José María Alunda

Practical Approach for Typing Strains of Leishmania infantum by Microsatellite Analysis

ABSTRACT Currently the universally accepted standard procedure for characterizing and identifying strains of Leishmania is isoenzyme analysis. However, in the Mediterranean area, despite their very wide geographical distribution, most Leishmania infantum strains belong to zymodeme MON-1. In order to increase our understanding of polymorphism in strains of L. infantum, we developed PCR assays amplifying 10 microsatellites and sequenced PCR products. The discriminative power of microsatellite analysis was tested by using a panel of 50 L. infantum strains collected from patients and dogs from Spain, France, and Israel, including 32 strains belonging to zymodeme MON-1, 8 strains belonging to zymodemes MON-24, MON-29, MON-33, MON-34, or MON-80, and 10 untyped strains. Five of the microsatellites were polymorphic, revealing 22 genotypes, whereas the five remaining microsatellites were not variable. In particular, MON-1 strains could be separated into 13 different closely related genotypes. MON-33 and MON-34 strains also gave two additional genotypes closely related to MON-1, while MON-29, MON-24, and MON 80 strains exhibited more divergent genotypes. Among the foci examined, the Catalonian focus displayed a high polymorphism, probably reflecting isoenzyme polymorphism, while the Israeli focus exhibited a low polymorphism that could be consistent with the recent reemergence and rapid spread of canine leishmaniasis in northern and central Israel. The strains originating from the south of France and the Madrid, Spain, area displayed significant microsatellite polymorphism even though they were monomorphic by isoenzyme analysis. In conclusion, microsatellite polymorphism exhibits a high discriminative power and appears to be suitable for characterization of closely related strains of L. infantum in epidemiological studies.

Diagnostic Techniques To Detect Cryptic Leishmaniasis in Dogs

ABSTRACT This study of several techniques for detecting cryptic leishmaniasis in dogs from areas in Spain where Leishmania infantum is highly endemic concludes that immunological techniques (enzyme-linked immunosorbent assay, immunofluorescence antibody test, Western blotting, delayed-type hypersensitivity reaction, and in vitro lymphocyte proliferation assay) do not clearly differentiate between noninfected and infected asymptomatic dogs and that culture and PCR are more reliable diagnostic tools.

Leishmania-specific lymphoproliferative responses and IgG1/IgG2 immunodetection patterns by Western blot in asymptomatic, symptomatic and treated dogs

Peripheral cell responses against Leishmania infantum and serology by IFAT and WB were determined in 87 untreated dogs from an endemic area (Madrid, Spain) and in 15 treated dogs (antimonials, allopurinol). All untreated symptomatic dogs (nine) did not show any lymphoproliferative response, whereas 21 out of 78 untreated asymptomatic dogs had a positive cellular response. Serum IgG(2) from dogs with clinical signs of patent leishmaniosis reacted with a variety of peptides (26, 29, 34-35.4, 42, 45, 50-57 and 67 kDa), but IgG(1) response was mainly directed against a 67-kDa peptide. Successfully treated dogs displayed a low immunoreactivity of both IgG(1) and IgG(2), particularly against 67 kDa, thus indicating the potential prognostic value of this region. Positive cellular response of dogs treated with good clinical progress was only observed up to 5-12 months post treatment. Untreated asymptomatic dogs with positive cell response showed a clear recognition by IgG(2) of approximately 67 and 45 kDa antigens, whereas IgG(1) did not recognise any antigen.

Follow-up of Leishmania infantum naturally infected dogs treated with allopurinol: immunofluorescence antibody test, ELISA and Western blot

Fourteen dogs naturally infected with Leishmania infantum and treated with allopurinol were monitored clinically and serologically with immunofluorescent antibody test (IFAT, amastigotes and promastigotes), enzyme linked-immunosorbent-assay (ELISA, IgG1 and IgG2) and Western blotting (WB). In all dogs therapy lead to clinical improvement together with decreasing specific antibodies in IFAT, ELISA and WB, demonstrating the usefulness of serology for follow-up. Although IgG1 and IgG2 varied considerably between individual animals, IgG2 of all dogs was predominantly in both ELISA and WB. This suggests the value of monitoring the IgG2 response (especially against 29 and 67 kDa antigens) in the follow-up of treated dogs.

ABOMASAL PARASITES IN WILD SYMPATRIC CERVIDS, RED DEER, CERVUS ELAPHUS AND FALLOW DEER, DAMA DAMA, FROM THREE LOCALITIES ACROSS CENTRAL AND WESTERN SPAIN: RELATIONSHIP TO HOST DENSITY AND PARK MANAGEMENT

A survey of abomasal parasites in cervids from Central Spain was conducted at 3 sites, Quintos de Mora (Toledo), Maluéñez de Arriba (Cáceres), and La Herguijuela (Cáceres). Commonly occurring helminths belonged to 3 polymorphic species of the Ostertagiinae: Spiculopteragia asymmetrica/S. quadrispiculata, Ostertagia leptospicularis/O. kolchida, and O. drozdzi/O. ryjikovi. Trichostrongylus axei was found in very few cases. Ostertagia drozdzi/O. ryjikovi and the minor male morphotype, S. quadrispiculata, are reported for the first time in red deer from Spain. The 3 ostertagiine species are also reported for the first time in fallow deer from Spain. These 3 species of Ostertagiinae are primarily parasites of cervids, and nematode species characteristic of domestic ruminants were not present. Prevalence of infection by gastrointestinal parasites in cervids was high, ranging from 97.5 to 100%, across the 3 areas sampled. Mean intensity of infection and abundance showed a positive relationship to the population density of red deer. Helminth burdens were higher in fallow deer than in the sympatric red deer and may reflect the gregarious social structure and different foraging patterns of fallow deer.

Vaccination of Manchego lambs against Haemonchus contortus with a somatic fraction (p26/23) of adult parasites

A low molecular weight fraction from adult Haemonchus contortus containing two peptides (p26/23) was used to vaccinate Manchego female lambs between 3.5 and 5 months of age. Immunizing injections were given three times on days 0, 14 and 28 of the experiment. On day 43, lambs were challenged with 400 third stage larvae/kg live weight. Vaccination induced a lengthening of prepatent periods, significant reduction (> 60%) in mean faecal egg counts and smaller variations in packed cell volume values. At necropsy, average worm burden in the vaccinated lambs was significantly lower (61.6%) than that found in unvaccinated challenged animals. A clear correlation was found between protection and serum antibody response in immunized lambs.

Value of Western Blotting in the Clinical Follow-Up of Canine Leishmaniasis

Specific serum antibody levels in Leishmania infantum-infected dogs treated with a combination of glucantime and allopurinol were estimated by indirect immunofluorescence and Western blotting. The sensitivity of Western blot was greater than that obtained with immunofluorescence titration. In general, both diagnostic methods concurred with the post-treatment clinical status of the animals. Clinical improvement of successfully treated dogs was related to lower immunofluorescence titers and simpler and/or less reactive immunodetection patterns in Western blotting. The recognition, by infected dogs, of certain low molecular weight antigens, particularly one of approximately 26 kDa, was restricted to pretreatment samples and a single animal in relapse thus apparently constituting an active infection marker.

Setting New Immunobiological Parameters in the Hamster Model of Visceral Leishmaniasis for In Vivo Testing of Antileishmanial Compounds

To establish suitable immunobiological parameters for in vivo testing of new antileishmanial compounds in the golden hamster model of visceral leishmaniasis, two groups of 8 animals were infected each with 105 or 107 stationary promastigotes by the intracardiac route and the clinical and immunoparasitological features were monitored up to day 155 after infection. All animals became infected at both doses, although significant differences were observed between parasite burdens in liver and spleen. The mean number of parasites in animals infected with 107 promastigotes increased by 9.5 times in liver and by 43.1 times in spleen compared with those infected with 105 promastigotes. In animals given the higher dose, the outcome of the disease occurred between days 75 and 90 after infection, whereas no signs of disease were apparent in those given the lower infecting dose. Positive antibody (IgG) responses were detected earlier (week 5–7 after infection) in animals infected with the higher dose than in those infected with the lower dose (week 8–10 after infection), but these responses did not correlate with individual parasitological loads in liver and spleen. An inverse correlation was observed between infecting doses and in vitro spleen lymphocyte proliferation against mitogens (ConA). The proportion of CD4+ and CD19+ spleen cell increased in animals given the higher infection, whereas it decreased in those given the lower infection compared to naive controls.

Anti-leishmanial activity of a new formulation of amphotericin B

The effectiveness of albumin microspheres loaded with amphotericin B was tested in an in vivo model of visceral leishmaniasis using the golden hamster. Free and encapsulated amphotericin B was tested at the dose of 1 mg/kg given by the intracardiac route on days 25, 26 and 27 post‐infection (p.i.) to treat animals previously infected with 107 stationary promastigotes by the intracardiac route. Encapsulated amphotericin was highly effective against infection causing a reduction of 88.8% and 87.2% in the early stage of infection (day 32 p.i.) and of 66.7% and 54% in a later stage of infection (day 135 p.i.) in liver and spleen parasite load respectively, compared with untreated animals, whereas free amphotericin was inactive. Lymphocyte proliferation was restored together with an increase in CD4+ subsets in animals treated with encapsulated amphotericin B, but not in those treated with the non‐encapsulated compound. Antibody responses did not increase after treatment with encapsulated amphotericin B with antibody levels remaining at base levels for most animals in contrast to those of untreated or treated with free amphotericin, where in most animals the antibody levels sharply increased. This new formulation could be a more economical alternative to liposomes for the treatment of visceral leishmaniasis with amphotericin B.

Vaccination of lambs against Haemonchus contortus infection with a somatic protein (Hc23) from adult helminths.

A somatic protein from adult Haemonchus contortus (Hc23), the most abundant component in a low molecular weight fraction with known immunizing effect against experimental haemonchosis, has been purified by immunochromatography. The immunoprophylactic value of Hc23 was tested in groups of 5-6 months old Assaf lambs using Al(OH)(3) or Escherichia coli lipopolysaccharide+inactivated Propionibacterium acnes as adjuvant and the results compared with uninfected control, uninfected and challenged or infected and challenged lambs. Immunization with Hc23 in either adjuvant elicited significant reductions in fecal egg counts after challenge with 15,000 L3s (70.67%-85.64%, respectively) and reduced (67.1% and 86%) abomasal worm counts (45 days post-challenge). Immunized lambs displayed higher peripheral eosinophil counts, were less anaemic and had weight gains than challenged controls. The results suggest that the Hc23 antigen can induce a partially protective response against haemonchosis in lambs.