Immune response to Leishmania infantum in healthy horses in Spain

Leishmania infantum infection has recently been described in horses in Europe. We report the results of a study on the immune response to L. infantum in horses living in an area endemic for leishmaniosis in NE Spain. Two ELISAs using protein A and anti-horse IgG conjugates were adapted to measure specific antibodies to L. infantum in horse sera. A lymphocyte proliferation assay (LPA) of peripheral blood mononuclear cells to L. infantum antigen was also performed to detect specific cellular immune response to Leishmania. Anti-L. infantum antibodies were detected in the serum of 16 of the horses studied (n=112) using the protein A assay but not in the assay using the anti-horse IgG conjugate. Specific lymphocyte proliferation was observed in 20 out of 55 horses. This study shows that horses in the area studied mount specific immune responses to L. infantum, and must therefore be considered among the species exposed to the parasite in this region. The infrequency of leishmaniosis in horses suggests that the immune response in this species is effective in controlling the infection.     

Rapid detection of Leishmania infantum infection in dogs: comparative study using an immunochromatographic dipstick rk39 test and direct agglutination

A rapid, sensitive and specific tool for detection of Leishmania infantum infection in dogs, would be highly desirable, because it would allow control interventions in endemic areas of Zoonotic visceral leishmaniosis (ZVL). In this study, we compared an immunochromatographic dipstick test with direct agglutination test (DAT) for detecting L. infantum infections in dogs from areas of ZVL endemic in Iran. The validity of the dipstick rk39 (Cypress Diagnostic Company, Belgium) for canine visceral leishmaniosis (CVL) was compared with a standard direct agglutination test on 116 clinically suspected dogs and 152 healthy controls from endemic areas of Ardabil and East Azerbaijan provinces, north-western of Iran for 1 year. A sensitivity of 70.9% and specificity of 84.9% were found at a 1:320 cut off titer when DAT confirmed cases were compared with healthy control. As the dipstick rk39 test is rapid, noninvasive and does not require much expertise or elaborate equipment, it can be used for screening and diagnosis of canine visceral leishmaniosis in remote endemic areas.     

Epidemiological aspects of canine visceral leishmaniosis in the Islamic Republic of Iran

An epidemiological study to examine the sero-prevalence of zoonotic visceral leishmaniosis (ZVL) among domestic and wild canines in endemic foci of Iran was carried out during 1999-2003 to assess the distribution of the disease and the possible association between infection in dogs, wild canines and people. Anti-leishmanial antibodies were detected by the direct agglutination test (DAT). Parasitological study was performed for all captured wild canines and were detected in some of the seropositive dogs with specific clinical signs (n=107). Serum samples (n=1568) were collected from domestic dogs in villages that are known endemic foci of human visceral leishmaniosis (HVL). Wild canine sera were collected from jackals (Canis aureus, n=10), foxes (Vulpes vulpes, n=10) and wolves (Canis lupus, n=10). Of the 1568 serum sampled collected from domestic dogs, 222 (14.2%) were positive by DAT (1:320 and above). No statistically significant difference was found between male (15.2%) and female (11.8%) sero-prevalence (P=0.083). Dogs of 8 years and above showed the highest sero-prevalence (40.6%). Only 23.9% of the seropositive domestic dogs had clinical signs. Parasitology and serology tests that were performed in 30 wild canines showed 10% these animals were infected by Leishmania infantum. Ten out of 11 Leishmania spp. isolated from the dogs and wild canines were identified as L. infantum and one other as L. tropica by molecular and biochemical techniques. For the first time in Iran, L. infantum and L. tropica were isolated from viscera of both a wolf and a domestic dog.     

Idiotype expression of IgG1 and IgG2 in dogs naturally infected with Leishmania infantum

Here we analyzed, by Western blot analysis, the idiotype expression of IgG1 and IgG2 in 109 canine sera corresponding to 50 dogs from endemic areas of leishmaniosis in order to detect markers related to Leishmania infantum infection and clinical condition (asymptomatic or symptomatic). Twenty-four dogs from an area free of leishmaniosis were used as controls. IgG1 and IgG2 responses in symptomatic and asymptomatic L. infantum infections differed mainly in subclass production (ELISA values), with higher IgG2 production occurring particularly in symptomatic dogs. Nevertheless, we observed little difference in the idiotype expression of these IgG subclasses, which, in general, recognized the same antigenic fractions. While early L. infantum infection was characterized by recognition of polypeptide fractions of low molecular weight, mainly fractions of 14, 16 and 18 kDa by IgG1 and 14 and 16 kDa by IgG2, symptomatology was associated with recognition by both IgG subclasses of a 24 kDa fraction and other antigens belonging to the AG24 family.     

Feline leishmaniosis due to Leishmania infantum in Italy

A case of leishmaniosis in domestic cats (Felis catus domesticus) is described. The subject showed a nodular lesion on the eyelid. The diagnosis was achieved by serological, parasitological, and light and electron microscopic investigations. By molecular techniques the aetiological agent was identified as belonging to Leishmania infantum, the species implicated in human and canine leishmaniosis in southern Europe. A preliminary study on the prevalence of asymptomatic feline leishmaniosis, performed in the areas where the infected cat was identified, revealed a low seroprevalence of infection: only 1 (0.9%) of the 110 cat sera examined by indirect fluorescent antibody test was positive for anti-Leishmania antibodies. Because clinical signs in feline leishmaniosis are unspecific and similar to those observed in other diseases commonly found in this species, leishmaniosis must be added to the differential diagnosis by feline veterinary practitioners and adequate serologic and histopathologic investigations must be performed in endemic areas.     

Study of efficacy of miltefosine and allopurinol in dogs with leishmaniosis

Visceral leishmaniosis is a life-threatening disease of medical, social and economic importance in endemic areas. It is an opportunistic infection in immunocompromised patients, including human immunodeficiency virus-positive subjects. Dogs are the main reservoir of Leishmania infantum. The aim of this study was to evaluate the efficacy of miltefosine and allopurinol for the control of human leishmaniosis using the dog as a model. The study included 28 sick dogs treated with miltefosine (2 mg/kg/day PO) administered concurrently with allopurinol (10 mg/kg/day, PO) for 30 days, and then with allopurinol alone, at the same dosage, for 1 year. Eight dogs (four of which relapsed) received a second cycle of miltefosine within 6 months of the first cycle. Efficacy was measured by real-time polymerase chain reaction assay on whole blood samples and lymph node aspirates, collected at baseline and every 3 months for 12 months. Of the total number of animals (28), two showed renal insufficiency and died after the start of therapy with miltefosine. Two other dogs presented some side effects to treatment, such as nausea, vomiting and reduction in white and red blood cell counts, and these animals were excluded from the follow-up. The results showed that the first cycle of therapy with miltefosine and allopurinol induced a drastic and progressive reduction of L. infantum load in lymph node aspirates but the second cycle did not eliminate the parasite.     

Identification of two highly performing Leishmania infantum antigens for serodiagnosis of canine leishmaniosis

In the aim of improving serodiagnosis of canine leishmaniosis, we analysed the humoral immune response of dog against Leishmania infantum parasite. The antigenic reaction of L. infantum polypeptides with sera from 31 dogs with parasitologically confirmed leishmaniosis was studied by using the immunoblot technique. Electrophoretic profile of the parasite extract showed more than 50 polypeptides, with molecular weights ranging from 12 to 170 kDa. Among these polypeptides, 37 antigen components, ranging from 14 to 91 kDa, were recognised by antibodies of L. infantum infected dogs. Three polypeptides (14, 16 and 76 kDa) reacted with all of the 31 serum samples. The other most frequently recognised antigens were those of 29.5, 32, 46, 59 and 66 kDa with a sensitivity of 87.1%, 93.6%, 96.8%, 87.1% and 80.6%, respectively. The 14 and 16 kDa bands were the most intense and remained detectable until a serum dilution of 1:6400. No reaction of these two major antigens was observed with sera collected from 50 Leishmania-free dogs, living in the leishmaniosis-free region of Rabat in Morocco, whereas the crude antigen used in IFAT or ELISA lead to three false positive results. Four antigen components of 29, 41, 55, and 70 kDa were recognised by some sera samples from negative controls. These results demonstrated the potential interest of the fractions of 14 and 16 kDa in immunodiagnosis of canine leishmaniosis.     

Evaluation of the presence of Leishmania spp. by real-time PCR in the lacrimal glands of dogs with leishmaniosis

Leishmania infantum infection is highly prevalent in endemic areas. Dogs with leishmaniosis may develop keratoconjunctivitis sicca (KCS). The goals of this study were (1) to quantify Leishmania amastigotes in the Meibomian glands (MG), main lacrimal gland (MLG) and nictitating membrane gland (NMG) from dogs with leishmaniosis; (2) to compare these results to immunohistochemistry (IHC), and (3) to explore the association between the Leishmania parasite load and the presence of ocular clinical signs. Twenty-five dogs diagnosed with leishmaniosis were included. MG, MLG and NMG from both eyes were collected. Histopathology, IHC and real-time PCR were performed. All specimens yielded positive real-time PCR results. For all three glands, samples from dogs with ocular clinical signs had mean ΔCt (cycle threshold) values significantly lower (higher parasite loads) than those from dogs without signs. Cut-off values of ΔCt<0, ΔCt<4 and ΔCt<4.9 for MG, MLG and NMG, resulted in a likelihood ratio of positives of 5.9, 6.38 and 6.38, respectively. Samples with ΔCt values below the reported cut-off were significantly more likely to display clinical signs related to KCS than those with results above the cut-off, for all three glands. Similarly, ΔCt values below the cut-off were significantly associated with positive IHC. In this study real-time PCR has been standardised for use in MG, MLG and NMG. A cut-off value established for each of these tissues may aid the clinician in the discrimination between ocular signs related to Leishmania from those associated with other causes of KCS.     

Nodular lesions of the tongue in canine leishmaniosis

In this case report, an atypical clinical presentation of leishmaniosis in a dog with multiple nodular lesions of the tongue is described. Haematological and biochemical analysis, serological test for Leishmania infantum antibodies and biopsy samples from several nodules of the tongue for histopathological examination were made. The final diagnosis of leishmaniosis was based upon the observation of amastigotes in the bioptic samples. It is recommended to consider leishmaniosis among the list of differentials of mucosal nodular lesions, at least in endemic areas.     

Infection of sandflies by a cat naturally infected with Leishmania infantum

Despite the recent reports of feline leishmaniosis from Southern Europe, cats are still regarded as unusual Leishmania hosts. A cat found chronically infected with Leishmania was submitted to xenodiagnosis. After being sedated, the animal was exposed to the bite of 100 laboratory-reared Phlebotomus perniciosus in a fine net cage for 90 min. Four out of 19 blood-fed sandflies (21%) showed motile promastigotes at the dissection. Parasites cultured from cat's lymph node and an infected fly were identical at PCR-RFLP genotyping and identified as Leishmania infantum MON-1, the main zymodeme responsible for human and canine leishmaniosis in Southern Europe. This is the first evidence of transmissibility of feline parasites to a proven vector, suggesting that cats may represent an additional domestic reservoir for L. infantum.     

Canine infection and the possible role of dogs in the transmission of American tegumentary leishmaniosis in Salta,Argentina

Some Leishmania species affect humans in two principal forms: visceral and cutaneous leishmaniosis (CL). Several studies have identified dogs as the main reservoirs of the visceral leishmaniosis (VL) caused by Leishmania infantum. The purpose of this work was to carry out a survey of the canine population associated with human cases of American tegumentary leishmaniosis (ATL), in order to establish the clinical, parasitological, serological and immunological characteristics of the canine disease, in an endemic region for both ATL and Chagas' disease in the province of Salta, in northwestern Argentina. Two hundred and eight dogs from the endemic area were examined and 41 (19.7%) of them presented lesions compatible with leishmaniosis. In order to investigate the presence of antibodies against Leishmania spp. and Trypanosoma cruzi, sera were screened by ELISA using two complex antigens from these parasites and, because of cross-reactions between them, a specific antigen for diagnosis of T. cruzi infection. Sixty-two (29.8%) of 208 dogs were positive for the complex antigen F45 from Leishmania and 50 (24%) were positive for the complex antigen F105 from T. cruzi. Nine dogs (4.3%) were positive for the specific Ag163B6-cruzipain suggesting that these dogs were truly infected with T. cruzi. Furthermore, three of these nine dogs presented Leishmania sp. in their skin lesions and therefore were considered as infected by both, T. cruzi and Leishmania parasites. The prevalence of Leishmania infection detected by lesions and/or positive serology was 27.4% (57/208). On the basis of previous observations regarding the clustered appearance of human ATL, the dog population was divided into two groups: zone A, dogs living within a 100 m radius from houses with human cases, and zone B, dogs living beyond this limit. The prevalence of ATL in dogs was significantly higher in zone A (34.6%) than in zone B (7.3%), suggesting a strong correlation between canine and human cases. The average time required for a parasitological diagnosis by microscopy was six times longer for dog samples than human ones, and the average number of parasites per 100 microscopic fields was 14-fold lower in canine samples. The high prevalence of Leishmania infection and the close association with human cases, demonstrated that dogs are a very susceptible host for Leishmania infection, but the scarcity of parasites in their lesions suggests that they may not be the main reservoir of the parasite in this endemic area.     

Little evidence of seasonal variation of natural infection by Leishmania infantum in dogs in Spain

Leishmania infantum, the etiological agent of canine leishmaniosis in the Mediterranean region, is vectored by Phlebotomus spp sandflies, which are active during the warmer months of the year. In order to determine whether seasonality in transmission induces seasonal changes in the prevalence of infection by L. infantum and of parasite-specific immune response, two groups of dogs, one in February (n=37) and another in October (n=42), were studied. Clinical signs compatible with leishmaniosis, as well as presence of microscopic skin lesions in the muzzle were recorded for all dogs. Assays were also performed for detection of L. infantum parasites in muzzle skin samples (PCR, immunohistochemistry and culture), specific serum antibodies (ELISA), and specific lymphocyte proliferation and interferon-gamma production. Although prevalence of non-specific clinical signs increased significantly after the sandfly season, this was not the case for Leishmania-specific markers: positivity by PCR (24% vs. 21%) or immunohistochemistry (3% vs. 2%) of muzzle skin samples, as well as lymphocyte proliferation (59% vs. 50%) or interferon-gamma production (21% vs. 27%) were similar in February and in October. Only prevalence of positive specific antibody titers increased noticeably in October (8% vs. 20%), although this was not statistically significant. Overall, the sandfly season did not have a marked impact on the prevalence L. infantum infection or parasite-specific immune responses analyzed in this study.     

Epidemiology of canine visceral leishmaniosis in the endemic area of Montes Claros Municipality,Minas Gerais State,Brazil

The Montes Claros City is located in an endemic area for visceral leishmaniosis in the Minas Gerais State, Brazil. With the implementation of a program for the control of visceral leishmaniosis in 1994, a sectional study was carried out to evaluate the infection by viscerotropic Leishmania in the population of dogs from Montes Claros, basically using indirect immunofluorescence antibody test (IFAT). Blood samples were collected on filter paper from 33,937 dogs, representing 96.1% of the canine local population. The prevalence for visceral leishmaniosis was found to be 9.7% in the municipality, being 9.9% in the urban area and 8.8% in the rural area. The annual incidence showed to be 64.3/1000 dogs. Prevalence of infection was not correlated with dogs age. The most affected breeds were: Boxer (24.6%) and Cocker (26.9%); Mongrel dogs had a prevalence of 7.8%. Short-hair animals had a prevalence of 11.9%, while long-furred animals had a prevalence of 8.9%. The isoenzymatic profile indicated that Leishmania (Leishmania) chagasi was the visceral leishmaniosis etiological agent in Montes Claros City, Minas Gerais State, Brazil. The main geographical areas for the parasite transmission were identified, and control measures were immediately started. The role of the dog as a reservoir for L. chagasi was confirmed. It was demonstrated that short-furred animals are at a higher risk of acquiring visceral leishmaniosis than the long-furred dogs.     

Detection of Leishmania infantum by real-time PCR in a canine blood bank

O bjectives : Risk for transmission of Leishmania infantum from blood products has been largely demonstrated in human and veterinary literature. Appropriate screening of canine blood donors is important especially in an endemic area such as Barcelona (Spain). The purpose of this study was to evaluate the presence of L infantum DNA parasites by real-time quantitative PCR in our canine blood bank.
M ethods : Samples from blood products obtained from 92 canine blood donors were assayed for L infantum by means of real-time PCR amplification and quantification.
R esults : The prevalence of quantitative PCR-positive blood samples among healthy seronegative blood donors was 19·6 per cent.
C linical S ignificance : The results of this study show that L infantum infection is common in canine blood donors and their blood products in an endemic area, despite a negative commercial serological screening for infectious diseases. Therefore, screening by PCR should be included in an integrated approach to evaluate L infantum infection among potential blood donors.     

Genetic diversity of human zoonotic leishmaniasis in Iberian Peninsula

Leishmaniasis caused by Leishmania (Leishmania) infantum is a zoonotic disease endemic in South Europe, from Portugal to the Middle East. The aim of the present study was to investigate the genetic diversity of L. infantum parasites in Iberian Peninsula. Twenty-four L. infantum strains isolated from immunocompetent patients with leishmaniasis from several localities of Portugal and Spain were studied. The use of kinetoplast DNA-PCR-restriction fragment length polymorphism as a molecular marker revealed intra-specific variation. No association was found between genotype and clinical form of the disease or patients age group. Two main clusters were identified with this marker: (i) zymodeme MON-1 strains and (ii) non-MON-1 strains. However, no association was found between strains variability and geographical distribution suggesting that parasite populations of different regions in the Iberian Peninsula are homogenous.     

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.     

Sero-epidemiological study of canine Leishmania spp. infection in the municipality of Alijó (Alto Douro, Portugal)

Visceral leishmaniosis caused by Leishmania infantum is a zoonotic disease in the Mediterranean basin. We report an epidemiological survey carried out in dogs from the municipality of Alijó in the endemic region of Alto Douro (north Portugal). Performance of the direct agglutination test (DAT) was assessed in 205 matching samples of blood collected on filter paper and serum. A high degree of agreement (97.6%; k = 0.83) was found between the results obtained from both types of samples. DAT was then used to test more blood on filter paper (B-FP) samples from other dogs of the same municipality. The detected sero-prevalence was 18.7% (288/1540), with values ranging from 0.0 to 81.1% in each of the 19 parishes of Alijó. Three distinct geographical zones of mean sero-prevalence could be defined: northwestern (2.5%), intermediate (11.4%) and southern (49.9%). No statistically significant difference was observed between male (19.1%) and female (17.8%) sero-prevalences (P = 0.560). Dogs of 9-11 years of age showed the highest sero-prevalence (28.4%), but all the other age-intervals (0-2, 3-5, 6-8 and 12-17 years) presented values (15.0-22.3%) not significantly different from the mean of the whole study population. Risk factors for canine Leishmania infection were age and geographical zone. Only 5.9% of the sero-positive animals had clinical signs of canine leishmaniosis and the overall prevalence of disease was 1.1%. This study validates the use of B-FP samples and confirms DAT as a simple and sensitive serological test to evaluate the level of canine Leishmania infection in areas of high sero-prevalence.     

Risk factors for canine neosporosis in farm and kennel dogs in southern Italy

Neosporosis by Neospora caninum causes losses to livestock production through abortion in cattle while, in dogs, it induces neuromuscolar disease. This study investigated neosporosis seroprevalence associated risk factors (including the seropositivity to leishmaniosis) in dogs of southern Italy, determined the prevalence of N. caninum oocyst shedding and examined the relationship between seroprevalence of neosporosis in farm dogs and cattle. Using an inhibition ELISA, 20.9% of 306 dogs had percent inhibition values >10 (indicative of exposure) and farm dogs had a significantly (p<0.001) higher seroprevalence than dogs in a rescue kennel. Whilst N. caninum seroprevalence was associated with increasing age in dogs (p< or =0.01) there was no association between seropositivity for N. caninum and for Leishmania infantum. Oocysts of N. caninum were not detected in faecal samples from 230 dogs including 160 farm dogs. The results indicated that neosporosis infection is common in southern Italy both in dogs and in cattle and that dogs at higher risk of exposure are free-ranging ones living in farms. The lack of correlation between canine seroprevalence for N. caninum and L. infantum assumes a particular significance in an endemic area for leishmaniosis.     

The importance of canine leishmaniosis in non-endemic areas, with special emphasis on the situation in Germany

This review article summarizes the situation of canine leishmaniosis in Germany. Published case studies on infections with Leishmania (L.) infantum in either humans or dogs are analyzed. Diagnosed cases of infections by Leishmania spp. in humans and animals are not a notifiable disease in Germany or other European countries. Taking this into consideration one may assume that there might be a significant gap between the analyzed and reported cases and the infectious status within the country. The reported case studies and results from surveys indicate that the majority of all L. infantum infections are acquired during travelling in endemic regions, predominantly the Mediterranean region. However there are cases reported from human infections and growing number of cases in dogs, where the case history may indicate an autochthonous infection within Germany, a country within a non-endemic region. The current data from entomological field studies proved the presence of two phlebotomine sand fly species. Phlebotomus (P.) mascittii, an anthropophilic sand fly species and P. perniciosus a proven vector of L. infantum. The impact from a growing leishmania-positive dog population within Germany, the distribution of at least two sand fly species, one with vector potential in the light of climate change and other non-vectorial transmissions are summarized.     

Importation of parasites by tourism and animal trading

International transport of animals is associated with an enhanced risk of import of a number of parasite species that are not endemic in Germany. Pets accompany tourists on journeys all over the world, but in particular to southern Europe, and dogs are increasingly imported from (sub-)tropical countries for reasons of animal welfare. Veterinary practitioners are thus confronted with parasites that are unusual in their patients. The specific transmission routes determine whether an imported parasite is able to become endemic. Although parasites eradicated in Germany such as Trypanosoma equiperdum and Tritrichomonas foetus may be principally imported they are unlikely to re-establish due to the current epidemiological conditions. Imported animals that harbour parasites which may occur naturally but are currently rare in Germany are a potential source of increased infection risk. Certain areas in Germany have probably become endemic for Babesia canis and the respective vector tick Dermacentor reticulatus has been found. Whether other vector borne parasitic diseases such as leishmaniosis or heartworm disease have the potential to spread into Germany is still a matter of debate.