seroprevalence of asymptomatic individuals in areas of disease transmission and analyze the environmental, socioeconomic and demographic risk factors in the municipality of Petrolina/PE, Brazil. METHODS: We approached 92 individuals in 19 localities with VL cases recorded in the Information System for Notifiable Diseases (SINAN) between 2016 and 2017. In each visit, two questionnaires were applied seeking to obtain clinical, social, economic and environmental variables. Viable blood samples collected during the visits (n=90) were submitted to the Indirect Immunofluorescence Reaction to identify anti-Leishmania antibodies. For data analysis, descriptive statistical tests were performed using Stata® 14.0 software. RESULTS: The presence of antibodies was detected in 2.2% (2/90) of the samples, in which one individual presented symptoms and the other was classified as asymptomatic. No risk factors related to the presence of anti-Leishmania antibodies were identified. CONCLUSION: The identification of one asymptomatic individual reinforces the importance of expanding human prevalence studies in endemic areas. Despite the low prevalence, it was possible to identify a possible case with manifestation of the clinical form of chronic VL that needs proper care.

Visceral Leishmaniasis in Ardabil Province, Northwest of Iran: A Retrospective Epidemiological, Clinical and Paraclinical Study (1985-2018).

Background:We aimed to investigate the epidemiological, clinical and paraclinical aspects of Visceral Leishmaniasis in Ardabil Province, from 1986 to 2018.Methods:The disease has been diagnosed by a serological validate test as Direct Agglutination Test, parasitological methods, and clinical manifestations. The data were collected and then analyzed.Results:Overall, 2824 out of 69007cases (4.1%) had anti-Leishmania antibodies at titers≥1:3200. The majority of VL cases belonged to Meshkin-Shahr, and then Germi County. The percentages of both male and female cases were 52.8 % and 46.6%, respectively. Approximately 78.1% of cases were less than 5 yr old. The high incidence rate of the disease was recorded in the years 1987 and 2003. 48.6%, 3.2%, and 1.9% of the patients had low titer (1:3200), high titer (1:102400), and suspicious titer (1:1600), respectively. Out of 1200 bone marrow aspiration, 23% were positive, and 77% were negative. Fever, paleness, and hepatosplenomegaly were the main symptoms in patients. Moreover, abdominal swelling, edema, weight loss, lymphadenopathy and jaundice were some other symptoms in the patients. Among biochemistry factors, the levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, triglyceride and cholesterol have been increased. The death rate was estimated to be 2.8%, mainly related to the Ardabil and Germi cases.Conclusion:Kala-Azar is still one of the most important infectious diseases in Ardabil Province, and is responsible for considerable death rate in infants. More attention should be done to parasite control and early diagnosis.

Validation of a mixture of rK26 and rK39 antigens from Iranian strain of Leishmania infantum to detect anti-Leishmania antibodies in human and reservoir hosts

Mediterranean type of visceral leishmaniasis (VL) is a zoonotic parasitic infection. Some provinces of Iran are endemic for VL while other parts are considered as sporadic areas. This study aimed to assess a combination of recombinant K26 and rK39 antigens as well as crude antigen (CA), derived from an Iranian strain of L. infantum, compared to direct agglutination test (DAT) for the detection of VL in humans and domestic dogs as animal reservoir hosts of the disease. A combination of rK26 and rK39 antigens and also CA was evaluated using indirect ELISA on serum samples of 171 VL confirmed humans (n = 84) and domestic dogs (n = 87) as well as 176 healthy humans (n = 86) and domestic dogs (n = 90). Moreover, 36 serum samples of humans (n = 20) and canines (n = 16) with other potentially infectious diseases were collected and tested for finding cross- reactivity. The results of ELISA were compared to DAT, currently considered as gold standard for the serodiagnosis of VL. The sensitivity and specificity, positive predictive and negative predictive values were calculated compared to DAT. The positive sera had previously shown a positive DAT titer ≥ 1:800 for humans and ≥ 1:80 for dogs. Analysis was done by MedCalc and SPSS softwares. Using the combination of rK26 and rK39 in ELISA, a sensitivity of 95.2% and a specificity of 93.0% % were found in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 98.9% and specificity of 96.7%% were found at a 1:80 (cut-off) titer compared with DAT. A good degree of agreement was found between the combined rK39 and rK26-ELISA with DAT in human (0.882) and dog serum samples (0.955) by kappa analysis (p < 0.05). The ELISA using the CA test showed 75% sensitivity in human and 93.1% in dog serum samples as well as 53.5% specificity in human and 83.3% in dog,s sera, respectively. The combination of rK26 and rK39 recombinant antigen prepared from Iranian strain of Leishmania infantum showed high accuracy for the serodiagnosis of VL in human and domestic dogs. Further extended field trial with a larger sample size is recommended.

A Challenging Case of Visceral Leishmaniasis

The term leishmaniasis includes multiple clinical syndromes: visceral, cutaneous, and mucosal leishmaniasis, resulting from an infection of macrophages throughout the reticuloendothelial system in the dermis and the naso-oropharyngeal mucosa, respectively. The clinical phenotype is mainly driven by the leishmania biologic characteristics and, ultimately, also by the host immune status. The disease is endemic in focal areas in the tropics, subtropics, and southern Europe, transmitted by the bite of female phlebotomine sandflies. Sandflies regurgitate the parasite’s flagellated promastigote stage into the host’s skin; promastigotes bind to receptors on macrophages are phagocytized and transformed within phagolysosomes into non-flagellated amastigotes which replicate and infect additional macrophages. Amastigotes ingested by sandflies transform back into infective promastigotes. Depending on the host’s innate and acquired immune status, systemic and visceral leishmaniasis can be characterized by irregular fever, weight loss, enlargement of the spleen and liver, and anaemia. We present a 42 year-old man with long-lasting type 1 autoimmune hepatitis under immunosuppressive treatment. In January 2017, the patient started to experience low-grade unresponsiveness to empiric antibiotic therapy. The patient developed severe anemia and progressive multilineage cytopenia accompanied by increased levels of inflammatory markers. FDG-PET revealed increased glucose uptake in the liver, spleen, and the whole bone marrow. The subsequently performed bone marrow biopsy evidenced Leishmania amastigotes inside macrophages, confirmed by serological positivity to anti-Leishmania antibody. Immunosuppressive therapy was suspended and replaced by treatment with amphotericin B at 4 mg/kg/day from day 1 to day 5, followed by a single infusion on days 10, 17, 24, 31, and 38. The bone marrow smear after treatment still evidenced few Leishmania amastigotes; in consideration of the patient’s immunosuppression status, two further doses of amphotericin B on days 45 and 52 were employed, leading to infection resolution. In real-life, as exemplified in this case, administering two additional doses of amphotericin B (concerning the guidelines) offered an additional therapeutic opportunity for a patient under long-term immunosuppressive treatment.

Leishmania exposure in dogs from two endemic countries from New and Old Worlds (Brazil and Portugal): evaluation of three serological tests using Bayesian Latent Class Models

BackgroundZoonotic leishmaniosis caused by Leishmania infantum is endemic in several countries of the Mediterranean Basin, Latin America, and Asia. Dogs are the main hosts and reservoirs of human infection. Thus, from a One Health perspective, early diagnosis of Leishmania infection in dogs is essential to control the dissemination of the parasite among other dogs and to humans. The aim of this study was to estimate the diagnosis accuracy of three serological tests to detect antibodies to Leishmania in dogs from two endemic settings using Bayesian latent class models (BLCM).MethodsA total of 378 dogs from two Portuguese and Brazilian endemic areas of leishmaniosis (194 animals from Portugal and 184 from Brazil) were screened. Detection of anti-Leishmania antibodies was performed using two commercial ELISA (L. infantum IgG-ELISA® and EIE-LVC®) and a rapid immunochromatographic test (DPP-LVC®). Bayesian latent class models were used to estimate Leishmania infection prevalence, together with sensitivities and specificities of the three diagnostic tests, in the two dog populations simultaneously. Predictive values were also calculated. Credibility intervals (CI) were obtained, considering different types of prior information.ResultsA posterior median Leishmania seroprevalence of 13.4% (95% CI 9.0–18.7) and of 21.6% (15.0–28.3) was estimated to the Portuguese and Brazilian dog subpopulations, respectively. The Bayesian analysis indicated that all tests were highly specific (specificity above 90%), and that the DPP-LVC® was more sensitive (96.6%; 83.1–99.9) than both ELISAs in the Portuguese subpopulation, while in the Brazilian subpopulation, EIE-LVC® and L. infantum IgG-ELISA®, had the highest sensitivity (88.2%; 73.7–97.0) and specificity (98.7%; 95.1–99.9), respectively.ConclusionsIn general, the levels of diagnosis accuracy of the three serological tests to detect Leishmania antibodies assessed by BLCM indicate their utility in canine epidemiological studies. The same approach should be used to assess the performance of these techniques in the clinical management of infected and sick dogs using representative samples from the wide spectrum of clinical situations, namely from subclinical infection to manifest disease. The low positive predictive value of the serological tests used in the current protocol of the Brazilian Ministry of Health suggests that they should not be used individually and may not be sufficient to target reservoir-based control interventions.Graphical

Association between feline immunodeficiency virus and Leishmania infantum infections in cats: a retrospective matched case-control study

BackgroundFeline leishmaniosis caused by Leishmania infantum is often associated with feline immunodeficiency virus (FIV) infection; however, the role and clinical significance of this coinfection remain unknown. This study aimed to assess whether FIV is associated with L. infantum infection in cats from canine leishmaniosis endemic areas and to report the clinical signs and hematological alterations associated with coinfection.MethodsA retrospective matched case-control study (ratio 1:2) was conducted. Data of clinical examination and complete blood count (CBC) were selected from a cohort of 705 cats examined for epidemiological studies on feline leishmaniosis conducted between 2012 and 2019. Ninety-one FIV seropositive cases and 182 FIV seronegative control cats were selected. Matching was done according to age, sex, lifestyle and geographic provenience of case cats. Rapid ELISA devices were mainly used to detect anti-FIV antibodies. Anti-Leishmania IgG antibodies were detected by indirect-immunofluorescence test (IFAT). Leishmania DNA was searched in blood, oral and conjunctival swabs by quantitative real-time PCR.ResultsFeline immunodeficiency virus seropositive cats had no hematological abnormalities suggestive of an advanced stage of FIV infection and were statistically more frequently IFAT positive, and their risk of being L. infantum antibody positive was 2.8 greater than in the FIV seronegatives. The association of FIV seropositivity with L. infantum antibody positivity was confirmed in the univariable model of logistic regression. A multivariate model found FIV infection and L. infantum PCR positivity as predictors of a positive L. infantum IFAT result. Male outdoor cats from rural or suburban areas were at risk for FIV and L. infantum antibody positivity. Clinical signs more frequently associated with the coinfection were oral lesions, pale mucous membranes and low body condition score (BCS).ConclusionsThis study documents that FIV seropositive cats with no hematological abnormalities suggestive of an advanced stage of FIV infection are more prone to be L. infantum seroreactive by IFAT in endemic areas. Therefore, FIV seropositive cats should be tested for L. infantum antibodies and treated for preventing sand fly bites. Pale mucous membranes, low BCS and oral lesions but no CBC abnormalities were significantly associated with the coinfection.Graphical abstract

Detection of specific antibodies against Leishmania infantum in canine serum and oral transudate using an in-house ELISA

BackgroundCanine leishmaniosis caused by the protozoan Leishmania infantum is a complex infection due to its variable clinical signs and laboratory findings. Therefore, a broad range of techniques is available for diagnosis. Testing for specific antibodies in serum is the most commonly used technique, although the testing of other body fluids, such as oral transudate (OT), can be an alternative as its collection is non-invasive and testing can be performed by untrained personnel. The aim of this study was to assess and compare the detection of L. infantum-specific antibodies in paired samples of serum and OT collected from apparently healthy dogs and dogs with clinical leishmaniosis using an in-house enyzme-linked immunosorbent assay (ELISA).MethodsSerum and OT were collected from 407 dogs, which varied in breed, sex, age, lifestyle and clinical status, by many practicing veterinarians in Spain. The main geographical areas of sampling included Barcelona (n = 110), Mallorca (n = 94), Cadiz (n = 54) and Asturias (n = 47). The majority of infected dogs were apparently healthy (89.9%) while 41 presented clinical signs and/or clinicopathological abnormalities compatible with L. infantum infection and subsequently diagnosed with leishmaniosis (10.1%). An in-house ELISA was performed to quantify the anti-Leishmania antibodies in serum and OT.ResultsThe L. infantum infection rate determined by the in-house ELISA was 37.1% in serum samples and 32.7% in OT samples. Serum and OT ELISA results showed a positive correlation (Spearman's correlation coefficient rs = 0.6687, P < 0.0001). The percent agreement between the serum and OT ELISA results was 84%, while agreement according to Cohen's kappa statistic (κ) was substantial (0.66) when all samples were analyzed. The highest percent agreement (92.1%) between both tests was found in dogs from low endemicity regions and from sick dogs, with both groups presenting almost perfect agreement according to Cohen’s κ agreement test (0.84). Few seronegative dogs (n = 23) tested positive by the OT ELISA. The agreement between serum and OT went from almost perfect to moderate when the geographical distribution and clinical status were analyzed.ConclusionsThe results of this study demonstrated an almost perfect to moderate agreement between OT and serum samples tested using the in-house ELISA. These results are particularly promising in sick dogs with high antibody levels while the results seem less optimal in apparently healthy dogs with low antibody levels.Graphical

Characterization of lesions in the temporal muscle and the male reproductive system (epididymis and testicle) of dogs experimentally infected with Leishmania infantum with different clinical stages

Leishmaniosis is a zoonotic disease with a very complex pathogenesis modulated by the interaction between the parasite, the vector and the host. Although the pathological characteristics have been extensively studied in the typically affected organs, some locations such as muscles and reproductive organs have been less studied. The objective of this study was to evaluate the presence of lesions in the temporal muscle and the male reproductive organs (testicle and epididymis) and correlate their characteristics with the presence of the parasite and with the clinical status of the dogs. The temporal muscle was studied in 25 infected beagle dogs (nine females and 16 males) and five uninfected control dogs (two females and three males) and the testicle and epididymis in the 19 males. Dogs were euthanized one year after infection and clinical signs, anti-Leishmania serum antibodies, and lymph node parasite load were assessed. Muscular and reproductive lesions were characterized by H&E and immunohistochemistry (IHC). The presence of the parasite in the lesions was evaluated using IHC and molecular techniques.Myositis was observed in 72% (18/25) of the dogs and was characterized by lymphoplasmacytic or histiocytic lesions. Mild and severe lesions were detected, the latter being statistically associated with the presence of the parasite and with the clinical status of the dogs. Orchitis was observed in 50% (8/16) of the dogs and was mainly mild and lymphoplasmacytic. No statistical relationship was found between testicular lesions and the presence of the parasite or the clinical status. Epididymitis was observed in 87.5% (14/16) of the dogs, and the lesions were often infiltrated by numerous histiocytes and neutrophils. Epididymal lesions were statistically associated with the clinical status of the dogs and with the presence of the parasite in the lesions. IgG and IgM immunoglobulins were found in all lesions, suggesting a local immune response with reactivation of the infection. Leishmania was more frequently detected in severe and histiocytic lesions, although some lesions had no detectable parasites.These results have shown that lesions in the temporal muscle, epididymis, and testicles are common in dogs infected by Leishmania infantum and that dogs may show a different response to infection. This response is characterized by varying degrees of cellular and immune responses associated with a variable presence of the parasite.

A Questionnaire-Based Survey on the Long-Term Management of Canine Leishmaniosis by Veterinary Practitioners.

Simple SummaryCanine Leishmaniosis is a chronic and potentially fatal disease, caused by Leishmania infantum, a zoonotic microorganism. In economically disadvantaged regions, costs associated with long-term patient monitoring may determine that some owners decline veterinary follow-up of their dogs, with potentially severe implications for animal welfare and public health. This online, questionnaire-based survey aimed to assess how Portuguese veterinary practitioners perform long-term patient management and recognize relapses. More than half of the respondents stated that most dog owners declare having financial restraints, which condition the use of diagnostic tests during long-term follow-up. Allopurinol ad aeternum or until disease remission and domperidone were the most prescribed treatment, and relapses were detected by the reappearance or worsening of clinical signs by most veterinary practitioners. The rate of relapse detection was higher in the most economically favored regions, probably because of a lesser constraint on the use of the appropriate diagnostic tests. This study confirms that owner financial restraints negatively influence veterinary follow-up and relapse recognition, potentially compromising clinical decision making and favoring the maintenance of Leishmania infantum infection endemic status in Portugal.Canine Leishmaniosis (CanL) is a chronic and potentially fatal disease. In economically disadvantaged regions, costs associated with long-term patient monitoring may determine that some owners decline veterinary follow-up of their dogs. This online, questionnaire-based survey aimed to assess how Portuguese veterinary practitioners perform long-term patient monitoring and recognize relapses. More than 50% of respondents reported that 50–100% of dog owners declared financial restraints. Hence, in these circumstances, most veterinary practitioners only performed clinical examination and serology. However, when owners did not declare financial restriction, other tests were additionally performed, such as renal and hepatic profiles, hemogram, serum protein electrophoresis and urine protein creatinine ratio. The mean number of exams performed when owners presented financial restraints was significantly lower than the number of exams performed without economic limitations. Most veterinary practitioners prescribed allopurinol ad aeternum or until disease remission and domperidone. CanL relapses were recognized by more than half of respondents “Always”, through the reappearance or worsening of clinical signs, whereas about a quarter detected an increase in anti-Leishmania antibody levels and identified abnormalities in the serum protein electrophoresis profile. The relapse rate was higher in the Lisbon Metropolitan Area and north, the most economically favored regions of Portugal. This study confirms that owner financial restraints negatively influence veterinary follow-up and relapse recognition, ultimately compromising clinical decision making and favoring the maintenance of Leishmania infantum infection endemicity.

Evaluation of five different rapid immunochromatographic tests for canine leishmaniosis in Spain

Canine leishmaniosis is a vector-borne disease caused by Leishmania parasites. Serological methods are the most common tests used for the diagnosis. This study aimed to evaluate and compare different serological commercial immunochromatographic rapid tests available in Spain to detect anti-Leishmania canine antibodies. The immunochromatographic tests were evaluated in different groups of dogs (healthy seronegative dogs (n = 21), naturally-sick dogs with moderate anti-Leishmania antibodies (n = 39), naturally-sick dogs with high anti-Leishmania antibodies (n = 37), dogs with the serological result of other pathogens infection (n = 20) and exposed dogs (n = 33)) admitted to the Veterinary Teaching Hospital of the University of Zaragoza (Spain) according to the clinical information sent with the sample to the laboratory for diagnostic purposes. The serology status was also routinely recorded through an in-house enzyme-linked immunosorbent assay (ELISA) and an in-house indirect immunofluorescence test (IFAT). The qualitative commercial serological immunochromatographic tests used were: FASTest LEISH, Uranotest Leishmania, Uranotest Leishmania 2.0, Speed Leish K, Witness Leishmania, and DFV Test Leishmania. Performance measures analyzed for each test were: sensitivity, specificity, and area under the receiver-operating (ROC) curve. The maximum specificity (1.00) was attained for Uranotest Leishmania and DFT Test Leishmania, followed by FASTest LEISH (0.98), Uranotest Leishmania 2.0 (0.98), Speed Leish K (0.98), and Witness Leishmania (0.95). The maximum sensitivity was attained for FASTest LEISH (1.00), followed by Uranotest 2.0 (0.97), Speed Leish K (0.97), Uranotest (0.96), and the lowest results with Witness (0.84) and DFV Test (0.59). Regarding the ROC curve, the maximum value was attained with the FASTest LEISH (0.99), followed by Uranotest (0.98), Uranotest 2.0 (0.97), Speed Leish K (0.97), Witness (0.90), and the lowest result with DFV Test (0.79). Efforts in the field of diagnosis should focus on establishing a commercial immunochromatographic test with high sensitivity and specificity with a reasonable cost-benefit balance.

Canine leishmaniosis in Tunisia: Growing prevalence, larger zones of infection.

BackgroundDiscovered by Nicolle and Comte in 1908 in Tunisia, Leishmania infantum is an intracellular protozoan responsible for zoonotic canine leishmaniosis (CanL) and zoonotic human visceral leishmaniasis (HVL). It is endemic in several regions of the world, including Tunisia, with dogs considered as the main domestic reservoir. The geographic expansion of canine leishmaniosis (CanL) has been linked to global environmental changes that have affected the density and the distribution of its sand fly vectors.Methodology/Principal findingsIn this study, a cross-sectional epidemiological survey on CanL was carried out in 8 localities in 8 bioclimatic areas of Tunisia. Blood samples were taken from 317 dogs after clinical examination. Collected sera were tested by indirect fluorescent antibody test (IFAT; 1:80) for the presence of anti-Leishmania infantum antibodies. The overall seroprevalence was 58.3% (185/317). Among positive dogs, only 16.7% showed clinical signs suggestive of leishmaniosis. Seroprevalence rates varied from 6.8% to 84.6% and from 28% to 66% by bioclimatic zone and age group, respectively. Serological positivity was not statistically associated with gender. The presence of Leishmania DNA in blood, using PCR, revealed 21.2% (64/302) prevalence in dogs, which varied by bioclimatic zone (7.3% to 31%) and age group (7% to 25%). The entomological survey carried out in the studied localities showed 16 species of the two genera (Phlebotomus and Sergentomyia). P. perniciosus, P. papatasi, and P. perfiliewi were the most dominant species with relative abundances of 34.7%, 25% and 20.4%, respectively.Conclusions/SignificanceThe present report suggests a significant increase of CanL in all bioclimatic areas in Tunisia and confirms the ongoing spread of the infection of dogs to the country’s arid zone. Such an expansion of infection in dog population could be attributed to ecological, agronomic, social and climatic factors that affect the presence and density of the phlebotomine vectors.

New insights on severe clinical manifestations and deaths from visceral leishmaniasis in free-living crab-eating foxes (Cerdocyon thous) in Brazil

Visceral leishmaniasis (VL) is a zoonotic and severe neglected tropical disease, with worldwide distribution, that still cause many deaths among dogs and humans. Brazil is the country responsible for about 97% of the cases of leishmaniasis in the Americas and the disease is still considered a serious public health concern. Among wild mammalians, studies demonstrate the involvement of Cerdocyon thous in the biological cycle of Leishmania. Nevertheless, several authors consider the clinical manifestation of the disease to be rare or mild in free-living animals. Herein, we demonstrate the occurrence of severe clinical sings and deaths caused by VL in free-living crab-eating foxes. Three specimens of foxes collected from periurban areas were diagnosed with VL. The animals presented cutaneous, ophthalmological, gastrointestinal, locomotor and hematological alterations, and died after clinical progression. We identified the presence of anti-Leishmania antibodies by immunochromatographic test in all specimens. We also observed intra and extracellular amastigotes in skin cytology and lymph node aspirate. Furthermore, Leishmania infantum DNA was identified in all samples by the polymer chain reaction technique. Additionally, we performed blood count and stool parasitological tests and observed hematological disorders common to VL, such as anemia and lymphopenia. Taken together, our data demonstrate that VL can induce clinical complications and even cause death in C. thous and corroborate that this crab-eating fox is an adequate host for L. infantum.

Live attenuated Leishmania major p27 gene knockout as a novel vaccine candidate: A study on safety, protective immunity and efficacy against canine leishmaniasis caused by Leishmania infantum

Canine leishmaniasis (CanL) is an important parasitic e disease caused by Leishmania infantum and is transmitted by female phlebotomine sand flies primarily between canines and secondarily to humans. Recently, we showed that immunization with Leishmania major p27 gene knockout (Lmp27−/−) as a live attenuated vaccine was safe, induced immunogenicity, and protected against the development cutaneous and visceral leishmaniasis in mice. The p27 protein is a component of the COX protein complex which is responsible for ATP production. In this study, we analyzed the Lmp27−/− candidate vaccine potential with this regard to the safety and induction of immunogenicity and protection against CanL. Variables such a clinical manifestation, anti-Leishmania antibodies using direct agglutination test (DAT), lymphocyte proliferation, delayed-type hypersensitivity (DTH), bone marrow aspiration (BMA) and parasite burden using parasitological and molecular examinations were measured. The results demonstrated that the Lmp27−/− vaccinated group showed no clinical signs after inoculation with Lmp27−/− mutant during a 12-month follow-up, and had significantly higher T-cell responses (Lymphocyte proliferation and DTH), lower seroconversion and parasite burdens following a challenge inoculation with L. infantum after 6-mounth. In conclusion, vaccination with Lmp27−/− parasites would be safe and provide significant immunoprotectivity and efficacy against infection with wild type (WT) L. infantum.

Serosurvey of anti-Leishmania (Leishmania) infantum antibodies in hunting dogs and hunters in Brazil.

Background and Aim:Although wild boar hunting activities and the hunting dog trade in the Atlantic Forest and Cerrado biomes of Brazil overlap both with endemic and with non-endemic areas for visceral leishmaniasis, no study to date has focused on Leishmania spp. exposure among hunting dogs and hunters. The aim of the present study was to assess the presence of Leishmania spp. antibodies in hunting dogs and hunters in different anthropized areas of two Brazilian biomes.Materials and Methods:Blood samples were collected from 170 hunting dogs and 46 hunters between October 2016 and May 2018. The presence of antibodies against Leishmania spp. in hunting dogs was screened through a dual-path platform immunochromatographic test (DPP rapid test; Bio-Manguinhos/Fundação Oswaldo Cruz, Rio de Janeiro, Brazil) and in hunters through an rK39-based rapid immunochromatographic test. Both tests were used in accordance with Brazilian Ministry of Health recommendations.Results:Overall, although antibodies were detected through the immunochromatographic test in 3/170 (0.02%) of these female asymptomatic hunting dogs, all living in anthropized areas of the Atlantic Forest biome in South Brazil, no sample was confirmed through the enzyme-linked immunosorbent assay. All the hunters were non-reactive in the rapid immunochromatographic test.Conclusion:Our study on three suspicious hunting dogs has suggested that Leishmania (Leishmania) infantum may circulate both in endemic and non-endemic areas in Brazil. In addition, a high rate of hunting dog replacement due to death and trade may have led to less chance of infection and transmission between animals and between animals and humans, which would corroborate the outcomes reported here. Further studies should be conducted to fully establish whether hunting dogs and hunters may be used as sentinels in other areas endemic for Leishmania spp.

Therapeutic effect of Prosopis strombulifera (LAM) BENTH aqueous extract on a murine model of cutaneous leishmaniasis

Background and aimProsopis strombulifera (Lam.) Benth is a rhizomatous shrub native from different zones of Argentine Republic. P. strombulifera aqueous extract (PsAE) has different effects and several biological activities have been reported. The goal of this study was to analyze the activity of PsAE on a murine model of cutaneous leishmaniasis caused by Leishmania amazonensis. Experimental procedurePsAE was orally administered at 150 mg/animal/day on BALB/c mice infected in the right footpad (RFP) with 1 × 105 promastigotes of L. amazonensis. As a chemotherapeutic control of treatment, animals receive a commercial form of meglumine antimoniate (MA) (Glucantime®, Aventis, Paris, France). Results and conclusionWe observe that the size of RFP lesions of infected mice without treatment showed a grade of inflammation, ulceration and necrosis at the site of infection much greater than that observed with PsAE or MA treatment. Moreover, PsAE was capable of decreasing parasite burden and splenic index. Furthermore, PsAE treated mice showed a significant decrease in O.D. of total anti-Leishmania IgG antibody responses against L. amazonensis. This decrease was similar to those observed when the reference drug, MA, was used. This would indicate that PsAE treatment inhibits or delays disease progression in mice. In conclusion, our findings suggest that PsAE could be a potential candidate to be used, as a new therapeutic strategy, to treat cutaneous leishmaniasis caused by L. amazonensis.

Early detection and persistent positivity of anti-Leishmania antibodies using a recombinant protein-based ELISA in naturally infected dogs in Brazil

BackgroundVisceral leishmaniasis (VL) is a zoonotic disease caused by Leishmania infantum, for which dogs constitute the main urban parasite reservoir. Control measures and the treatment of canine visceral leishmaniasis (CVL) are essential to reduce VL cases. Early and accurate detection of L. infantum-infected dogs is crucial to the success of VL control. To improve the serological detection of L. infantum-exposed dogs, we evaluated the early diagnosis capacity of a recombinant protein (rLci5) in an immunosorbent assay (ELISA) to detect naturally infected dogs. Additionally, we evaluated the persistence of the positive results obtained by rLci5 ELISA in comparison to other conventional diagnostic test methods.MethodsSerum samples obtained from 48 L. infantum-infected dogs involved in a cohort study were evaluated using different diagnostic methods (qPCR, EIE-LVC, DPP-LVC and splenic culture). The results were compared to rLci5 ELISA to determine its capacity to diagnose L. infantum infection at earlier infection time points. The persistence of positive diagnostic test results was also compared for each dog evaluated.ResultsrLci5 ELISA presented higher rates of positive results at early time points compared to the other diagnostic tests employed in the cohort study, as early as 24 months prior to detection by other tests. rLci5 ELISA positivity was 52.1% (25/48) at baseline, while qPCR was 35.4% (17/48), DPP-LVC 27.1% (13/48), EIE-LVC 22.9% (11/48) and culture only 4.2% (2/48). In at least one of the time points of the 24-month cohort study, rLci5 ELISA was positive in 100% (48/48) of the dogs, versus 83% (40/48) for qPCR, 75% (36/48) for DPP-LVC, 65% (31/48) for EIE-LVC and 31% (15/48) for culture. Investigating clinical signs in association with diagnostic test positivity, rLci5 ELISA successfully detected CVL in 62.9% (95/151) of the clinical evaluations with a score of 0–3, 64.3% (45/70) with scores between 4 and 7, and 73.7% (14/19) with scores > 7, providing higher rates of positivity than all other methods evaluated. Moreover, rLci5 ELISA presented the greatest persistence with respect to test positivity: 45.8% of the dogs evaluated.ConclusionFour diagnostic tests were compared to rLci5 ELISA, which presented earlier infection diagnosis and a greater persistence of positive test results. Accordingly, the use of the rLci5 ELISA can improve CVL diagnostic performance by detecting infected dogs sooner than other testing methods, with enhanced persistence of positive results over the course of the infection.Graphic abstract

Immunosuppression by UVB radiation exacerbates Leishmania mexicana skin lesions in mice

Cutaneous leishmaniasis is the most common form of leishmaniasis in humans. The disease is caused by several species, such as Leishmania mexicana, a protozoa parasite. Several major risk factors are associated with this disease, including poverty, poor housing, inadequate domestic hygiene, malnutrition, mobility, and occupational exposure. Solar radiation (UVB) has not been considered a risk factor because there is no scientific evidence demonstrating a correlation with increased susceptibility to cutaneous leishmaniasis. In this study, the shaved skin of the back of C57BL/6 mice was irradiated with 24.2 mJ/cm2 of UVB. A delayed-type hypersensitivity (DTH) reaction was used to assess UV-induced immune suppression. Skin lesions were quantitated, and parasite burden and the presence of anti-Leishmania mexicana antibodies in serum and germinal centers in draining lymph nodes were determined. We found an increased in the lesion size and parasitic load in UVB-irradiated mice compared to the WT mice and B lymphocyte activation in draining lymph nodes and increased IgG1 production. Our results show an important role of UVB-induced suppression in cutaneous leishmaniasis through local production of IL-10 and systemic IgG1antibodies. This is the first study that demonstrates the effects of UVB radiation on cutaneous leishmaniasis by Leishmania mexicana.

Development and Clinical Evaluation of Serum and Urine-Based Lateral Flow Tests for Diagnosis of Human Visceral Leishmaniasis.

Visceral leishmaniasis (VL), a fatal parasitic infection, is categorized as being neglected among tropical diseases. The use of conventional tissue aspiration for diagnosis is not possible in every setting. The immunochromatography-based lateral flow assay (LFA) has attracted attention for a long time due to its ability to give results within a few minutes, mainly in resource-poor settings. In the present study, we optimized and developed the LFA to detect anti-Leishmania antibodies for VL diagnosis. The performance of the developed test was evaluated with serum and urine samples of Indian VL patients and Brazilian sera. The new test exploits well-studied and highly-sensitive purified antigens, LAg isolated from Leishmania donovani promastigotes and protein G conjugated colloidal-gold as a signal reporter. The intensity of the bands depicting the antigen–antibody complex was optimized under different experimental conditions and quantitatively analyzed by the ImageJ software. For the diagnosis of human VL in India, LFA was found to be 96.49% sensitive and 95% specific with serum, and 95.12% sensitive and 96.36% specific with urine samples, respectively. The sensitivity and specificity of LFA were 88.57% and 94.73%, respectively, for the diagnosis of Brazilian VL using patients’ sera infected with Leishmania infantum. LFA is rapid and simple to apply, suitable for field usage where results can be interpreted visually and particularly sensitive and specific in the diagnosis of human VL. Serum and urine LFA may improve diagnostic outcomes and could be an alternative for VL diagnosis in settings where tissue aspiration is difficult to perform.

Antibodies to Leishmania in naturally exposed domestic ferrets (Mustela putorius furo) in Spain

Zoonotic leishmaniosis due to Leishmania infantum is a vector-borne disease endemic in southern Europe and dogs are the main reservoir for this infection. Seasonal variations in antibody titers in this species in areas where phlebotomine vectors have seasonal patterns of activity are important for epidemiological, preventive and clinical studies related with canine leishmaniosis. It has been suggested that cats, rabbits and ferrets may act as peridomestic reservoirs and not only as accidental hosts. The aim of this study was to determine if seropositive ferrets (Mustela putorius furo) to Leishmania could be affected by seasonal variations of anti-Leishmania antibodies. A group of seropositive clinically healthy ferrets (n = 21) were included in this study. A significant reduction in anti-Leishmania infantum antibodies was detected during non-transmission period (December 2020-February 2021) in comparison to transmission period (April-October 2020). This study describes for the first time a seasonal variation in the anti-Leishmania antibodies detected in domestic ferrets following natural exposure during sand fly transmission period and the following non-sand fly transmission period in a Mediterranean area considered as an area where L. infantum is endemic.