Leishmania infantum in wild animals in endemic areas of southern Italy

Leishmania infantum infection in wildlife is increasingly reported in Europe, but scant data are available in Italy so far. This study aimed to investigate the circulation of L. infantum among sylvatic hosts in Sicily (southern Italy), a highly endemic area for canine leishmaniosis, through serological and molecular tools. Target tissues (skin, spleen, lymph nodes) collected from 71 European rabbits, 2 European hares, 7 red foxes, 11 European wildcats and 1 pine marten, were qPCR analysed for the detection of L. infantum DNA. Additionally, 40 rabbits, older than one year, were serologically screened for specific anti-Leishmania antibodies. Leishmania infantum was molecularly diagnosed in 5.4% (n = 5) of the examined animals (3/71 European rabbits, 2/7 red foxes). In many of the qPCR positive animals (4/5), the parasite DNA was more prevalent in visceral than cutaneous tissues. None of the positive animal showed signs of disease and/or macroscopic alterations of organs; low parasitic burden in all positive tissue samples was also recorded. Only one rabbit serum (i.e., 2.5%) tested positive for anti-Leishmania antibodies. The seropositive rabbit was in good health status and no amastigotes were observed in lymph-node aspirate and blood smears.This study provides first evidence of L. infantum infection in wild animals from Sicily (southern Italy). Despite the low prevalence of infection here reported, the circulation of the Leishmania in wild reservoirs in Sicily remains worthy of future investigations for a better understanding of their role in the epidemiology of the disease as well as to fine-tune control strategies in the area.     

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.     

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.     

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.     

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.     

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.     

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.     

Strength and medium-term impact of HisAK70 immunization in dogs: Vaccine safety and biomarkers of effectiveness for ex vivo Leishmania infantum infection

HisAK70 candidates have successfully been tested in cutaneous (CL) and visceral leishmaniosis (VL) mouse models. Here, we analyse different biomarkers in dog trials after a heterologous immunization strategy with a HisAK70 candidate (plasmid DNA plus adoptive transfer of peripheral blood-derived dendritic cells (DCs) pulsed with the same pathoantigen and CpG ODN as an adjuvant) to explore the antileishmanial activity in an ex vivo canine co-culture system in the presence of Leishmania infantum parasites. In the canine model, the heterologous HisAK70 vaccine could decrease the infection index in the DC-T cell co-culture system by up to 54% after 30 days and reach almost 67% after 100 days post-immunization, respectively, compared to those obtained in the control group of dogs. The observed security and potential to fight ex vivo L. infantum infection highlight a HisAK70 heterologous immunization strategy as a promising alternative to evaluate its effectiveness against canine VL.     

Exposure of client-owned cats to zoonotic vector-borne pathogens: Clinic-pathological alterations and infection risk analysis

Zoonotic Vector-Borne Diseases (VBDs) represent a relevant health issue for pets and humans. Italy is a major epidemiological hub for feline VBDs, because of suitable conditions for vector biology and disease transmission patterns. The present study investigated the exposure to major zoonotic arthropod-borne pathogens of cats in Italy, along with the evaluation of clinic-pathological features and a risk factor analysis. Out of 167 examined cats, 52 (31.1%) were seropositive for at least one vector-borne pathogen, being positivity for Bartonella henselae the most recorded (18%). Also, various cats seroreacted for Rickettsia felis (10.8%) and Rickettisa typhi (4.2%), Leishmania infantum (3%), Anaplasma phagocytophilum (2.4%) and Ehrlichia canis (2.4%). Forty-six cats were tested also for antibodies against D. immitis and two (4.3%) scored positive. The statistical analysis showed a positive association between flea infestation and seropositivity to B. henselae, other than an association between the administration of monthly ectoparasiticide treatments and seronegativity for Rickettsia spp.; seropositive cats were older than negative animals and the lifestyle (i.e. indoor vs outdoor) was not correlated with exposure to vector-borne pathogens. The majority of seropositive cats appeared clinically healthy or showed aspecific clinical signs. Around 80% of seropositive cats had one or more biochemical and/or complete blood count abnormalities. The present data confirm the endemicity of zoonotic feline VBDs in Italy and indicate that awareness on arthropod infections and transmitted pathogens should be kept high and possible implemented, towards the protection of animal and human health with adequate surveillance plans.     

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.     

Canine lymphoma and vector‐borne diseases: Molecular and serological evaluation of a possible complicity

Lymphoma is the most common haematological malignancy in dogs and its aetiology is largely unknown. The presence of canine vector‐borne agents (CVBD) in lymphoma tissues has been described and its causative effects questioned. We intended to evaluate the presence and extent of Leishmania infantum, Ehrlichia canis, Anaplasma phagocytophilum and Bartonella henselae infection in dogs with lymphoma. Sixty‐one dogs, living in the Lisbon metropolitan area, with a diagnosis of lymphoma were enrolled. Immunofluorescence assays were used to detect serum IgG's. The presence of DNA from CVBD agents in tumour tissue was assessed by PCR. All dogs tested negative for B. henselae, A. phagocytophilum and E. canis by both serology and PCR. Regarding L. infantum, 8.2% (n = 5) of the dogs had a positive serologic result. L. infantum DNA was detected in two samples of diffuse large B‐cell lymphoma (DLBCL). These results show an increased, but not significant, seropositivity (8.2% vs 7.9%) and molecular detection (3.3% vs 1.2%) for L. infantum in dogs with lymphoma, when compared to the reported canine population in the same geographical area. We could not identify an association between lymphoma and E. canis, A. phagocytophilum, B. henselae or Leishmania infantum infection in the studied population. Nevertheless, further studies, following dogs trough their CVBD disease evolution, are worthwhile and may help clarify a possible role of CVBD agents in lymphomagenesis.     

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.     

Alternative strategy for visceral leishmaniosis control: HisAK70-Salmonella Choleraesuis-pulsed dendritic cells

Here, we describe a novel approach that exploits an attenuated mutant of Salmonella enterica serovar Choleraesuis as carrier to deliver a plasmid encoding protein HisAK70. Subsequently, dendritic cells (DCs) were pulsed with this vaccine vector. The aim of this study was to evaluate the effectiveness of the prepared HisAK70-S. Choleraesuis-pulsed DCs (HisAK70-SAL DCs) against visceral leishmaniosis (VL). In our ex vivo model of infection, the prepared formulations could decrease parasite growth by up to 80% by augmenting the production of IL-12p40 and by reducing arginase activity (ARG). Also, BALB/c mice when immunised with this formulation showed significant reduction in parasite burden in both spleen (20% of reduction) and liver (75% of reduction). The balance of the immune ratios IFN-γ/IL-10, TNF-α/IL-10, and IgG2a/IgG1 reflected the acquisition of an improved resistant phenotype in HisAK70-SAL DCs vaccinated mice compared to control mice. Our results suggest that HisAK70-SAL DCs could be a promising alternative approach for vaccine delivery that has the potential to fight Leishmania infantum (L. infantum) infection.     

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.     

Cellular immunophenotypic profile in the splenic compartment during canine visceral leishmaniasis

To determine the role of the spleen in the pathogenesis of canine visceral leishmaniasis (CVL), we analyzed cellular immunophenotypic profiles of 52 dogs naturally infected with Leishmania infantum, clinically classified as follows: asymptomatic dogs-I (AD-I), seronegative/PCR+; asymptomatic dogs-II (AD-II), seropositive/PCR+; oligosymptomatic dogs (OD) and symptomatic dogs (SD). Seven non-infected dogs (CD) were included as a control group. AD-II presented higher levels of CD8+ T splenocytes and lower TCD4+/TCD8+ ratio in comparison with CD. OD and SD showed lower percentages of CD21+ as compared with AD-II. All seropositive dogs presented lower levels of CD45RA+ than CD. Regardless of the stimuli used, the proliferation index from splenocytes in vitro was inversely correlated with clinical status. After LSA stimulation, there was a higher percentage of specific CD8+ T in AD-II than CD and non-stimulated culture. In contrast, splenocytes from SD under in vitro LSA stimulation induced decreased MHC-II+ expression in comparison with all groups, and non-stimulated culture. In conclusion, the role of CD8+ T splenocytes seems to be important for an effective immunological response, a hallmark of asymptomatic CVL, whereas the pronounced loss of MHC-II expression upon LSA stimulation is a biomarker of symptomatic CVL.     

Genetic diversity and phylogenetic relationships between Leishmania infantum from dogs,humans and wildlife in south‐east Spain

Leishmania infantum causes human and canine leishmaniosis. The parasite, transmitted by phlebotomine sand flies, infects species other than dogs and people, including wildlife, although their role as reservoirs of infection remains unknown for most species. Molecular typing of parasites to investigate genetic variability and evolutionary proximity can help understand transmission cycles and designing control strategies. We investigated Leishmania DNA variability in kinetoplast (kDNA) and internal transcribed spacer 2 (ITS2) sequences in asymptomatically infected wildlife (n = 58) and symptomatically and asymptomatically infected humans (n = 38) and dogs (n = 15) from south‐east Spain, using single nucleotide polymorphisms (SNPs) and in silico restriction fragment length polymorphism (RFLP) analyses. All ITS2 sequences (n = 76) displayed a 99%–100% nucleotide identity with a L. infantum reference sequence, except one with a 98% identity to a reference Leishmania panamensis sequence, from an Ecuadorian patient. No heterogeneity was recorded in the 73 L. infantum ITS2 sequences except for one SNP in a human parasite sequence. In contrast, kDNA analysis of 44 L. infantum sequences revealed 11 SNP genotypes (nucleotide variability up to 4.3%) and four RFLP genotypes including B, F and newly described S and T genotypes. Genotype frequency was significantly greater in symptomatic compared to asymptomatic individuals. Both methods similarly grouped parasites as predominantly or exclusively found in humans, in dogs, in wildlife or in all three of them. Accordingly, the phylogenetic analysis of kDNA sequences revealed three main clusters, two as a paraphyletic human parasites clade and a third including dogs, people and wildlife parasites. Results suggest that Leishmania infantum genetics is complex even in small geographical areas and that, probably, several independent transmission cycles take place simultaneously including some connecting animals and humans. Investigating these transmission networks may be useful in understanding the transmission dynamics, infection risk and therefore in planning L. infantum control strategies.     

Identification of Babesia species infecting dogs using reverse line blot hybridization for six canine piroplasms,and evaluation of co-infection by other vector-borne pathogens

Canine infection by vector-borne hemoparasites is frequent in tropical and sub-tropical areas where exposure to hematophageous ectoparasites is intensive. A reverse line blot (RLB) assay was designed to improve the simultaneous detection of all named canine piroplasm species combined with other vector-borne pathogens of dogs including Ehrlichia canis, Hepatozoon canis and Leishmania infantum common in the Mediterranean basin. Blood samples of 110 dogs from Spain (n = 21), Portugal (n = 14) and Israel (n = 75) were analyzed. The study evaluated 2 groups of dogs, 49 dogs with piroplasm infection detected by blood smear microscopy from Portugal, Spain and Israel, and 61 dogs surveyed from rural areas in Israel, for which infection status with vector-borne pathogens was unknown. Among the dogs previously diagnosed with piroplasmosis, infection with Babesia canis, Babesia vogeli, Babesia gibsoni and Theileria annae was detected in the Iberian dogs while only B. vogeli was found in Israeli dogs. These differences are attributed to the absence of tick vectors for some piroplasm species such as Dermacentor reticulatus in Israel. Eleven (79%) of the Babesia-positive dogs from Portugal were co-infected with other pathogens including L. infantum, H. canis and E. canis. Eight of 61 (13%) rural Israeli dogs were co-infected with two or more pathogens including B. vogeli, L. infantum, E. canis, and H. canis. Triple infections were demonstrated in 2 dogs. The RLB detection limit for Babesia was 50-fold lower than that of PCR. This study presents a RLB to simultaneously detect and separate the major vector-borne dog pathogens in southern Europe and the Middle East.     

Molecular and serological detection of animal and human vector-borne pathogens in the blood of dogs from Côte d’Ivoire

In Côte d’Ivoire, limited information are available on vector-borne pathogens, their prevalence and distribution. Here, we assess the occurrence and diversity of canine vector-borne diseases (CVBDs) in Abidjan and Yamoussoukro cities. Blood from a total of 123 dogs were tested for Leishmania infantum and Ehrlichia canis antibodies and screened for Leishmania and Trypanosoma spp., Piroplasmida, Filariidae and Anaplasmataceae by PCR and sequencing. Among dogs, 39 % were positive for at least one pathogen. Seroprevalences were: 15.4 % and 12.2 % for L. infantum and E. canis, respectively. DNA of L. infantum and T. congolense (4.1 %), Baabesia vogeli (1.6 %), Filariidae (Dirofilaria immitis, D. repens and Acanthocheilonema reconditum) (10.6 %) has been detected. Anaplasmataceae were detected in (17.1 %) and E. canis was the only identified specie. Co-infections were observed in 13.8 % of dogs: E. canis-L. infantum co-infection was the most prevalent (4.9 %). Age, breed and sex of dogs do not seem to influence infections. Village dogs were more susceptible to CVBDs than kennel dogs (PV = 0.0000008). This study reports for the first time the presence of L. infantum, B. vogeli, A. reconditum, D. immitis and D. repens in dogs from Côte d’Ivoire and determines the prevalence and diversity of CVBD pathogens. The results indicate that human and animal pathogens are abundant in Ivoirian dogs which requires attention of veterinarians, physicians and authorities against these diseases, especially against major zoonosis such as visceral leishmaniasis (L. infantum).