J.M. Alunda

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.

The effect of the host's age on the pathology of Eimeria stiedai infection in rabbits

The pathology of experimental Eimeria stiedai infection in 2- and 4-month-old rabbits raised coccidia-free was studied. Over 50 days of infection, the serum activities of aspartate (AST) and alanine (ALT) aminotransferases, alkaline phosphatase (AP) and gamma-glutamyltranspeptidase (GGTP) were analyzed as indicators of hepatic lesions and total serum globulins with respect to the immune response of the host; parasite development was followed by oocyst output; weight gain, food intake and relative liver weight were analyzed to control the performance of infected animals. The age of the host strongly affected parasite development and consequently the biochemical and zootechnical parameters differed more markedly in the younger animals; however, the enzyme ALT showed an increase independent of parasite development, and infection did not affect GGTP activity. The increase in age was responsible for resistance of rabbits to hepatic coccidiosis and older animals were less affected than young ones.

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.

A quantitative study of Eimeria infections of goats from central Spain

A survey on coccidial infections in goats from central Spain was carried out. Fifty-five goat farms from 28 localities belonging to the three bioclimatic subregions of the area were visited. Individual samples (702) were obtained from the rectum of 130 young (under 1 year of age) and adult (572 over 1 year of age) goats. Coproscopical analyses showed a 100% prevalence and a moderate average intensity (7606.48 +/- 17918.78 Eimeria oocysts per gram of faeces), although wide variations were seen among herds and among individuals within each goat flock. Statistical analysis showed a strong age-related reduction in oocysts output in goats up to 4 years of age. The intensity of coccidial infections (young and adult goats, herds) was independent of the prevailing bioclimatic conditions. Heavier infections in young goats were found in bigger herds, whereas adult goats from these same big flocks (over 300 animals) showed the lowest oocyst output. Under our conditions, neither the female restocking rate nor the age structure seemed to play a significant role in the intensity of the Eimeria infections.

Preventive treatment of rabbit coccidiosis with α-difluoromethylornithine

Abstract The effect of α-difluoromethylornithine (DFMO) administered in drinking water (0.2 and 0.5%) against liver and intestinal coccidioses in experimentally infected rabbits was studied. Zool-technical and clinical parameters were used to assess the efficacy of the compound. In both coccidioses the lower dose not show any important effect. The higher dosage (0.5%) however, reduced the lesions induced by coccidia to an important extent. The animals infected and treated with 0.5% DFMO showed similar weight gain, food intake and relative liver weight figures to the non-infected controls. Serum transaminase levels in the rabbits infected with Eimeria stiedai and treated with 0.5% DFMO were close to those of the non-infected animals. Infected rabbits treated with this dosage also showed reductions (over 80%) in faecal oocyst output.

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.

Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs

Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 μM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 μM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.

Effect of allicin on promastigotes and intracellular amastigotes of Leishmania donovani and L. infantum

Anti-leishmanial activity of allicin (=diallyl thiosulphinate) has been tested in vitro against promastigotes and intracellular amastigotes of Leishmania donovani and Leishmania infantum. Macrophage infections have been carried out in vitro in the murine cell line J774 and ex vivo with peritoneal macrophages from BALB/c mice with a modified method to isolate metacyclic promastigotes. The compound has shown a significant in vitro effect on the multiplication of promastigotes of L. donovani and L. infantum in a time- and dose-dependent manner. It has been shown for the first time the inhibition of multiplication of intracellular amastigotes of Leishmania by allicin. Inhibitory concentrations of the compound were in the micromolar range (10-30 μM) for both Leishmania species. Antileishmanial effect of allicin apparently was not related to products of degradation of the molecule as assessed by mass spectrometry analysis. Inhibitory activity of allicin against promastigotes and intracellular amastigotes increased when the compound was added to the cultures every 24 h. Two administrations of 5 μM allicin inhibited by ca. 50% the proliferation of Leishmania amastigotes. Low toxicity for mammalian cells of this compound suggests the interest of exploring the value of allicin in combined therapy against leishmanial infections.