anti-Leishmania Antibodies Research Articles

Rectal Administration of Leishmania Cells Elicits a Specific, Th1-Associated IgG2a Response in Mice: New Perspectives for Mucosal Vaccination against Leishmaniasis, after the Repurposing of a Study on an Anti-Viral Vaccine Candidate.

The mucosal immune system plays a pivotal role in the control of infections, as it represents the first line of defense against most pathogens, from respiratory viruses to intestinal parasites. Mucosal vaccination is thus regarded as a promising strategy to protect animals, including humans, from infections that are acquired by ingestion, inhalation or through the urogenital system. In addition, antigens delivered at the mucosal level can also elicit systemic immune responses. Therefore, mucosal vaccination is potentially effective also against systemic infections acquired through non-mucosal routes, for example, through the bite of hematophagous insects, as in the case of leishmaniasis, a widespread disease that affects humans and dogs. Here, we explored the potential of antigen rectal administration for the generation of anti-Leishmania immunity. Mice were immunized through rectal administration of whole cells of the model parasite Leishmania tarentolae (using a clone engineered to express the spike protein of the SARS-CoV-2 virus generated in a previous study). A specific anti-Leishmania IgG antibody response was detected. In addition, the recorded IgG2a/IgG1 ratio was higher than that of animals injected subcutaneously; therefore, suggesting a shift to a Th1-biased immune response. Considering the importance of a Th1 polarization as a protective response against Leishmania infections, we suggest that further investigation should be focused on the development of novel types of vaccines against these parasites based on rectal immunization.

Detection of Leishmania spp. in Cats: Analysis of Nasal, Oral and Conjunctival Swabs by PCR and HRM.

Feline leishmaniasis (FeL) is caused by several species of parasites of the genus Leishmania. The disease can occur with the presence or absence of clinical signs, similar to those observed in other common infectious diseases. In endemic regions for FeL, the infection has been associated with dermatological lesions. Therefore, considering the search for less invasive and more effective diagnostic techniques, we aimed to investigate the presence of Leishmania spp. in domestic cats through Polymerase Chain Reaction (PCR) and high-resolution melting (HRM) analyses of conjunctival, oral, and nasal epithelial cells, and we detected the presence of anti-Leishmania IgG antibodies from serological techniques of the Immunofluorescent Antibody Test (IFAT) and ELISA. The PCR and HRM for detection of Leishmania spp. were performed on 36 samples of epithelial cells from the conjunctiva of male and female cats, collected using sterile swabs. The serological tests IFAT and ELISA were also performed. The prevalence of Leishmania donovani infection was 11.1% (4/36) by PCR assay, and those results were confirmed for Leishmania species using the HRM technique. Twenty-four cats (24/36 = 66.7%) were reactive to the IFAT and twenty-two cats were reactive by the ELISA technique (22/36 = 61.1%). The use of conjunctival swabs was shown to be a non-invasive, practical, and easy-to-perform technique, and in addition to the genetic sequencing and HRM, it was able to identify the parasitic DNA of L. donovani in cats. This technique can be used for screening diagnosis in future epidemiological surveys of FeL and can be used as a complement to clinical and/or serological tests, as well as associating the clinical history of the animal, for the diagnostic conclusion.

Serological survey of Leishmania infantum in apparently healthy dogs in different areas of Spain.

Canine leishmaniosis caused by Leishmania infantum is an endemic disease in Spain. The dog is considered the main reservoir, and the detection of specific serum antibodies against L. infantum antigen is the most used technique for diagnosing this infection. The LEISCAN LEISHMANIA ELISA test is a commercialized enzyme-linked immunosorbent assay for the detection and measurement of canine anti-Leishmania serum antibodies. The aim of this study was to assess seroprevalence results of apparently healthy dogs in different areas of Spain using LEISCAN. Collection of sera from 5451 apparently healthy dogs was performed between 2020 and 2021 in different areas of Spain. Dogs were of adult age (≥12 months), were not previously diagnosed with clinical leishmaniosis or vaccinated against Leishmania and did not present clinical signs compatible with L. infantum infection. LEISCAN was performed following the manufacturer's protocol. The overall seroprevalence was 5.5%. The highest seroprevalences were found in the Southeast of Spain: Comunidad Valenciana (14%) and Región de Murcia (14%), whereas the lowest seroprevalences were found in Northern Spain: Galicia (1%), Navarra (2%) and Castilla y León (2%) (p-value <0.001). In conclusion, the seroprevalence for L. infantum in apparently healthy dogs in Spain varied from almost no infection to being over 10%.

Increased CCL-5 (RANTES) Gene Expression in the Choroid Plexus of Dogs with Canine Leishmaniosis

Visceral canine leishmaniasis (CanL) can cause several clinical manifestations, including neurological lesions. Few reports have characterized the lesions observed in the central nervous system (CNS) during CanL; however, its pathogenesis remains unclear. The choroid plexus (CP) is a specialized structure responsible for the production and secretion of cerebrospinal fluid (CSF) and considered an interface between the peripheral immune system and CNS. It can allow the passage of inflammatory cells or pathogens and has the potential to act as a source of inflammatory mediators in several diseases. Thus, this study aimed to evaluate the role of CP as a possible route of inflammatory cells in the development of brain lesions in dogs with CanL, as well as its association with blood–CSF barrier (BCSFB) dysfunction. Samples were collected from 19 dogs that were naturally infected with CanL. We evaluated the histopathological lesions in the brain and investigated the gene expression of the cytokines. Capture enzyme-linked immunosorbent assay (ELISA) was used to evaluate the presence of the same cytokines in the CSF. Biochemical analysis was performed to compare the presence of albumin in the serum and CSF. Indirect ELISA was performed to measure the presence of anti-Leishmania antibodies in the CSF, which would suggest the disruption of the BCSFB. Histopathological evaluation of the dogs’ brains revealed mild-to-severe inflammatory infiltrates, mainly in the CP and meninges. We also detected the presence of anti-Leishmania antibodies and albumin in the CSF, as well as Leishmania DNA in the CP. The gene expression of CCL-5 was increased in the CP of infected dogs compared with that of controls, and there was a tendency for the increase in the gene expression of CXCL-10. Thus, our findings confirm the disfunction of the BCSFB during CanL and suggest that the chemokines CCL-5 and CXCL-10 can be responsible for the recruitment of inflammatory cells found in CP.

Signalment, Immunological and Parasitological Status and Clinicopathological Findings of Leishmania-Seropositive Apparently Healthy Dogs.

Canine leishmaniosis caused by Leishmania infantum is a disease with a wide range of clinical manifestations. Epidemiological serosurveys performed in Europe often lack a thorough assessment of clinical health status of studied dogs. The aim of this study was to evaluate signalment, immunological and parasitological status and clinicopathological findings of L. infantum-seropositive apparently healthy dogs (n = 212) living in endemic areas. Routine laboratory tests, endpoint in-house ELISA to quantify the anti-Leishmania antibodies, blood Leishmania qPCR and IFN-γ ELISA were performed. All dogs enrolled were L. infantum-seropositive and were classified as healthy (n = 105) or sick (n = 107) according to LeishVet guidelines. The sick group presented a higher proportion of medium to high antibody levels and positive qPCR and lower IFN-γ concentration compared to the healthy group. Sick dogs were mostly classified in LeishVet stage IIa. Biochemical alterations (98%) were the most common clinicopathological findings, with fewer urinary tract (46%) and hematological (40%) alterations. Apparently healthy L. infantum-seropositive dogs can be classified between truly healthy dogs and sick dogs with clinicopathological findings. Sick dogs presented medium to high seropositivity and parasitemia and low IFN-γ concentrations, and their most common clinicopathological abnormalities were serum protein alterations followed by proteinuria and lymphopenia.

Immunological profile of two canine breeds in an endemic region of Leishmania infantum.

Visceral leishmaniosis is the one of the most important protozoal zoonoses in Europe, and it is caused by Leishmania infantum, an intracellular protozoan parasite. The disease is endemic in dogs in the Mediterranean area. The main goal of this work is to correlate the levels of several cytokines linked to immune response against L. infantum infection in two canine breeds. Thirty-one Boxer and twenty-eight Ibizan Hound dogs living in the Valencian Community (East coast of Spain) were analyzed for the presence of anti-Leishmania antibodies in serum by IFAT test. Cytokines IFN-γ, TNF-α, IL-2, IL-6, IL-8, and IL-18 were determined by ELISA commercial tests. The levels of IFN-γ, IL-2, and IL-18 in our study, cytokines linked to a cellular immune response, were higher (p<0.05) in the Ibizan Hound breed; IL-6 levels were higher, although not significant, and only levels of IL-8 were higher in Boxer than in Ibizan Hound. No expression of TNF-α was found. These results corroborate that Ibizan Hound can develop a protective response against canine leishmaniosis, while Boxer is a susceptible breed. The study of immunological aspects in the different canine breeds may represent a useful tool in the prediction of the disease.

Live attenuated Leishmania infantum centrin deleted mutant (LiCen-/-) as a novel vaccine candidate: A field study on safety, immunogenicity, and efficacy against canine leishmaniasis

This study was designed to evaluate the safety, immunogenicity, and efficacy of a single dose of L. infantum (LiCen-/-) live attenuated candidate vaccine against canine leishmaniasis (CanL). Eighteen healthy domestic dogs with no anti-Leishmania antibodies and negative leishmanin skin test (LST) were randomly inoculated intravenously with either L. infantum (LiCen-/-) vaccine candidate in 10 dogs or phosphate-buffered saline (PBS) in 8 dogs. The safety, immunogenicity, and efficacy rate of L. infantum (LiCen-/-) vaccine candidate against CanL were evaluated by different criteria, including clinical manifestations, injection-site lesion, hematology and biochemistry values, anti-Leishmania antibodies using direct agglutination test (DAT), delayed-type hypersensitivity (DTH) using LST, and CD4+ and CD8+ T-cells subsets, as well as by measuring interferon (IFN-γ), interleukin (IL-23), IL-17, and IL-10 cytokines. Spleen aspiration and detection of Leishmania parasite using parasitological examinations (microscopy and culture) were performed in both vaccinated and control groups. Two months after intervention, each dog was challenged intraperitoneally (IP) with wide type (WT) L. infantum. Two-month follow-up post vaccination showed no clinical signs and serious side effects associated with the vaccination. A significant increase was found in the expression of IL-17, CD4+, and CD8+ gene transcripts in PBMCs, as well as increased levels of Th1 cytokines, and reduction of Th2 cytokine. The efficacy of the vaccine candidate was calculated to be 42.85%. While the time window for assessing the vaccine's effectiveness was too limited to draw any real conclusions but the preliminary results showed a moderate efficacy rate due to inoculation a single dose of L. infantum (LiCen-/-) vaccine candidate. Further investigations with more sample sizes and multiple doses of the vaccine candidate using natural challenges in the endemic areas of CanL are recommended.

Seroprevalence of Visceral Leishmaniasis in Children Up To 12 Years Old of Rural Areas from Kermanshah Province, Western Part of Iran.

After the earthquake in 2017 a few new cases of visceral leishmaniasis (VL) were reported from SarPol-e-Zahab district of Kermanshah Province, western part of Iran. This study was conducted to determine the seroprevalence in Kermanshah Province. This descriptive cross-sectional study was conducted on children up to 12 years of age from SarPol-e-Zahab County, Kermanshah Province, western part of Iran in 2021. For each individual, a questionnaire including age, sex, clinical features, history of the disease, and contact with canines as reservoir hosts of VL were completed, separately. To determine VL seroprevalence, blood samples were collected from the children and after centrifugation, the sera samples were separated and tested using Direct Agglutination Test (DAT) for detection of anti-L. infantum antibodies. Statistical analyses were performed using SPSS16. Totally, 13 persons were seropositive; 7 samples with titer 1:800, 3 samples had 1:1600, 2 samples had 1:3200 and 1 sample had 1:6400. None of the seropositive cases had a history of kala-azar. There was no significant difference between males and females at titers of anti-Leishmania specific antibodies. L. infantum infection is being circulated with low prevalence in children up to 12 years old from SarPol-e-Zahab County but it is necessary that the surveillance system is regularly monitored among physicians and public health managers in the studied areas.

TLR9 agonist CpG ODN 2395 promotes the immune response against Leishmania donovani in obesity and undernutrition mice

As important immunomodulators, CpG ODNs have broad application prospects in the treatment and prevention of leishmaniasis. In order to explore the immunomodulatory effect of CpG ODNs on mice infected with Leishmania parasites in different nutritional status, TLR9 agonist CpG ODN 2395 or TLR9 antagonist CpG ODN 2088 was injected into normal, obesity and undernutrition BALB/c mice infected with Leishmania donovani, respectively. Subsequently, spleen and liver parasite loads, spleen and liver immune gene expression, spleen T cell subsets proportion and PD-1 expression, serum lipids, serum cytokines, and anti-Leishmania antibodies were measured to assess the immune response of mice with different nutritional status. The results displayed that at the 8th week after infection, the spleen parasite load of obesity and undernutrition mice was significantly higher than that of normal mice, but the liver parasite load showed no statistical difference among the three groups. The treatment of CpG ODN 2395 or CpG ODN 2088 significantly reduced the spleen parasite load of obesity and undernutrition infected mice, but did not reduce that of normal infected mice. In obesity infected mice, CpG ODN 2395 promoted the up-regulation of TCR, ICOS and TLR4 in spleen, promoted the secretion of IFN-γ and anti-Leishmania total IgG and IgG1 antibodies, and increased the content of serum HDL-C. In undernutrition infected mice, CpG ODN 2395 promoted the up-regulation of spleen CD28 and TLR9, increased the proportion of spleen CD3+ T cells, and decreased the content of serum IL-10. Our results demonstrated that CpG ODN 2395 enhanced the immune response and clearance of Leishmania parasites in obesity and undernutrition mice, which might be used as a therapeutic agent for obesity and undernutrition leishmaniasis patients in the future.

Antiparasitic Activity of Two Natural Terpenes from Salvia cuspidata against Leishmania amazonensis.

Leishmaniasis is a neglected disease caused by flagellated parasites of the Leishmania genus affecting more than 10 million people worldwide. Current treatments for leishmaniasis involve the administration of poorly tolerated drugs with toxic side effects in patients. There is an imperative necessity for novel compounds to treat this disease. One of the most used strategies in the search for different antiparasitic compounds is the screening of purified plant molecules. The diterpenes 12-hydroxy-11,14-diketo-6,8,12-abietatrien-19,20-olide (HABTO) and 5-epi-icetexone (ICTX) isolated from Salvia cuspidata were shown to be effective against Leishmania amazonensis in vitro and in vivo. They displayed an antiproliferative effect against L. amazonensis promastigotes. They also induce an increase in ROS levels and affect the mitochondrial activity of parasites. HABTO and ICTX in an in vivo model of cutaneous leishmaniasis decrease footpad swelling, parasite load, and splenic index. Moreover, they induce significant reduction in the O.D. of total anti-Leishmania IgG and IgG1 subtype antibody responses against L. amazonensis compared to the PBS group but maintain high levels of IgG2a. This suggests that in HABTO- or ICTX-treated mice, there is a slowdown in the progression of the disease. These terpenes could be considered as possible novel antileishmanial agents against L. amazonensis and thus treat cutaneous leishmaniasis.

A Sero-Epidemiological Study on Visceral Leishmaniasis among Volunteer Children and Adults in Rural Areas of Shahroud, Iran 2018-2019.

Visceral leishmaniasis (VL) also known as Kala-azar is considered as one of the zoonotic infections in Mediterranean countries. The disease reservoir and vectors are dogs and sandflies respectively. Due to reported sporadic cases of Kala-azar in the past five years in Shahroud County, Semnan Province, Iran, this study aimed to investigate the status of this infection in this area and to determine its seroepidemiology to take required measurements for infection control and treatment. This study was conducted on 504 subjects residing in seven villages in Shahroud County. Blood samples were randomly selected using the cluster sampling method and were collected from subjects aged up to 13 years old (90%) and adults over 13 years old (10%) from September to May 2019. After separating sera from whole blood, samples were subjected to direct agglutination test (DAT) to detect anti-Leishmania infantum antibodies. A range of 1:10 to 1:800 dilutions were prepared from the samples. Results of 1:800 titration indicated that no sample was positive for antibodies against L. infantum. After the secondary screening, 10 cases (1.98%) showed the antibody titer of 1:100, while four cases (0.79%) showed the antibody titer of 1:400. Of 14 cases with the L. infantum antibodies, all were detected from the children <13 years old. According to clinical findings, no patient was suffering from fever, weight loss, splenomegaly, hepatomegaly, and cachexia and therefore did not show the Kala-azar symptoms. The results of the current study indicate that Kala-azar is not prevalent in Shahroud County.

Assessment of Risk of Exposure to Leishmania Parasites among Renal Disease Patients from a Renal Unit in a Sri Lankan Endemic Leishmaniasis Focus.

Leishmania donovani causes both cutaneous and visceral leishmaniasis (CL and VL) in Sri Lanka, where chronic kidney disease (CKD) and kidney transplant recipients' (KTR) geographical areas overlap. This study aimed to determine the risk of exposure to Leishmania infection among renal patients. This cross-sectional study in a renal unit assessed clinical symptoms and signs of CL and VL in recipients of blood/kidney or immunosuppressives. Sera were tested with Leishmania-specific DAT and rK-39 ELISA. There were 170 participants. A total of 84.1% (n = 143) were males (CKD: 101, KTR; 42, mean age 45) and 27 were females (females: CKD: 23, KTR: 4, mean age 39 years). Recipients of blood transfusion/s within last 2 years: 75.9% (CKD: 115, KTR: 14), on immunosuppressive therapy: 34.1% (CKD: 13, KTR: 45). Two CKD patients repeatedly showed clear positive titres (1: 12,800 and 1: 3200) with Leishmania-DAT and another two (CKD) became marginally positive with rK39-ELISA. Prevalence of anti-Leishmania antibodies: 2.4% (4/170). All four patients were clinically asymptomatic and were recipients of recent blood transfusions. Attributable risk of exposure to Leishmania infection through blood transfusions was 0.032, OR 2.99 (95% CI = 0.16 to 56.45, p = 0.47). Therefore, routine screening of kidney/blood donors and CKD and KTR patients in Sri Lanka may not be necessary.

Seroprevalence and Risk Factors Associated with Leishmania Infection in Dogs from Portugal.

Canine leishmaniosis (CanL) caused by Leishmania infantum is an important zoonosis in southwestern European countries where this disease is endemic, and dogs, as domestic animals in close contact with humans, are the reservoir hosts for the parasite. In Portugal, CanL is of relevant veterinary concern. The previous national study revealed an overall seroprevalence of 6.3%. Since then, new prophylactic measures, such as vaccines, have been introduced in Europe. The aim of this study was to update seroprevalence for Leishmania infection and reassess risk factors in Portugal. A cross-sectional study was conducted from January-March 2021 with 1860 client-owned dogs from continental Portugal. A questionnaire and whole blood samples on filter paper were collected and a direct agglutination test was used to calculate anti-Leishmania antibody titres. True seroprevalence was 12.5% (95% CI 10.3-13.2%). Potential risk factors associated with L. infantum infection in dogs were age ≥ 2 years (aOR = 1.68, 95% CI 1.1-2.6) and residing in the interior regions of the country (aOR = 1.92, 95% CI 1.3-2.9) and non-use of repellents (aOR = 1.75, 95% CI 1.2-2.5). The key to controlling CanL and its impact on Public Health in endemic areas lies in continuous implementation of prophylactic measures, through the correct use of repellents/insecticides and vaccines and early detection and monitoring of infected dogs.

Prevalence of mountain-zoonotic type visceral leishmaniasis in Yangquan City of Shanxi Province from 2015 to 2020

To investigate the prevalence of mountain-type zoonotic visceral leishmaniasis (MT-ZVL) in Yangquan City, Shanxi Province from 2015 to 2020, so as to provide the scientific evidence for formulating the MT-ZVL control strategy. The epidemiological data pertaining to MT-ZVL cases in Yangquan City from 2015 to 2020 were collected and descriptively analyzed. A Joinpoint regression model was created to analyze the trend in the MT-ZVL incidence in Yangquan City from 2015 to 2020 using annual percent change (APC). The sandflies surveillance data and the prevalence of Leishmania infections in dogs were collected in Yangquan City in 2020, and the regional distribution of sandflies density and sero-prevalence of Leishmania infections in dogs were calculated. In addition, the associations of sandflies density and sero-prevalence of Leishmania infections in dogs with the incidence of human MT-ZVL were examined using the linear correlation analysis. A total of 162 MT-ZVL cases were reported in Yangquan City, Shanxi Province from 2015 to 2020, with annual mean incidence of 1.9/105, and there were 4, 7, 16, 27, 33 cases and 75 cases with MT-ZVL reported from 2015 to 2020, appearing a tendency towards a rapid rise (APC = 72.79%, t = 11.10, P < 0.01). MT-ZVL cases were reported across the five counties (districts) of Yangquan City, and the cases predominantly occurred in Jiaoqu District (35.2%, 57/162) and Pingding County (33.3%, 54/162). MT-ZVL cases were predominantly detected in residents at ages of 15 years and older (71.6%, 116/162) and at ages of 0 to 2 years (22.2%, 36/162), with farmers (37.4%, 61/162) and diaspora children (24.5%, 40/162) as predominant occupations. The mean density of Phlebotomus chinensis was 6.3 sandflies per trap per night in Yangquan City from during the period from May to September, 2020, with the highest density observed in Jiaoqu District (12.6 sandflies per trap per night) and the lowest in Yuxian County (1.1 sandflies per trap per night), and there was a region-specific mean density of Ph. chinensis in Yangquan City (H = 17.282, P < 0.01). The sero-prevalence of serum anti-Leishmania antibody was 7.4% (2 996/40 573) in domestic dogs in Yangquan City, with the highest sero-prevalence seen in Jiaoqu District (16.6%, 1 444/8 677), and the lowest in Yuxian County (2.3%, 266/11 501), and there was a region-specific sero-prevalence rate of anti-Leishmania antibody in domestic dogs in Yangquan City (χ2 = 1 753.74, P < 0.01). The sero-prevalence of anti-Leishmania antibody was significantly higher in stray dogs (20.0%, 159/794) than in domestic dogs (χ2 = 176.63, P < 0.01). In addition, there were significant associations among the sandflies density, sero-prevalence of anti-Leishmania antibody in domestic dogs and the incidence of human MT-ZVL (r = 0.832 to 0.870, all P values < 0.05). The prevalence of MT-ZVL appeared a tendency towards a rapid rise in Yangquan City from 2015 to 2020, and systematic interventions are urgently needed for MT-ZVL control.

Nanoformulations with Leishmania braziliensis Antigens Triggered Controlled Parasite Burden in Vaccinated Golden Hamster (Mesocricetus auratus) against Visceral Leishmaniasis.

Leishmaniasis is a widespread vector-borne disease in Brazil, with Leishmania (Leishmania) infantum as the primary etiological agent of visceral leishmaniasis (VL). Dogs are considered the main reservoir of this parasite, whose treatment in Brazil is restricted to the use of veterinary medicines, which do not promote a parasitological cure. Therefore, efficient vaccine development is the best approach to Canine Visceral Leishmaniasis (CVL) control. With this in mind, this study used hamsters (Mesocricetus auratus) as an experimental model in an anti-Leishmania preclinical vaccine trial to evaluate the safety, antigenicity, humoral response, and effects on tissue parasite load. Two novel formulations of nanoparticles made from poly(D, L-lactic) acid (PLA) polymer loading Leishmania braziliensis crude antigen (LB) exhibiting two different particle sizes were utilized: LBPSmG (570 nm) and LBPSmP (388 nm). The results showed that the nanoparticles were safe and harmless to hamsters and were antigenic with the induction in LBSap, LBPSmG, and LBPSmG groups of total anti-Leishmania IgG antibodies 30 days after challenge, which persists 200 days in LBSap and LBPSmP. At the same time, a less pronounced hepatosplenomegaly in LBSap, LBPSmG, and LBPSmP was found when compared to control groups, as well as a less pronounced inflammatory infiltrate and granuloma formation in the spleen. Furthermore, significant reductions of 84%, 81%, and 90% were observed in spleen parasite burden accessed by qPCR in the LBSap, LBPSmG, and LBPSmP groups, respectively. In this way, LBSap, LBPSmG, and LBPSmP formulations showed better results in vaccinated and L. infantum-challenged animals in further reducing parasitic load in the spleen and attenuating lesions in liver and splenic tissues. This results in safe, harmless nanoformulation vaccines with significant immunogenic and infection control potential. In addition, animals vaccinated with LBPSmP had an overall reduction in parasite burden in the spleen, indicating that a smaller nanoparticle could be more efficient in targeting antigen-presenting cells.

Study of ocular manifestations and humoral immune response in eyes of dogs with leishmaniasis.

Ocular manifestations in dogs with leishmaniasis are frequent and complications in affected tissues can lead to blindness. Immune processes play a very important role in the pathogenesis of ocular inflammation. Therefore, the immunology of ocular manifestations in dogs with leishmaniasis remains complex and poorly understood. Estimation and characterisation of ocular and periocular manifestations in dogs naturally infected with Leishmania infantum and investigation of the production site of specific anti-Leishmania infantum IgG. The present investigation used 53 confirmed dogs infected with Leishmania infantum, presenting ocular and periocular lesions, and 10 control non-infected dogs. Complete macroscopic ophthalmic examination of eyelids and globes was performed. Both total and anti-Leishmania infantum IgG antibodies were studied in sera and aqueous humour (AH) of all dogs by ELISA technique. A Goldmann-Witmer coefficient (C value) was calculated. The main ophthalmological findings were keratoconjunctivitis (71.7%; 38/53), hyperplasia of conjunctival lymphoid follicles (54.7%; 29/53), blepharitis (50.9%; 27/53) and uveitis (20.7%; 11/53). Ocular production of anti-Leishmania infantum IgG was detected in 73.6% (39/53) of infected dogs. There was no correlation between the antibody levels in AH and sera of the same dog. The mean anti-Leishmania infantum IgG in AH was higher in uveitis, followed by lesions affecting only the adnexa (p < 0.0001). The highest mean C values were observed for uveitis, conjunctivitis and keratitis. Our findings suggest that production of anti-Leishmania IgG in dogs infected with Leishmania infantum with ocular manifestations begin in situ and follows by a transfer of antibodies from the bloodstream to the AH.

Detection of Leishmania (Mundinia) macropodum (Kinetoplastida: Trypanosomatidae) and heterologous Leishmania species antibodies among blood donors in a region of Australia with marsupial Leishmania endemicity

ObjectivesThe Australian Leishmania (Mundinia) macropodum parasite causes cutaneous leishmaniasis among marsupial species. Although cutaneous leishmaniasis is a major public health burden worldwide, it is not clear if humans are naturally exposed to the unique L. macropodum. To assess whether humans have an immunoglobulin (Ig) G response to L. macropodum, we examined anti-Leishmania antibodies among humans residing in a region of marsupial Leishmania endemicity in Australia. MethodsUsing a serological enzyme-linked immunosorbent assay, we characterized Leishmania-specific IgG and IgG subclass responses to soluble Leishmania antigen from L. macropodum, and other Leishmania species (L. donovani, L. major, and L. mexicana) in 282 blood donor samples. ResultsWe found that 20.57% of individuals demonstrated a positive total IgG response to L. macropodum. For individuals with antibodies to soluble Leishmania antigen from one Leishmania species, there was no increased likelihood of recognition to other Leishmania species. For samples with detectable L. macropodum IgG, IgG1 and IgG2 were the prevalent subclasses detected. ConclusionIt is not yet clear whether the IgG antibody detection in this study reflects exposure to Leishmania parasites or a cross-reactive immune response that was induced against an unrelated immunogen. Future studies should investigate whether L. macropodum can result in a viable infection in humans.

Seroepidemiological Study of Visceral Leishmaniasis (Kala-Azar) in Children under 12 Years Old in North of Iran: An Observational Study in 2019-2020.

Visceral leishmaniasis (VL) caused by the Leishmania donovani complex that is transmitted by the bites of female sandflies. Mediterranean type of VL caused by L. infantum. While, Roudbar County of Guilan Province has been introduced as a suspected cutaneous leishmaniasis focus; there are no published data on the seroprevalence of VL in Guilan Province. We aimed to investigate the sero-prevalence of this disease in Roudbar County. This descriptive cross-sectional study was carried out in 2019-2020 among children less than 12 years of age to determine the seroprevalence of VL by direct agglutination test (DAT). Blood samples were randomly collected from 918 children under 12 years of age refers to the public health center in the clusters. Out of 918 children, 14 (1.52%) showed anti-Leishmania antibodies, with 4 (0.43%), 2 (0.21%), 8 (0.87%) anti-L. infantum antibodies at titers 1:800, 1:1600 and ≥1: 3200 respectively. All children with anti-Leishmania antibody titers of ≥1:800 were evaluated by a physician. Clinical manifestation of VL including fever, anemia and hepatosplenomegaly observed in a 6-year-old boy from Defraz village with anti-Leishmania antibody of titers ≥102400.This patient was admitted to the pediatric hospital in Rasht, capital of Guilan province, Iran and was successfully treated. VL is being circulated with low prevalence in children up to 12 years old in Roudbar, northern part of Iran. Accordingly, it is critical to improve the awareness of physicians and public health supervisors about the importance of this fatal disease in Guilan province and especially in Roudbar area.

Domestic dogs carriers of Leishmania infantum, Leishmania tropica and Crithidia fasciculata as potential reservoirs for human visceral leishmaniasis in northeastern Iran.

In recent years, cases of human visceral leishmaniasis (HVL) have been reported in some districts of Golestan Province, northeastern Iran, particularly in rural areas. Recent epidemiological evidence in Leishmania infantum endemic regions of in Iran indicates approximately 50%-80% of seropositive dogs are asymptomatic for Leishmania infection. The goal in this study was to determine Leishmania species infecting domestic dogs in Golestan Province, Iran. Between 2015 and 2016, blood samples were obtained from 100 domestic dogs in rural regions of Golestan Province, northeastern Iran. All samples were tested for anti-Leishmania antibodies using a direct agglutination test (DAT), and for Leishmania spp. kinetoplast DNA (kDNA) using PCR. Seven (7%) dogs were antibody positive and 25 dogs (25%) were Leishmania spp. DNA positives by PCR positive for leishmaniasis. Four of the seven (71%) antibody-positive dogs and 19 of the 25 (76%) PCR-positive dogs were asymptomatic. The rate of infection detected by PCR was significantly higher in male dogs (21/75, 28%) than that in female dogs (4/25, 16%). The ITS1 PCR-RFLP assay identified the presence of L. infantum, L. tropica or Crithidia spp. in the 25 PCR-positive samples. The high proportion of asymptomatic dogs in the study areas represent they act as potential reservoirs in the transmission cycle of Leishmania spp. and also Crithidia fasciculata as an emerging agent for the first time. Moreover, our data showed that PCR is a more reliable assay than DAT for detecting Leishmania spp. infection among asymptomatic dogs.

Absence of DNA and anti-leishmanial antibodies in dogs (Canis familiaris) in Plateau state, Nigeria.

Dogs are important sentinels for the surveillance of some zoonotic diseases including human leishmaniasis. To obtain information on the role of dogs in the epidemiology of leishmaniasis in Nigeria, 98 sera and 204 DNA samples obtained from dogs were screened for anti-leishmania antibodies and DNA of Leishmania spp. using the enzyme linked immunosorbent assay (ELISA) and PCR, respectively. Initially, three out of the 98 sera samples had ELISA borderline optical density (OD) values and were retested. Two out of the three samples turned out to be negative while one sample gave yet a borderline OD value on a retest. A real time polymerase chain reaction (RT-PCR) targeting the 120-bp fragment of the minicircle kDNA of Leishmania spp. run on DNA extracted from EDTA preserved blood of the borderline positive serum failed to amplify the 120-bp sequence of Leishmania spp. In the second phase of the study, 204 DNA from dog blood samples were subjected to conventional PCR targeting the 300-350 bp of the internal transcribe spacer region 1 (ITS1) of Leishmania spp. None of the samples could be amplified (n = 204, 0%). Our study suggests that L. infantum is not prevalence in Jos, Plateau State, Nigeria and this should be confirmed using a larger sample of local dogs tested using PCR methods in lymphoid tissue samples.