Detection of anti-Leishmania infantum antibodies in sylvatic lagomorphs from an epidemic area of Madrid using the indirect immunofluorescence antibody test

An outbreak of human leishmaniosis was confirmed in the southwest of the province of Madrid, Spain, between July 2009 and December 2012. Incidence of Leishmania infection in dogs was unchanged in this period, prompting a search for alternative sylvatic infection reservoirs. We evaluated exposure to Leishmania in serum samples from animals in the area with an indirect immunofluorescence test (IFAT). Using promastigotes from six culture passages and a 1/25 threshold titer, we found anti-Leishmania infantum seroreactivity in 9.3% of cats (4 of 43), 45.7% of rabbits (16/35) and 74.1% of hares (63/85). Use of promastigotes from >10 in vitro passages resulted in a notably IFAT lower titer, suggesting antigenic changes during extended culture. Postmortem inspection of seropositive animals showed no clinical signs of infection. The results clearly suggest that asymptomatic hares were the main reservoir in the outbreak, and corroborate IFAT as a sensitive serological surveillance method to detect such cryptic Leishmania infections.     

Evidence for widespread Leishmania infantum infection among wild carnivores in L. infantum periendemic northern Spain

Leishmania spp. infection was investigated in tissue samples of wild carnivores from the Spanish Basque Country (BC), by PCR and DNA sequencing. The region is at the northern periphery of Leishmania infantum endemic Iberian Peninsula and infection in the dog (reservoir) or other species has not been previously reported. Leishmania kinetoplast DNA was detected by real-time PCR (rtPCR) in 28% (44/156) of animals. Specifically, in 26% of Eurasian badgers (n = 53), 29% of foxes (n = 48), 29% of stone martens (n = 21) and in 25–50% of less numerous species including genets, wild cats, pole cats, European mink and weasels. Infected animals particularly badgers, were most prevalent in the southernmost province of the BC (Araba) in areas dominated by arable land. Subsequent amplification and sequencing of a fragment of the rRNA internal transcribed spacer 2 (ITS2) from a subset of rtPCR positives samples confirmed the species as L. infantum, showing a high sequence homogeneity with ITS2 sequences of L. infantum from dogs and humans from southern Spain. In summary, this study reports for the first time L. infantum infection in wild carnivores from the BC including in stone martens, pole cats and minks in which infection has not been previously described. It supports the need to study infection in dogs and people in this region and is an example of the value of infection surveillance in wildlife to assess potential risks in the domestic environment and their role in spreading infections in non-endemic areas.     

Leishmania infantum secreted iron superoxide dismutase purification and its application to the diagnosis of canine Leishmaniasis

Leishmania spp. are digenetic parasites whose infection occurs inside the mononuclear phagocitary system. The iron superoxide dismutase secreted (Fe-SODe) by promastigotes of Leishmania spp. seems to plays an important role in the defense to environmental detoxification and neutralization of oxidative stress damage caused by reactive oxygen species (ROS) produced by macrophages during the infection. Parasites Fe-SODe is involved in establishing the infection and manifestation of Leishmaniasis. Its high immunogenicity makes it a useful molecular marker in diagnosing trypanosomatids infections. The aim of this study is demonstrate that purified Fe-SODe from Leishmania infantum is much more sensitive than un-purified Fe-SODe for diagnosis canine Leishmaniasis. We have purified a Fe-SODe of L. infantum using an ion exchange and a molecular sieve chromatographies and its application in diagnosis of canine Leishmaniasis was tested. One hundred and forty-five dogs’ sera from Andalusia Autonomous Community, Spain were tested by ELISA and Western blot and the antigen Fe-SODe purified is compared with two different antigens: the total parasites soluble lysate and the unpurified Fe-SODe. To validate the results obtained using the Fe-SODe purified we tasted 10 L. infantum infected dogs’ sera from Lombardy, Italy as positive control.     

Disseminated Leishmania infantum infection in two sibling foxhounds due to possible vertical transmission

Two sibling foxhounds born to a Leishmania seropositive bitch were presented after testing seropositive for Leishmania. Leishmania infantum infection was detected via histopathology, culture, and quantitative polymerase chain reaction (q-PCR). This is the first report of natural infection with Leishmania infantum with the possibility for vertical transmission in North America.     

Detection of Leishmania infantum DNA by real-time PCR in canine oral and conjunctival swabs and comparison with other diagnostic techniques

The use of non invasive sampling, such as collection of conjunctival swabs, as a diagnostic tool for the detection of Leishmania DNA is of interest. The purpose of this study was to evaluate the diagnostic utility of detecting Leishmania infection with the use of conjunctival swab samples in dogs living in a highly endemic area for leishmaniosis and to investigate, for the first time, the presence of Leishmania DNA in oral swabs in the same population. One hundred sixty-three dogs living outdoor and recruited in various provinces of Sicily were studied. Leishmania infantum indirect fluorescent antibody test (IFAT), delayed-type hypersensitivity reaction to leishmanin (DTH) and real-time PCR of blood (BL), lymph node (LN), conjunctival (CS) and oral swab (OS) samples were performed. The positive PCR percentages in LN, CS, OS and BL samples were: 24.5%, 22.1%, 8.7% and 5.5%, respectively. Serological and DTH positive percentages were 27.0% and 73.8%, respectively. Seropositive and LN-PCR positive dogs had a high likelihood to be positive by CS-PCR. The similar positive PCR percentages found in CS and LN samples suggest the use of CS-PCR as non-invasive alternative technique to LN-PCR for the detection of Leishmania infection in dogs. In addition, this study demonstrated, for the first time, the presence of Leishmania DNA in oral swabs in dogs.     

Evaluation of the circadian rhythm of anti-Leishmania IgG2 and IgA antibodies in serum and saliva of dogs with clinical leishmaniosis

In this study, the circadian rhythm of IgG2 and IgA specific antibodies in serum and saliva samples of 6 dogs experimentally infected with Leishmania infantum was assessed. Sampling was performed at 8.00, 12.00, 16.00, 20.00, and 00.00 h on two consecutive days. Anti-Leishmania antibody levels in serum were expressed without any correction, whereas in saliva were shown in different ways: without any correction, adjusted by protein concentration and corrected by the salivary flow rate. No significant differences in anti-Leishmania IgG2 antibody levels in serum and saliva samples with or without correction were found. Significant differences were found when anti-Leishmania IgA levels were corrected by the salivary flow rate. In addition, a greater intra-individual variation of antibody levels was observed in saliva than in serum. However, this variation did not modify the serological status of the dogs. Therefore, it could be concluded that there is no circadian rhythm in serum and saliva samples and sampling can be performed at any time of the day.     

Course of experimental infection of canine leishmaniosis: Follow-up and utility of noninvasive diagnostic techniques

This study compares the utility of a molecular diagnosis of experimental CanL on non-invasive samples (urine, conjunctival (CS), oral (OS) and vulvar (VS) swabs) with that of traditional invasive techniques during the course of infection. Eight dogs were experimentally infected with Leishmania infantum and followed monthly for 12 months to assess clinical, clinicopathological, immunological and parasitological variables. Active infection was produced in 100% of the dogs. The animals showed positive bone marrow (BM) cytologies and cultures, clinical signs, clinicopathological abnormalities and a high specific humoral immune response. The infection was detected at 90 days post-infection (p.i.) by real-time quantitative PCR (rtQ-PCR) on BM in all dogs and in blood in 2 dogs, while anti-L. infantum antibody seroconversion occurred between Days 120 and 180 days p.i. The tissue with the highest L. infantum kDNA load, as detected by rtQ-PCR, was BM (range 381.5–70,000 parasites/ml at the study end), this sample type showing greater sensitivity than peripheral blood (PB). The vulvar swabs used here for the first time to quantify parasite loads in dogs revealed a greater load than oral and conjunctival swabs at one year p.i. Urine samples showed the lowest concentrations of L. infantum DNA (maximum: 8.57 parasites/ml). Our results suggest that for the early detection of infection, adding to serology a test such as rtQ-PCR on OS or VS improves sensitivity and specificity.     

Detection of Leishmania (L.) infantum in stray dogs by molecular techniques with sensitive species-specific primers

Background: Canine visceral leishmaniasis (CVL) is a worldwide parasitic zoonosis caused by Leishmania (Leishmania) infantum around the world. Canids are the definitive hosts and sand flies the intermediate hosts.

Objective: To test the hypothesis that a new species-specific primers (Lch14:Lch15, targeting a multiple alignment for L. infantum kDNA minicircle) is an efficient diagnostic tool for L. infantum.

Methods: The presence of L. infantum DNA was assessed in blood samples of 69 stray dogs using the conventional PCR (cPCR) and quantitative PCR (qPCR). Additional 50 lymph nodes and 50 bone marrow samples (positive and negative samples for parasitological tests) from dogs from endemic and nonendemic areas for CVL were also used.

Results: L. infantum strains, and all positive lymph node and bone marrow samples for parasitological test gave positive results for cPCR and qPCR, presenting analytical sensitivity of ～10⁰ parasite mL^?1. For the blood samples, 40/69 (58%; CI 95%; 46%–69%) resulted positive for L. infantum in both tests. All positive samples were confirmed by sequencing.

Conclusion: This study showed the importance of the specific detection of L. infantum based on species-specific primers by molecular techniques, highlighting the application as a confirmation method in epidemiological studies and to adopt the best control measures.     

Phenotype evaluation of human and canine isolates of Leishmania infantum

Human visceral leishmaniasis (VL) and canine leishmaniasis (CanL) in countries of South and Central America are caused by Leishmania infantum and has been endemic in Brazil for several years. The parasite biodiversity as well as the pharmacologic properties of drugs and the host species, are involved in the efficacy or inefficacy of leishmaniasis treatments. Although there are substantial number of reports describing the genetic characterization of the clinical field isolates of L. infantum,the phenotypic parameters have been less studied. In this study isolates from human and canine leishmaniasis (Hum1 and Can1) obtained in Campinas, São Paulo state, Brazil were identified as L. infantum. The Hum1 and Can1 isolates exhibited typical promastigote growth pattern. Regarding morphological features Can1 isolate differed in cell size. The infectivity in vitro of both isolatesis lower compared to the reference strain of L. infantum. Moreover, the in vivo infectivity of the three parasites is similar in Balb/c mice. The Hum1 isolate is more sensitive to leishmanial drugs (amphotericin B, miltefosine and glucantime) than the Can1 isolate when inside human macrophages, but not when inside canine macrophages. These findings indicated that L. infantum isolates differs in some phenotypic characteristics.     

A survey on canine leishmaniasis and phlebotomine sand flies in central Italy

Zoonotic visceral leishmaniasis (ZVL) is a vector-transmitted zoonosis caused by the parasitic protozoan Leishmania infantum. Bloodsucking sand flies of the subfamily Phlebotominae are the obligatory insect hosts, and the dog is the only domestic reservoir.This study reports data from a survey of canine infection and sand fly phlebotomine monitoring in the province of Perugia in central Italy. The overall seroprevalence in a total of 100 dogs tested was 8% (95% confidence interval: 3.8-15.6%). Data analysis revealed that serological positivity was statistically associated with age (p-value = 0.03) and the area where dogs lived. Standard blacklight traps employed for sampling Culicoides midges in bluetongue disease surveillance were used in phlebotomine monitoring. A total of 5698 sand flies were collected and the two species, Leishmania competent vectors, were identified, Phlebotomus perfiliewi (50%) and Phlebotomus perniciosus (30%).     

Cryptic Leishmaniosis by Leishmania infantum, a feature of canines only? A study of natural infection in wild rabbits, humans and dogs in southeastern Spain

An epidemiological study was carried out to investigate asymptomatic Leishmania infantum infection by PCR and ELISA in wild rabbits, humans and domestic dogs in southeastern Spain. Seroprevalence was 0% (0/36) in rabbits, 2% (13/657) in humans and 7% (14/208) in dogs. The prevalence of PCR-positives was 0.6% (1/162) in rabbits tested in a wide range of tissue samples, 2% (8/392) in humans analysed in blood samples and 10% (20/193) and 67% (29/43) in dogs analysed in blood and lymphoid tissue samples, respectively. Results suggest that wild rabbits have a very low risk of becoming chronically infected with L. infantum, and provide further evidence that cryptic L. infantum infection is widespread in the domestic dog population and is also present in a comparatively smaller proportion of healthy humans. The epidemiological and clinical implications of these findings are discussed.     

Anti-Leishmania IgA in urine samples from dogs with clinical leishmaniasis

Recently, anti-Leishmania IgG has been detected in urine samples from Leishmania-infected dogs and its concentrations have been correlated with impairment of renal function. The presence and relationship with other anti-Leishmania Ig isotypes in urine have not yet been investigated. The current study analyzed the concentrations of anti-Leishmania IgA and IgG in sera (Ig-S) and urine (Ig-U) samples by ELISA in 64 untreated dogs with clinical leishmaniasis. All 64 serum samples tested were positive for anti-Leishmania IgG. Fifty of them (78.1%) were also positive for anti-Leishmania IgA. The results showed the presence of anti-Leishmania IgA-U in 38% of the 50 dogs that were positive for specific IgA-S. Thirty-eight of the 64 dogs positive for Leishmania-specific IgG-S (59.4%) were also positive for Leishmania-specific IgG in urine (IgG-U). The concentrations of anti-Leishmania IgA-U were significantly correlated with urine protein/creatinine (uP/C) ratio (ρ = 0.542; P < 0.001) and with serum biochemical parameters, such as γ-globulins, urea and creatinine. Goldmann–Witmer coefficient (C value) indicated that detection of specific IgA in urine samples from dogs with leishmaniasis might not only be due to impairment of filtration of the glomerular barrier but also be due to local production of this isotype, which might reflect a local immunological response to the presence of the parasite in the genitourinary tract. Anti-Leishmania IgG-U concentrations were highly correlated with uP/C ratio (ρ = 0.779; P < 0.001) and C value did not support in any case local production of this isotype. IgG isotype might be a more suitable and specific tool to evaluate renal damage due to the lower IgA-U sensitivity and correlation coefficients and evidence of IgA local production. However, dogs found positive for both Ig isotypes in urine presented significantly higher specific IgG-U concentrations and higher uP/C ratios than dogs found positive only for IgG-U, thus suggesting that the first group suffered more severe renal damage. This fact makes it necessary to evaluate the prognosis of dogs showing both anti-Leishmania IgA-U and IgG-U in future studies.     

Zoonotic intestinal parasites and vector-borne pathogens in Italian shelter and kennel dogs

This study investigated the presence of zoonotic parasites and vector-borne pathogens in dogs housed in kennels and shelters from four sites of Italy. A total of 150 adoptable dogs was examined with different microscopic, serological and molecular methods.Overall 129 dogs (86%) were positive for one or more parasites and/or pathogens transmitted by ectoparasites. Forty-eight (32%) were positive for one infection, while 81 (54%) for more than one pathogen. The most common zoonotic helminths recorded were hookworms, roundworms and Capillaria aerophila, followed by mosquito-borne Dirofilaria spp. and Dipylidium caninum. One hundred and thirteen (77.9%), 6 (4.1%) and 2 (1.4%) dogs were positive for Rickettsia spp., Leishmania infantum and Anaplasma spp., respectively.The results show that dogs living in rescue facilities from the studied areas may be infected by many zoonotic internal parasites and vector-borne pathogens, and that control measures should be implemented.     

Detection of Leishmania infantum DNA by real-time PCR in saliva of dogs

This study developed a real-time quantitative PCR (qPCR) assay to detect L. infantum kinetoplast DNA (kDNA) in canine saliva. The qPCR showed an efficiency of 93.8%, a coefficient of correlation of 0.996 and a detection limit of 0.5 fg/reaction (0.005 parasites), although it detected until 0.25 fg/reaction (0.0025 parasites). When samples from 12 dogs experimentally infected with L. infantum were collected, L. infantum kDNA was detected at 16-weeks post-infection (wpi) in 41.7% and 91.7% of saliva and bone marrow samples, respectively, and at 47-wpi in 75% of both samples. L. infantum kDNA can be detected by qPCR in canine saliva, with lower sensitivity in the early stages of infection and a lower parasite load estimation compared to bone marrow. However, saliva had similar sensitivities to bone marrow in the later stages of the infection and could be used to detect L. infantum kDNA being aware of its limitations.     

Recent advances in leishmaniosis in pet animals: epidemiology, diagnostics and anti-vectorial prophylaxis

The leishmanioses are diseases caused by protozoa of the genus Leishmania, parasites infecting numerous mammal species, including humans, and transmitted by the bite of phlebotomine sand flies. They are a large group of diseases ranging over inter-tropical zones of America and Africa, and extend into temperate regions of Latin America, Europe and Asia. Pet animals are found infected with different Leishmania species but Leishmania infantum is the most widespread being dogs the main reservoir of zoonotic visceral leishmaniosis (ZVL). Dogs are very susceptible to this parasite and may suffer from a complex syndrome, canine leishmaniosis (CanL), one of the major zoonoses globally causing severe fatal disease in this animal. Infections in cats and horses have also been reported in areas where CanL is diagnosed. In Europe dogs and cats are common companion animals and their health is of great concern, therefore management of leishmaniosis in pets generally follows that of human ZVL. The recent spread of Leishmania infections in non-endemic territories has been monitored by means of canine surveys, which represent a suitable approach because of the dog's role as a sentinel host. New tools have been developed for the surveillance and control of ZVL. A number of insecticide-based preparations have been specifically registered for dog protection against sand fly bites, with elevated efficacy for both individual and mass protection.     

The development of a real-time PCR assay for the quantification of Leishmania infantum DNA in canine blood

Recent research has demonstrated the high sensitivity of real time PCR (qPCR) in the diagnosis of Leishmania infantum infection. The goal of this study was to develop and evaluate a qPCR detection system for the diagnosis of visceral leishmaniosis (VL) in dogs. Specific primer sets were developed for the Leishmania donovani complex, in which a fragment of 132 bp of kDNA from L. infantum was amplified. The reaction was performed using the ABI PRISM 7000 system with ABI PRISM software used to carry out the analysis. When canine blood samples were assessed using this system the detection limit of the method was found to be 0.07 parasites per reaction, the efficiency was 94.17% (R² = 0.93, slope = −3.47) and the sensitivity and specificity were 100% and 83.33% respectively. The use of such a sensitive, reproducible and rapid qPCR-based assay will be useful in the diagnosis and control of L. infantum infection in endemic areas, where serological surveys often underestimate true disease prevalence.     

Pancytopenia in a cat with visceral leishmaniasis

Abstract: A 4‐year‐old, domestic shorthair, female spayed cat was presented for decreased appetite and depression. Severe pancytopenia with erythrocyte autoagglutination was found. The cat was seronegative for feline immunodeficiency and leukemia viruses. Immune‐mediated hemolytic anemia was suspected but no response to treatment with a blood transfusion, enrofloxacin, and prednisone was observed. Blood and bone marrow smears obtained 11 days later contained Leishmania amastigotes in the cytoplasm of neutrophils and macrophages, respectively. Serologic and PCR testing of peripheral blood confirmed infection with Leishmania infantum. Despite treatment, the cat worsened clinically and was euthanized. At necropsy, visceral dissemination of the parasite was confirmed. The findings in this case indicate that visceral leishmaniasis should be considered as a differential diagnoses in cats with pancytopenia in areas endemic for Leishmania. In addition, amastigotes may be observed in peripheral blood neutrophils.     

A systematic review of the efficacy of prophylactic control measures for naturally-occurring canine leishmaniosis,part I: Vaccinations

Canine leishmaniosis (CanL) is an important zoonotic disease; however, the efficacy of available vaccines for the prevention of naturally-occurring Leishmania infantum (L. infantum) infection in dogs remains unclear.     

A systematic review of the efficacy of prophylactic control measures for naturally occurring canine leishmaniosis. Part II: Topically applied insecticide treatments and prophylactic medications

The objective of this study was to systematically review the efficacy of topically applied insecticide treatments of dogs (impregnated collars, spot-ons), and prophylactic medications to prevent natural Leishmania infantum (L. infantum) infection in dogs.     

Epidemiology of Leishmania infantum,Toxoplasma gondii,and Neospora caninum in Rattus rattus in absence of domestic reservoir and definitive hosts

Isolated environments are privileged settings to study transmission of infection. Montecristo is a small island where no wild or domestic carnivores are present. Invasive Black rats Rattus rattus (n = 78) were captured and tested by PCR for Leishmania infantum, Toxoplasma gondii and Neospora caninum. We wanted to test, for these parasites, the existence of a sylvatic cycle independent of reservoir or definitive hosts. None of the rats tested positive by PCR for either T. gondii or N. caninum. We recorded a 15.5% prevalence (CI95% 8–26%) of L. infantum in the rats and Phlebotomus mascittii was captured in Montecristo, leading us to identify it as possible vector of the parasite.