Montserrat Cuquerella

Serum antibody response of Castellana sheep to Haemonchus contortus infection and challenge: relationship to abomasal worm burdens.

Primary and secondary serum antibody responses to Haemonchus contortus were studied in Castellana sheep. Ten-month-old sheep were infected (200 L3/kg live weight (lw)) and challenged (400 L3/kg lw) or uninfected and equally challenged with H. contortus. Primary infections induced a partially protective response upon challenge, characterized by higher serum protein levels, longer prepatent periods, lower fecal egg counts, and significant reduction in the establishment rate of the parasite and abomasal adult and L4 worm burdens. The resistant status of the infected and challenged sheep was not clearly related either to the serum specific antibody levels (IgG: IgG1, IgG2; IgM; IgA) estimated by ELISA or to immunodetection patterns in the Western blots.

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.

Biological characterization and pathogenicity of three Haemonchus contortus isolates in primary infections in lambs

The biological characterization and differential pathogenicity of three isolates of Haemonchus contortus, one autochthonous (Aran 99) and two allochthonous (Moredun Research Institute, MRI, and Merck Sharp and Dohme, MSD) were studied by primary experimental infection of Manchego lambs. Thus, six female lambs (5.5 months old) were infected with 12,000 L3 larvae of each helminth isolate. Parasitological (pre-patent period, parasite egg shedding dynamics), biopathological (packed cell volume (PCV), haemoglobin concentration, plasma proteins, serum pepsinogen) and zootechnical parameters (live weight gain, thoracic perimeter) were measured throughout the study. After sacrifice (85 days post-infection (pi)), lamb carcasses were inspected for parasite burden and development (establishment rate, male/female ratio, degree of parasite development), and the average carcass weight of the experimental groups was compared. The autochthonous combination (Manchego lambs-Aran 99) had a longer pre-patent period (28 days) and a significantly different pattern of egg elimination (maximum elimination on day 80 pi). The establishment rate and parasite burden (average values of 8.18% and 988 adult helminths, respectively) were both low, with no significant differences between isolates. There were no significant differences in parasitic nematode development in terms of size and weight (1264.66 microm and 149.45 microg for male worms and 2093.33 microm and 411.46 microg for females, respectively), although Aran 99 females weighed less (p<0.05). All isolates induced a slight but significant reduction of PCV values from day 23 pi onwards. Inter-isolate differences were found, with the effects in the case of MSD being more pronounced. Variations of serum protein levels were minimal in all lamb groups. The live weight gain of MSD- and Aran 99-infected animals was significantly lower (p<0.05) than for MRI-infected lambs and uninfected control animals. Carcass yield from the lambs infected with the autochthonous isolate (Aran 99) was lower. The MSD isolate therefore showed a higher comparative pathogenicity.

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.

Identification and partial purification of a 26 kilodalton antigen of adult Haemonchus contortus

Adult H. contortus soluble extracts were fractionated by means of gel filtration (S-200) and anion exchange chromatography (DEAE-Sephacel). Fractions from both analyses were checked by ELISA and western blotting (WB) with sera from lambs infected with H. contortus, monospecific heterologous sera (anti-Trichostrongylus colubriformis and anti-Teladorsagia circumcinta) and sera from naturally infected sheep with mixed trichostrongylid infections. High cross reactivity was seen between H. contortus and heterologous sera, particularly with the anti-T. colubriformis serum, when fractions from gel filtration were checked by ELISA. Individual fractions containing the highest positive/negative and positive/heterologous ratios were pooled and analysed by SDS-PAGE. One of the pools (A4) containing 2 regions around 48-55 and 25-27 kDa were strongly recognized by homologous sera in WB. Similar results were obtained with the first peak eluted in the DEAE-Sephacel chromatography with NaCl 0.1 M. The pooled fraction A4 from gel filtration was further fractionated by anion exchange chromatography and the peak obtained with the NaCl gradient contained a ca. 26 kDa antigen apparently specific for the diagnosis of H. contortus infections in lambs.

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.

Improvement of 96-well microplate assay for estimation of cell growth and inhibition of Leishmania with Alamar Blue

The value of resazurin-based Alamar Blue redox indicator to determine multiplication of Leishmania promastigotes in 96-well microtiter plates was examined. In addition, assay was validated with amphotericin B (AmB) and allicin. The method was tested on L.donovani and L.infantum promastigotes under different culture conditions (variable air-phase, presence of phenol red, initial cell density, incubation time, use of Hepes buffer). Results showed that the gas-phase of promastigote cultures was critical. The method yielded consistent results with initial plating cell densities of 2.5 × 10⁵ promastigotes/well, up to 72 h incubation and 5% CO₂ atmosphere or reduced air availability (sealed plastic bags, film-sealed microplates). Detection of low numbers of promastigotes and earlier results could be obtained using fluorimetry instead of spectrophotometry. The addition of 20 mM Hepes improved the results. Fluorescence intensity correlated to promastigotes number in both Leishmania spp. Inhibitory concentration (IC₅₀) values for AmB and allicin using cell counting and fluorimetry were comparable. Under these conditions this one-step, low-cost redox indicator can be used in drug sensitivity assays and studies of differential proliferation rates of Leishmania isolates or strains in a 96-well format.

Cross antigenicity among ovine trichostrongyloidea. Preliminary report.

A preliminary trial on the extent of cross-antigenicity among the sheep strongylids Haemonchus contortus, Trichostrongylus colubriformis, Teladorsagia circumcincta and Nematodirus battus in 2.5- to 4-month-old lambs has been carried out using ELISA and Western blotting (WB). Cross antigenicity was tested using soluble extracts from adult and third stage larvae (L3) of H. contortus as antigenic source probed with sera from lambs with monospecific heterologous infections. There was cross-antigenicity between L3 of H. contortus and Trichostrongylus colubriformis in ELISA and WB. Immunodetection results with adult H. contortus antigen showed a closer relationship to Teladorsagia circumcincta. Certain heterologous sera reacted with H. contortus antigens more strongly than the homologous one, but sera from the H. contortus-infected animals had reactivity around the 25 kDa region from adult antigens which could have potential diagnostic use.

Lamb serum recognition of infective larvae and adult Haemonchus contortus antigens.

Sixteen- to eighteen-week-old lambs were infected with 2500 3rd stage larvae (L-3) of Haemonchus contortus or kept as uninfected controls. Two months later all animals were challenged with 5000 L-3 of this parasite. Soluble antigens of H. contortus L-3 and adult worms were analyzed by SDS-PAGE and Western blotting during experimental infection and challenge. Antigens from both sources, particularly of low molecular weight under reducing conditions, were recognised by the pooled sera of infected lambs. No single L-3 antigen was recognised by all infected lambs, whereas two peptides having around 25 and 26 kDa from adults were recognised by infected animals during the patency and could be of potential use in the diagnosis of lamb haemonchosis.

In Vitro Synergistic Effect of Amphotericin B and Allicin on Leishmania donovani and L. infantum

ABSTRACT Current monotherapy against visceral leishmaniasis has serious side effects, and resistant Leishmania strains have been identified. Amphotericin B (AmB) has shown an extraordinary antileishmanial efficacy without emergence of resistance; however, toxicity has limited its general use. Results obtained showed, using a fixed-ratio analysis, that the combination of diallyl thiosulfinate (allicin) and AmB ranged from moderately synergic to synergic at low concentrations (0.07 μM AmB plus 35.45 μM allicin induced 95% growth inhibition). None of the treatments, alone or in combination, had noticeable adverse effects on macrophages (Mϕ) in the concentration range examined (allicin, 0.5, 1, 5 and 10 μM; AmB, 0.05, 0.075, and 0.1 μM). Allicin, AmB, or the combination did not affect the infection rate (percentage of infected Mϕ) of Leishmania. Allicin enhanced the activity of AmB on intracellular amastigotes of Leishmania donovani and L. infantum (ca. 45% reduction of amastigote burden with 0.05 μM AmB plus 10 μM allicin); this represented nearly a 2-fold reduction in the 50% inhibitory concentration (IC50) of the antibiotic added alone. Results point toward the possible utility of testing this combination in vivo to reduce the toxicity associated with monotherapy with AmB.