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.     

Longitudinal monitoring of anti‐saliva antibodies as markers of repellent efficacy against Phlebotomus perniciosus and Phlebotomus papatasi in dogs

A 2‐year longitudinal study of enzyme‐linked immunosorbent assay (ELISA) antibodies against Phlebotomus perniciosus and Phlebotomus papatasi (Diptera: Psychodidae) sandfly saliva was performed in 32 Beagle dogs treated preventively with an imidacloprid–permethrin topical insecticide in an endemic area in Spain. Dogs were grouped into three sandfly exposure groups according to the time of inclusion in the study. Assays analysed immunoglobulin G (IgG) against salivary gland homogenates (SGH) of both species and recombinant P. papatasi rSP32 and P. perniciosus rSP03B proteins in serum. The dogs were participating in a Leishmania infantum (Kinetoplastida: Trypanosomatidae) vaccine trial and were experimentally infected with the parasite in the second year. No dog acquired natural L. infantum infections during the first year, but most developed anti‐saliva antibodies, and median log‐transformed optical densities (LODs) were seasonal, mimicking those of local sandflies. This indicates that the repellent efficacy of the insecticide used is below 100%. Multi‐level modelling of LODs revealed variability among dogs, autocorrelation and differences according to the salivary antigen and the dog's age. However, dog seroprevalence, estimated using pre‐exposure LODs as cut‐offs, was relatively low. This, and the fact that dogs did not become naturally infected with L. infantum, would support the efficacy and usefulness of this imidacloprid–permethrin topical insecticide in canine leishmaniasis control.     

Canine antibody response to Phlebotomus perniciosus bites negatively correlates with the risk of Leishmania infantum transmission

Background

Phlebotomine sand flies are blood-sucking insects that can transmit Leishmania parasites. Hosts bitten by sand flies develop an immune response against sand fly salivary antigens. Specific anti-saliva IgG indicate the exposure to the vector and may also help to estimate the risk of Leishmania spp. transmission. In this study, we examined the canine antibody response against the saliva of Phlebotomus perniciosus, the main vector of Leishmania infantum in the Mediterranean Basin, and characterized salivary antigens of this sand fly species.

Methodology/Principal Findings

Sera of dogs bitten by P. perniciosus under experimental conditions and dogs naturally exposed to sand flies in a L. infantum focus were tested by ELISA for the presence of anti-P. perniciosus antibodies. Antibody levels positively correlated with the number of blood-fed P. perniciosus females. In naturally exposed dogs the increase of specific IgG, IgG1 and IgG2 was observed during sand fly season. Importantly, Leishmania-positive dogs revealed significantly lower anti-P. perniciosus IgG2 compared to Leishmania-negative ones. Major P. perniciosus antigens were identified by western blot and mass spectrometry as yellow proteins, apyrases and antigen 5-related proteins.

Conclusions

Results suggest that monitoring canine antibody response to sand fly saliva in endemic foci could estimate the risk of L. infantum transmission. It may also help to control canine leishmaniasis by evaluating the effectiveness of anti-vector campaigns. Data from the field study where dogs from the Italian focus of L. infantum were naturally exposed to P. perniciosus bites indicates that the levels of anti-P. perniciosus saliva IgG2 negatively correlate with the risk of Leishmania transmission. Thus, specific IgG2 response is suggested as a risk marker of L. infantum transmission for dogs.     

Transmission of visceral leishmaniasis in a previously non‐endemic region of Tunisia: Detection of Leishmania DNA in Phlebotomus perniciosus

Visceral leishmaniasis (VL) has been endemic in northern Tunisia and has occurred sporadically in the center of Tunisia. Recently, there have been several cases from areas known to be free of VL. We report in this work all human and canine cases of VL recorded between 2003 and 2011 and an entomological study of phlebotomine fauna in a previously non‐endemic region. Sixty‐three cases of VL were diagnosed and identified as L. infantum using several different methods. Eight species of 179 sand flies were caught and identified by both morphological and molecular methods. Two genera were present, Phlebotomus and Sergentomya, with an abundance of the subgenus Phlebotomus (Larrousius) spp., a classic vector of VL in Tunisia. Moreover, Leishmania DNA was detected in seven unfed Phlebotomus pernicousus and L. infantum was identified in three of them. This result confirms the establishment of a transmission cycle of VL in the studied region by the coexistence of infected vectors with infected hosts.     

Potential distributions of phlebotomine sandfly vectors of human visceral leishmaniasis caused by Leishmania infantum in Morocco

Zoonotic visceral leishmaniasis is a common vector-borne systemic disease caused by Leishmania infantum (Kinetoplastida: Trypanosomatidae). In Morocco the situation is complex: many sandfly species have been collected in areas in which the disease is endemic, but only Phlebotomus ariasi, Phlebotomus perniciosus and Phlebotomus longicuspis (Diptera: Psychodidae) have been confirmed to have vectorial roles. The objective of the present study was to ascertain the potential distribution of L. infantum and its vectors in Morocco, using ecological niche modelling. Vector records were obtained from field collections of the Laboratory team and from previously published entomological observations. Epidemiological data for L. infantum modelling were obtained from Moroccan Ministry of Health reports. The jackknife test indicated that the bioclimatic variables with the greatest influence on model development for all species were annual precipitation and precipitation in the driest quarter of the year. MaxEnt model representations for sandfly species that act as vectors of L. infantum showed the widespread geographic distribution of these species in Morocco, specifically in northern and central Morocco, where foci of visceral leishmaniasis are found. The ecological niche modelling points out areas in which the probability of occurrence of these species is higher. This information should be considered as a starting point for further research to fully elucidate the ecology and epidemiology of these species, as well as of the pathogens they transmit.     

Leishmania (Sauroleishmania) tarentolae isolation and sympatric occurrence with Leishmania (Leishmania) infantum in geckoes,dogs and sand flies

The trypanosomatid protist Leishmania tarentolae is a saurian-associated parasite vectored by the Sergentomyia minuta sand fly. This study aimed to confirm the circulation of L. infantum and L. tarentolae in sand flies, reptiles and dogs and to isolate new strains of these protists. Reptilian and sheltered dog blood samples were collected, and sand flies were captured. Samples were tested for Leishmania spp. using duplex real-time PCR (dqPCR) and real-time PCR (qPCR); the origin of blood meal was identified in engorged sand flies by conventional PCR. The reptilian blood and intestinal content of sand fly females were cultured. Dog sera were tested by IFAT using both Leishmania species. Four Tarentola mauritanica geckoes were molecularly positive for L. infantum or L. tarentolae, with no co-infections; moreover, amastigote-like forms of L. infantum were observed in the bone marrow. 24/294 sand flies scored positive for Leishmania spp. by dqPCR, 21 S. minuta and two Phlebotomus perniciosus were positive for L. tarentolae, while only a single Ph. perniciosus was positive for L. infantum. Blood meal analysis confirmed reptile and dog in S. minuta, dog and human in Ph. perniciosus and dog in Phlebotomus neglectus. Two axenic strains of L. tarentolae were obtained. Twelve of 19 dogs scored positive for L. infantum and L. tarentolae by IFAT and three of them also for L. infantum by dqPCR, and six by qPCR. These data confirm the sympatric circulation of L. infantum and L. tarentolae in geckoes, sand flies, and dogs, and suggest that geckoes may be infected with L. infantum.     

Enhanced attraction of sand fly vectors of Leishmania infantum to dogs infected with zoonotic visceral leishmaniasis

BackgroundThe sand fly Phlebotomus perniciosus is the main vector of Leishmania infantum, etiological agent of zoonotic visceral leishmaniasis in the Western Mediterranean basin. Dogs are the main reservoir host of this disease. The main objective of this study was to determine, under both laboratory and field conditions, if dogs infected with L. infantum, were more attractive to female P. perniciosus than uninfected dogs.Methodology/Principal findingsWe carried out a series of host choice experiments and found that infected dogs were significantly more attractive to P. perniciosus than uninfected dogs in the laboratory as well as in the field. Significantly more P. perniciosus fed on infected dogs than on uninfected dogs. However, the fecundity of P. perniciosus fed on infected dogs was adversely impacted compared to uninfected dogs by lowering the number of laid eggs. Phlebotomus perfiliewi, the second most abundant sand fly species in the field site and a competent vector of L. infantum had similar trends of attractivity as P. perniciosus toward infected dogs under field conditions.ConclusionsThe results strongly suggest that L. infantum causes physiological changes in the reservoir host which lead to the host becoming more attractive to both male and female P. perniciosus. These changes are likely to improve the chance of successful transmission because of increased contact with infected hosts and therefore, infected dogs should be particularly targeted in the control of zoonotic visceral leishmaniasis in North Africa.     

Molecular detection of Leishmania (Sauroleishmania) tarentolae in human blood and Leishmania (Leishmania) infantum in Sergentomyia minuta: unexpected host-parasite contacts

The detection of atypical Kinetoplastida in vertebrate hosts and vectors might suggest unexpected host-parasite contacts. Aside to major vectors of Leishmania (Leishmania) infantum in Italy (e.g. Phlebotomus perniciosus and Phlebotomus perfiliewi), the sand fly fauna also includes Sergentomyia minuta, herpetophilic and proven vector of Leishmania (Sauroleishmania) tarentolae, in which records of blood meal on mammals and detection of L. infantum DNA are increasing. This study was conducted in Central Italy aiming to molecularly detect potential atypical Leishmania host-vector contacts. Detection of Leishmania spp. DNA was performed by polymerase chain reaction (SSU rRNA, ITS1 targets) on field-collected sand fly females (N = 344), blood samples from humans (N = 185) and dogs (N = 125). Blood meal identification was also performed on engorged sand flies. Leishmania spp. DNA was found in 13.1% sand flies, 3.7% humans and 14.4% dogs. Sequence analysis identified L. infantum in S. minuta (4.4%), P. perniciosus (9.1%), humans (2.2%) and dogs (14.4%). Leishmania tarentolae was detected in S. minuta (12.6%), P. perfiliewi (6.6%) and human (1.6%) samples. Of 28 S. minuta examined for blood meal, 3.6 and 21.4% scored positive for human and lizard DNA, respectively. These results indicate the importance of one-health approach to explore new potential routes of transmission of leishmaniasis involving S. minuta.     

An Autochthonous Case of Canine Visceral Leishmaniasis in Korea

A 12-year-old spayed female mixed-bred dog presented with nasal bleeding of 2 days duration and a skin nodule in the left flank. No abnormalities were found in coagulation profiles and blood pressure. Cytological evaluation of the nodule revealed numerous characteristic round organisms having a nucleus and a bar within macrophages and in the background, consistent with leishmaniasis. In vitro culture was unsuccessful but PCR of the nodular aspirate identified the organisms as Leishmania infantum, and the final diagnosis was canine leishmaniasis. No history of travel to endemic countries was noted. Because the dog had received a blood transfusion 2 years before the illness, serological screening tests were performed in all donor dogs of the commercial blood bank using the commercial Leishmania ELISA test kit, and there were no positive results. Additional 113 dogs with hyperglobulinemia from Seoul were also screened with the same kits but no positive results were obtained. To the best of the author''s knowledge this is the first autochthonous case of canine leishmaniasis in Korea.     

Evidence for an impact on the incidence of canine leishmaniasis by the mass use of deltamethrin-impregnated dog collars in southern Italy

Dogs are the domestic reservoir of Leishmania infantum Nicolle (Kinetoplastida: Trypanosomatidae), the agent of zoonotic human visceral leishmaniasis. In southern Europe, where canine leishmaniasis (CanL) is widespread due to L. infantum, killing seropositive dogs is considered unacceptable and drug treatment has low efficacy in preventing transmission. We made a field evaluation of the efficacy of deltamethrin dog collars in a CanL focus of southern Italy, Mount Vesuvius area of Campania region, where the vector is Phlebotomus perniciosus Newstead (Diptera: Psychodidae), by assessing their impact on the incidence of CanL in an intervention town, compared to that in dogs of control towns where no collars were fitted. During two consecutive transmission seasons, collars were fitted to 350 (1998) and 354 (1999) dogs from San Sebastiano al Vesuvio (70% of the canine population). Control dogs (371 and 264 in the 2 years, respectively) were from four towns of the same area. Before each transmission season, the CanL seroprevalence in the intervention and control towns was evaluated by cross-sectional surveys and found to be similar (about 15% in 1998 and 10% in 1999, respectively). After each transmission period, incidence rates of seroconversions were determined in adult dogs that were serologically negative before the season under evaluation, and in puppies. After the 1998 season, 2.7% of the dogs in the intervention town seroconverted compared to 5.4% in the control towns (50% protection, P = 0.15). After the 1999 season, 3.5% of collared dogs seroconverted compared to 25.8% of control dogs (86% protection, P < 0.001). The increase in seroconversion rates recorded in control dogs suggests an increase in the Leishmania force of infection in the canine reservoir during the 1999 sandfly season, as supported by the concomitant increase of human cases in control towns and in the whole Campania region. Our results suggest that the impact of mass use of deltamethrin-impregnated dog collars on the incidence of CanL may be negligible during low transmission seasons, or probably in low endemic foci, but can be very strong when the force of transmission is high.     

Canine Antibodies against Salivary Recombinant Proteins of Phlebotomus perniciosus: A Longitudinal Study in an Endemic Focus of Canine Leishmaniasis

BackgroundPhlebotomine sand flies are vectors of Leishmania parasites. During blood feeding, sand flies deposit into the host skin immunogenic salivary proteins which elicit specific antibody responses. These anti-saliva antibodies enable an estimate of the host exposure to sand flies and, in leishmaniasis endemic areas, also the risk for Leishmania infections. However, the use of whole salivary gland homogenates as antigen has several limitations, and therefore, recombinant salivary proteins have been tested to replace them in antibody detection assays. In this study, we have used for the first time sand fly salivary recombinant proteins in a longitudinal field study on dogs.ConclusionsThese results suggest that P. perniciosus rSP03B protein is a valid alternative to whole saliva and could be used in large-scale serological studies. This novel method could be a practical and economically-sound tool to detect the host exposure to sand fly bites in CanL endemic areas.     

Canine visceral leishmaniasis caused by Leishmania infantum in Senegal: risk of emergence in humans?

In the context of global warming and the risk of spreading arthropod-borne diseases, the emergence and reemergence of leishmaniasis should not be neglected. In Senegal, over the past few years, cases of canine leishmaniasis have been observed. We aim to improve the understanding of the transmission cycle of this zoonosis, to determine the responsible species and to evaluate the risk for human health. An epidemiological and serological study on canine and human populations in the community of Mont Rolland (Thiès area) was conducted. The data showed a high seroprevalence of canine leishmaniasis (>40%) and more than 30% seropositive people. The dogs’ seroprevalence was confirmed by PCR data (concordance > 0.85, Kappa > 0.7). The statistical analysis showed strong statistical associations between the health status of dogs and seropositivity, the number of positive PCRs, clinical signs and the number of Leishmania isolates. For the first time, the discriminative PCRs performed on canine Leishmania strains clearly evidenced that the pathogenic agent is Leishmania infantum. The results obtained show that transmission of this species is well established in this area. That the high incidence of seropositivity in humans may be a consequence of infection with this species is discussed.     

Opportunistic feeding behaviour and Leishmania infantum detection in Phlebotomus perniciosus females collected in the human leishmaniasis focus of Madrid,Spain (2012–2018)

BackgroundAn outbreak of human leishmaniasis due to Leishmania infantum has been registered in an urban area of southwestern Madrid, Spain, since 2010. Entomological surveys carried out in the municipalities of Fuenlabrada, Leganés, Getafe and Humanes de Madrid showed that Phlebotomus perniciosus is the only potential vector. In this work, an intensive molecular surveillance was performed in P. perniciosus females captured in the region between 2012 and 2018.Methodology/Principal findingsA total of 1805 P. perniciosus females were analyzed for Leishmania infection, and 1189 of them also for bloodmeal identification. Eleven different species of vertebrate were detected by amplification and subsequent sequencing of the 359 bp cytb fragment. The most prevalent blood source identified was hare (n = 553, 46.51%), followed by rabbit (n = 262, 21.95%). Less frequent were cat (n = 45, 3.80%), human (n = 34, 2.90%), pig (n = 14, 1.20%), horse (n = 11, 0.93%), sheep (n = 3, 0.25%), rhea (n = 3, 0.25%), partridge (n = 1, 0.09%) and chicken (n = 1, 0.09%). The distribution of the blood meal sources varied between the different locations. Regarding L. infantum detection, PCR amplification of a fragment of kDNA, cpb gene and ITS1 region showed 162 positive specimens (8.97%). The highest infection rate was found in the municipality of Leganés (15.17%).ConclusionsThe results of this molecular survey in P. perniciosus, the only leishmaniasis vector in the outbreak occurred in southwestern Madrid region, showed its opportunistic blood-feeding behaviour, high infection rates and the differences between the different points. This study was an essential part of the intensive surveillance plan in the area and the results obtained have supported the implementation of control measures in the outbreak.     

Cutaneous leishmaniasis caused by Leishmania infantum transmitted by Phlebotomus tobbi

Transmission of cutaneous leishmaniasis (CL) caused by Leishmania infantum was studied in South Anatolia, Turkey. Small, non-ulcerating lesions prevailed and patients were negative in rK39 tests for antibody detection for human visceral leishmaniasis (VL). The most abundant sand fly species, Phlebotomus tobbi, was found positive for Leishmania promastigotes with a prevalence of 1.4% (13 out of 898 dissected females). The isolated strains were identical with those obtained from patients with CL and were typed as L. infantum. Phylogenetic analysis revealed similarity to MON-188 and a clear difference from the MON-1 clade. Blood-meal identification showed that P. tobbi feeds preferentially on cattle and humans. This finding, the high number of CL patients and relative scarcity of dogs in the focus, suggests that the transmission cycle could be anthroponotic.     

Gentamicin-Attenuated Leishmania infantum Vaccine: Protection of Dogs against Canine Visceral Leishmaniosis in Endemic Area of Southeast of Iran

An attenuated line of Leishmania infantum (L. infantum H-line) has been established by culturing promastigotes in vitro under gentamicin pressure. A vaccine trial was conducted using 103 naive dogs from a leishmaniosis non-endemic area (55 vaccinated and 48 unvaccinated) brought into an endemic area of southeast Iran. No local and/or general indications of disease were observed in the vaccinated dogs immediately after vaccination. The efficacy of the vaccine was evaluated after 24 months (4 sandfly transmission seasons) by serological, parasitological analyses and clinical examination. In western blot analysis of antibodies to L. infantum antigens, sera from 10 out of 31 (32.2%) unvaccinated dogs, but none of the sera from vaccinated dogs which were seropositive at >100, recognized the 21 kDa antigen of L. infantum wild-type (WT). Nine out of 31 (29%) unvaccinated dogs, but none of vaccinated dogs, were positive for the presence of Leishmania DNA. One out of 46 (2.2%) vaccinated dogs and 9 out of 31 (29%) unvaccinated dogs developed clinical signs of disease. These results suggest that gentamicin-attenuated L. infantum induced a significant and strong protective effect against canine visceral leishmaniosis in the endemic area.     

Visceral Leishmaniasis in Rural Areas of Alborz Province of Iran and Implication to Health Policy

Visceral leishmaniasis (VL) or kala-azar mainly affects children in endemic areas. This study was conducted to determine the seroprevalence of VL using direct agglutination test (DAT) in children living in rural districts of Alborz Province located 30 km from Tehran capital city of Iran. Multi-stage cluster random sampling was applied. Blood samples were randomly collected from 1,007 children under 10 years of age in the clusters. A total of 37 (3.7%) of the studied population showed anti-Leishmania infantum antibodies with titers of ≥1:800. There was a significant association between positive sera and various parts of the rural areas of Alborz Province (P<0.002). Two children with anti-Leishmania infantum antibodies titers of ≥1:3,200 indicated kala-azar clinical features and treated with anti-leishmaniasis drugs in pediatric hospital. The findings of this study indicated that Leishmania infection is prevalent in rural areas of Alborz Province. Therefore, it is necessary to increase the awareness and alertness among physicians and public health managers, particularly in high-risk rural areas of the province in Iran.     

Chemiluminescence activity in whole blood phagocytes of dogs naturally infected with Leishmania infantum

Dogs are the domestic reservoir of Leishmania infantum, a vector-borne intracellular protozoan agent of human visceral leishmaniasis. The role of polymorphonuclear leukocytes (PMNs) in the immune defence against this parasite has been poorly studied. We have investigated the function of peripheral blood PMNs in naive beagle dogs that have been naturally exposed to phlebotomine vectors in an area highly endemic for canine leishmaniasis, and found infected by Leishmania at the end of the transmission season. Whole blood phagocyte oxidative metabolism was assessed by a rapid method that determines a luminol-amplified chemiluminescence (CL) emission. This was evaluated using either a soluble stimulant, phorbol mirystate acetate (PMA), or phagocytic stimuli, such as zymosan unopsonized (ZYM) or opsonized with autologous serum (OPZ). In blood samples taken 2 months after exposure to Leishmania transmission, data on CL emission revealed a significant decrease of reactive oxygen intermediates (ROI) production in the presence of both PMA and ZYM, compared with blood samples obtained from dogs before exposure. On the contrary, no variations in CL emission were detected in presence of OPZ. Our data indicate that immunological changes occur early in canine leishmaniasis and confirm that the role of PMNs and their products need to be clarified. Copyright © 2000 John Wiley & Sons, Ltd.     

Molecular screening of Leishmania spp. infection and bloodmeals in sandflies from a leishmaniasis focus in southwestern Turkey

Leishmaniasis is an arthropod‐borne disease that affects approximately 2 million people worldwide annually. The aims of this study were to detect the presence of Leishmania (Kinetoplastida: Trypanosomatidae) DNA and the feeding preferences of probable vector species in an endemic focus of Leishmania infantum in Turkey. Entomological sampling was performed in August and October 2015 in Ayd?n province, where cases of human and canine leishmaniasis have been reported previously. A total of 1059 sandfly specimens comprising nine species belonging to two genera, Phlebotomus and Sergentomyia (both: Diptera: Psychodidae), and five subgenera of the Phlebotomus genus (Phlebotomus, Paraphlebotomus, Larroussius, Adlerius and Transphlebotomus) were collected in five villages. Among all Phlebotomus specimens, Phlebotomus neglectus (39%) was noted as the most abundant species, followed by Phlebotomus tobbi (18%). Leishmania DNA was detected in pools from P. neglectus, P. tobbi and Sergentomyia dentata by kDNA polymerase chain reaction (PCR). Leishmania DNA from Phlebotomus specimens was identified as L. infantum, but Leishmania DNA from Sergentomyia spp. could not be identified to species level by ITS‐1 real‐time PCR. The detection of Leishmania DNA in wild‐caught P. neglectus and the high percentage (24.2%) of human DNA in engorged specimens suggests that P. neglectus is probably an important vector species for L. infantum in Ayd?n province.