Soledad Castillejo

Serological and parasitological follow-up in dogs experimentally infected with Leishmania infantum and treated with meglumine antimoniate.

Six healthy beagle dogs were infected with Leishmania infantum (MCAN/ES/92/BCN-83/MON-1) by intravenous inoculation of 5 x 10(7) promastigotes and two others were used as controls. When animals showed clinical signs of disease at 29, 37, 41 and 45 weeks post-infection (p.i.), they were treated with meglumine antimoniate (20.4 mg Sb/kg/12 h) subcutaneously for two periods of 10 days each. Sera were tested periodically for Leishmania antibodies by Dot-ELISA, ELISA and Western blot (WB). Aspirates of popliteal lymph node (PLN), peripheral blood sample (PB) and healthy skin were cultured in NNN and Schneiders medium. PLNs were positive between 8 and 20 weeks p.i. and in one animal PB was positive 6 weeks p.i. Samples of healthy skin, obtained before treatment, were also positive. Dot-ELISA and ELISA detected specific antibodies at an early stage between 4 and 12 weeks p.i and surpassed the cut-off between 16-24 weeks p.i., while the WB was positive between 10-19 weeks p.i. The pattern of bands revealed during the first stages of infection was variable and only in two cases did the positivity start with bands of low molecular weight (12-14 kD); the number of bands increased until 15-24 weeks p.i., after which sera revealed a complete pattern of bands, from 12 to 85 kD, in the antigen of Leishmania. After treatment the clinical improvement of the animals was accompanied by a decrease in antibody titers (Dot-ELISA and ELISA) although the parasites remained in the PLN. This was reflected in the WB by a decrease in the intensity of bands, especially those in the region of 12-30 kD. A new increase in the antibody levels between 3 and 5 months after terminating the therapy was detected in the WB by a restoration of the initial complete pattern of bands.

Epidemiology of canine leishmaniosis in Catalonia (Spain): The example of the Priorat focus

An epidemiological survey of canine leishmaniosis was conducted in the Priorat, a rural region in the Northeast of Spain, for 10 years (1985-1994). Seroprevalence throughout the region, determined by dot-ELISA and IFI, was 10.2% (8-12%). Forty percent of the dogs studied had a low level of anti-Leishmania antibodies, whereas only 50% were seronegative. Only one-third of the seropositive dogs had evident symptoms of the disease. Annual incidence of the disease was 5.7% and the level of endemicity was stable during the study. Four Leishmania zymodemes (MON-1, MON-29, MON-77, MON-105) were present in the focus, and their distribution in the different hosts is discussed. Apart from dogs and foxes, no other reservoir host has been found in the region.

Leishmania infantum-specific IgG, IgG1 and IgG2 antibody responses in healthy and ill dogs from endemic areas: Evolution in the course of infection and after treatment

The expression of IgG, IgG1 and IgG2 specific antibodies for Leishmania infantum was studied in five groups of dogs in Catalonia (Spain): I, 99 asymptomatic dogs (infected and uninfected) from a highly endemic area for leishmaniosis; II, 139 untreated dogs with clinically patent leishmaniosis; III, 11 naturally infected asymptomatic dogs monitored for up to 5 years since they were found seropositive to Leishmania antigen and without treatment; IV, 25 naturally infected dogs with clinically patent leishmaniosis and treated with either meglumine antimoniate and allopurinol or allopurinol alone and V, six experimentally infected dogs, treated with meglumine antimoniate and controlled for 5 years. The levels (ELISA units) of IgG, IgG1 and IgG2 in asymptomatic dogs (group I) were very variable (24+/-33, 32+/-31 and 26+/-31, respectively), and, as expected, lower than in ill dogs (group II) (168+/-34, 84+/-71 and 172+/-31, respectively). In both groups, the correlation between IgG and IgG2 levels (r=0.95, P<0.001 in group I and r=0.63, P<0.001 in group II) was higher than between IgG and IgG1 levels (r=0.01, P>0.05 in group I and r=0.31, P<0.001 in group II). In group III, IgG and IgG2 expression increased during infection, while IgG1 expression remained the same. In dogs of group IV, IgG levels after 1 year of treatment decreased more in responsive (mean values, 163+/-42 before treatment (b.t.) and 100+/-36 after treatment (a.t.)) than in unresponsive dogs (158+/-29 b.t. and 124+/-51 a.t.), especially for IgG1 (94+/-89 b.t. and 20+/-21 a.t. in responsive dogs and 35+/-25 b.t. and 22+/-13 a.t. in unresponsive dogs) rather than for IgG2 (156+/-16 b.t. and 114+/-45 a.t. in responsive and 151+/-11 b.t. and 125+/-36 a.t. in unresponsive dogs). Similar results were observed in the evolution of experimentally infected animals after consecutive and specific treatments. Overall results show the great variation in Leishmania-specific IgG1 expression in asymptomatic and symptomatic dogs, their lack of correlation with that of IgG2 and chemotherapy is more effective in dogs with initially high expression of IgG1.

Serologic Diagnosis of Canine Leishmaniasis by Dot-ELISA

A dot-enzyme-linked immunosorbent assay (ELISA) using protein A-peroxidase was evaluated as a diagnostic test for canine leishmaniasis. The test results were in agreement with parasitologic diagnosis and indirect immunofluorescence assay results. The sensitivity of the test calculated on 31 dogs with positive parasitologic examination was 90% when a titer of 1/800 was established as a cutoff and 100% when a titer of 1/400 was established. The specificity calculated on the canine population from nonendemic areas was 100% when both titers were established. Nevertheless, in endemic areas titers near the cutoff need careful interpretation. The results of this study demonstrate that dot-ELISA protein A using a bio-dot apparatus is highly suitable for seroepidemiologic field work.

First survey on canine leishmaniasis in a non classical area of the disease in Spain (Lleida, Catalonia) based on a veterinary questionnaire and a cross-sectional study.

The Spanish distribution of canine leishmaniasis (CanL) is heterogeneous and very few data are available for the north of the country, including the province of Lleida (Catalonia, Spain). This work describes the results obtained from a questionnaire sent to veterinarians throughout the province of Lleida. The majority of veterinarians (25/32, 78.1%) believed CanL cases were increasing and that the dogs had been infected locally (30/32, 93.8%). Also, a cross-sectional study was performed on the seroprevalence of CanL in kennel dogs, with and without compatible clinical signs, in the county of Pallars Sobirà (Pyrenees of Lleida), where an autochthonous case of CanL had been previously detected. Four serological tests were used (IFAT, ELISA, Western blot, ICF) and dogs that tested positive with at least two immunological methods were considered seropositive and probably infected. 33.1% (48/145) of the dogs were seropositive. The results of a mixed logistic regression model showed that the risk of seropositivity increased with age (OR=1.35, p-value=0.002), among dogs living in the southern part of Pallars Sobirà (OR=6.20, p-value=0.025) and among dogs whose owners considered their animals to be at risk of leishmaniasis infection (OR=1.26, p-value=0.024) and who were unaware of anti-sand fly preventive methods (OR=11.6, p-value=0.009). The risk decreased when dogs lived in an urban-periurban habitat (OR=0.17, p-value=0.002). The information gathered in the veterinary questionnaires helped us to define the knowledge, perception and awareness of the disease in a naïve region, supporting the hypothesis of an existing CanL focus in Pallars Sobirà, which was confirmed by the seroepidemiological survey. The seroprevalence study carried out on kennel dogs of local origin proved useful for detecting an autochthonous focus of leishmaniasis through the analysis of a small number of animals.

Application of molecular techniques in the study of natural infection of Leishmania infantum vectors and utility of sandfly blood meal digestion for epidemiological surveys of leishmaniasis

Epidemiological studies on the distribution of leishmaniasis caused by Leishmania infantum Nicolle, 1908 (Kinetoplastida: Trypanosomatidae) have been based principally on serological surveys of the canine reservoir. This methodology is useful due to the facility of sampling, the rapidity in obtaining results, its consistency and because it allows the detection of heterogeneous foci of canine leishmaniasis (CanL) even in small areas. Other investigations have analysed Leishmania parasitism in sandflies (Diptera: Psychodidae: Phlebotominae) by using classical dissection techniques. These techniques allow the vector species to be incriminated in different foci, although they suffer from being very time consuming. Lately, studies in this field are increasingly using molecular techniques, which are faster and easier to perform. In the present work, we applied a nested-PCR in a study of natural infection of sandflies by Leishmania in three isolated farms where serological data on canine leishmaniasis of local dogs were also obtained. The analysis allowed the detection of 38.7% of females with positive nested-PCR (78%, 18% and 0%, respectively, in the different isolated farms). The positive Leishmania DNA samples were genotyped and identified as L. infantum. The results of this work provide new data for the vectorial capacity of Phlebotomus ariasi in a Pyrenean area, which can be considered at risk of becoming a new focus of CanL. The females with positive nested-PCR displayed blood in the midgut at different degrees of digestion, and/or were gravid. According to the multivariate logistic regression analysis, the risk of nested-PCR-positivity increased significantly with the degree of blood digestion (OR = 1.3; P value = 0.025). The Phlebotomus species and the presence of eggs were not statistically associated with nested-PCR positivity (P value of >0.05). The correlation of positive nested-PCR results with the presence of seropositive dogs in the farm confirms the utility of this technique in the study of the distribution and intensity of leishmaniasis foci. Also, the importance of sandfly blood-meal digestion for epidemiological surveys of leishmaniasis foci has been demonstrated.

Temporal trends in canine leishmaniosis in the Balearic Islands (Spain): a veterinary questionnaire. Prospective canine leishmaniosis survey and entomological studies conducted on the Island of Minorca, 20 years after first data were obtained.

Leishmaniosis is present in the Mediterranean region of Europe, where Leishmania infantum is responsible for the disease, dogs are the main reservoir, and sand flies of the Phlebotomus genus, subgenus Larroussius, are proven vectors. Some areas, including Minorca in the Balearic Islands, are considered free of the disease, despite the presence of vectors. However, in the context of the current expansion of canine leishmaniosis in parts of Europe, an epidemiological study using a veterinary questionnaire was carried out to establish the current situation of the disease in the Balearic Islands. While 50% of veterinarians thought that the incidence of canine leishmaniosis had not changed over time, 26.2% perceived an increasing trend, mainly those from Minorca, where most of the veterinarians polled (88.1%) considered the new diagnosed cases as autochthonous. A cross-sectional serological study performed in this island gave a seroprevalence rate of 24%. Seroprevalence among animals of local origin and with no history of movements to endemic areas was 31%. The presence of autochthonous canine leishmaniosis in Minorca was not correlated with an increase in vector density. The environmental and climatic factors that influenced the distribution and density of Phlebotomus perniciosus on the island and the possible causes of the apparent emergence of canine leishmaniosis in Minorca are discussed.