Cristina Riera

The Ibizian hound presents a predominantly cellular immune response against natural Leishmania infection

Veterinarians working in the Balearic Islands (Mallorca), an endemic region of canine leishmaniosis, have reported very few cases of leishmaniosis in Ibizian hounds while concurrently observing that dogs of other breeds had a high incidence of clinical canine leishmaniosis. To further investigate this observation, two populations of dogs from the Balearic Islands were examined for the presence of Leishmania-specific cellular immunity using a delayed type hypersensitivity test (DTH) to leishmanin and for the presence of Leishmania-specific humoral immunity using an ELISA. Fifty-six asymptomatic dogs, 31 Ibizian hounds and 25 dogs belonging to other breeds were examined. Seventy-seven percent of the dogs demonstrated a specific immune response against Leishmania, either humoral or cellular. This finding suggests that the infection rate (77%) was higher than previously considered. For Ibizian hounds 81% were DTH positive while only 48% of the other dogs were DTH positive. A statistical association between Ibizian hounds and positive DTH response was found. A specific humoral response was found in 48% of Ibizian hounds and in 56% of the other dogs. No statistical association relative to the Leishmania-specific IgG1 and IgG2 levels were found between the two groups. The Ibizian hound has been reported to be more resistant to Leishmania infection and we found that the Ibizian hound mounts a significant cellular response to infection. Thus, the Ibizian hound may be an interesting canine model for the investigation of protective anti-Leishmania immune response.

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.

Detection of Leishmania infantum cryptic infection in asymptomatic blood donors living in an endemic area (Eivissa, Balearic Islands, Spain) by different diagnostic methods

The extent of cryptic leishmaniasis in blood donors from a Spanish endemic area, (Eivissa Island) was studied using various immunological and parasitological methods. Sera from 656 blood donors were analysed: 16 (2.4%) were positive by ELISA and 50 (7.6%) by Western blot. Peripheral blood mononuclear cells (PBMC) and buffy coat (BC) samples, were analyzed by culture and nested-PCR. DNA of L. infantum was amplified in 27 (22.1%) of 122 PBMC. Parasites were isolated in 3 (4.5%) of 67 BC cultures and the strains were identified as L. infantum zymodeme MON-28. No parasites were isolated in PBMC culture. After 12 months, a second blood sample was obtained from 18 blood donors who were positive by nested-PCR in the first extraction; nine of them remained positive. Delayed type hypersensitivity (DTH) tests on 15/67 donors (22.3%) were positive. Comparison of results obtained by ELISA, WB and DTH; ELISA, WB and nested-PCR and nested-PCR and BC culture showed a significant association (Pearson test, P < 0.05). L. infantum zyodeme MON-28 was identified in three strains isolated from asymptomatic donors, which suggests a low virulence capacity of these strains. The detection of Leishmania DNA in a high number of asymptomatic subjects supports the need to monitor it in blood donors endemic areas.

Clinical profile of Trypanosoma cruzi infection in a non-endemic setting: Immigration and Chagas disease in Barcelona (Spain)

BACKGROUND Chagas disease is no longer limited to Latin America and is becoming frequent in industrialised countries in Europe and United States. METHODS A descriptive study of Latin American immigrants in Barcelona attending two centres for imported diseases during a period of 3 years. The main outcome was the identification of Trypanosoma cruzi-infected individuals in a non-endemic country and the characterization of their clinical and epidemiological features. RESULTS A total of 489 Latin American patients participated in the study. Forty-one percent were infected by T. cruzi, and the most frequent country of origin was Bolivia. All T. cruzi infected patients were in chronic stages of infection. 19% of cases had cardiac disorders and 9% had digestive disorders. CONCLUSIONS A high percentage of participants in this study were infected by T. cruzi and various factors were found to be associated to the infection. It is important to improve clinical and epidemiological knowledge of T. cruzi infection in non-endemic countries and to develop appropriate screening and treatment protocols in these settings.

Asymptomatic infection by Leishmania infantum in blood donors from the Balearic Islands (Spain)

BACKGROUND: Visceral leishmaniasis (VL) caused by Leishmania infantum is a zoonotic disease endemic throughout the Mediterranean basin. The existence of asymptomatic human infection entails the risk of transmission by blood transfusion.

Evaluation of a latex agglutination test (KAtex) for detection of Leishmania antigen in urine of patients with HIV-Leishmania coinfection: value in diagnosis and post-treatment follow-up

The usefulness of antigen detection in urine as an alternative tool for diagnosis of leishmaniasis and post-treatment follow-up in patients with Leishmania-HIV coinfection was evaluated with a latex agglutination test (KAtex; Kalon Biological, UK). Forty-nine HIV-infected patients with visceral leishmaniasis were included in the study. Antigen detection in urine (ADU) was positive in 42 of 49 (sensitivity, 85.7%) samples obtained during a primary episode. After treatment, a follow-up study in 23 patients was performed by simultaneous ADU and culture of peripheral blood mononuclear cells in 148 determinations. The two methods gave concordant results in 94 cases, 38 of which were positive and 56 negative. In five cases, ADU was negative and culture of peripheral blood mononuclear cells was positive: two of these cases corresponded to clinical relapses. In 49 cases, culture of peripheral blood mononuclear cells was negative and ADU was positive. In the absence of clinical symptoms, the detection of parasite antigens in 71 of 130 (54.6%) urine samples was not associated with clinical disease. The Kaplan-Meier estimates of the probability of relapse at 6, 12, 18, and 24 months were 16% (95%CI, 15–17%), 20% (95%CI, 18–22%), 31% (95%CI, 27–35%), and 71% (95%CI, 52–89%), respectively, in patients with a positive ADU result. In contrast, when ADU was negative, the probability of relapse was 5% at 6 months (95%CI, 2–8%) (only 2 of 11 patients who relapsed had a negative test). ADU by KAtex is appropriate for primary diagnosis of visceral leishmaniasis, for monitoring the efficacy of treatment, and for detection of subclinical infection.

Nested PCR for diagnosis of canine leishmaniosis in peripheral blood, lymph node and bone marrow aspirates

A nested polymerase chain reaction (PCR) assay was developed using primers selected from the genomic DNA of Leishmania infantum and applied to the diagnosis of leishmaniosis in peripheral blood in dogs. Blood of 39 dogs of different breeds, all sampled in Catalonia (Spain), were tested for leishmaniosis by enzyme linked immunosorbent assay (ELISA), western blotting (WB) and peripheral blood mononuclear cell (PBMC) culture and nested PCR. Twenty negative controls (healthy dogs less than 1-year-old that had not been exposed to a sandfly season) were also studied. Nineteen of the 39 dogs studied were positive by ELISA and/or WB, and 18 of these had a positive PBMC nested PCR. PBMC nested PCR was negative in all the remaining animals that were negative by serological examination, including the 20 negative controls. Parasitological examination and nested PCR of bone marrow and lymph node aspirate from the 19 dogs positive by serological examination, were also positive. These results indicate that PBMC nested PCR is a sensitive and specific tool to diagnose leishmaniosis in dogs. The use of PBMC has the advantage over bone marrow and lymph node aspirates in that it is a less invasive sample.

A nested polymerase chain reaction for diagnosis and follow-up of human visceral leishmaniasis patients using blood samples

A nested polymerase chain reaction (PCR) assay for the diagnosis of human visceral leishmaniasis (VL) due to Leishmania infantum infection was developed using primers selected from the parasites genomic deoxyribonucleic acid (DNA). The assay, which is based on the use of leucocytes separated from blood samples by Ficoll-Paque centrifugation, was compared with culture in vitro. Blood samples were collected from 17 patients in Spain with a history of clinical VL, 15 of whom were also infected with human immunodeficiency virus (HIV) (13 samples during the VL episode and 31 samples during post-treatment monitoring) and one sample was collected from each of 28 patients with HIV infection and fever but no history of VL. The nested PCR using blood detected all the cases of parasitologically confirmed, clinically active VL, while culture detected 92%. The nested PCR detected Leishmania DNA in 18% of the HIV-infected patients with fever and no history of VL, none of whom gave a positive culture. Follow-up examination of the VL patients by nested PCR and culture demonstrated the persistence of L. infantum in blood for a long time after treatment.