Resumen de Fasciolosis ovina: estudios clínicos y desarrollo de nuevos métodos de diagnóstico y control
- español
El presente estudio se centra en el desarrollo de diversos métodos para el control de la infección experimental y natural por Fasciola hepática que puede detectarse en muestras fecales de ovinos a partir de la segunda y tercera semanas post-infección. En primer lugar se estudia la administración parenteral de Lippolysaccharide (LPS) de Ochrobactrum intermedium que supune una reducción de la infección y un incremento de la resistencia inespecífica del hospedador definitivo frente a la infección por Fasciola hepática. En segundo lugar se analiza la administración de dietas basadas en la suplementación con aceite de linaza y vitamina E donde se demuestra una reducción de la excreción fecal de huevos, la carga parasitaria y el tamaño de las fasciolas. Los ovinos así alimentados presentan unos índices hemáticos y bioquímicos menos afectados por la infección, con menor gravedad de la anemia y con funcionalidad hepática superior
- English
The main goals of this PhD Thesis were as follows:
1.- Comparison of three different techniques to diagnose Fasciola hepatica infection in experimentally and naturally infected sheep:
The aim of this study was to compare three different techniques for the early diagnosis of the infection by Fasciola hepatica in experimentally and naturally infected sheep. The experimental group consisted of 7 sheep infected with 200 metacercariae; faecal samples were taken weekly until 12 week post-infection (wpi). Under natural conditions, 45 individual faecal samples and 2 pools of faeces were collected from three different flocks with a history of F. hepatica infection. The results obtained by a coprological method were compared with a commercial immunoassay and with two PCR assays in faecal samples. Faecal eggs were detected by 9 wpi in experimental infection. On the other hand, only 24 out of 45 sheep were positive in naturally infected flocks. By means of a sandwich-ELISA kit, the infection was first detected by 4 wpi in the 57.1% experimentally infected sheep and this percentage reached 100% by 8 wpi. All naturally infected animals were positive with this method. Regarding PCR, a specific 423 bp fragment of mitochondrial DNA was amplified in faecal samples. The F. hepatica infection was detected from 3 wpi with a standard PCR, and from 2 wpi with a nested-PCR. Only 37 sheep out of 45 were positive by the standard PCR although the infection was diagnosed in all animals by the nested-PCR. In conclusion, the sensitivity of the nested-PCR described in our study is higher than the detection of eggs in faeces as well as the commercial immunoassay. Moreover, no cross reactions were described with gastrointestinal nematodes.
2.- Effect of Lipopolysaccharide (LPS) from Ochrobactrum intermedium on sheep experimentally infected with Fasciola hepatica:
The effects of the lipopolysaccharide (LPS) from Ochrobactrum intermedium was evaluated in sheep experimentally infected with Fasciola hepatica. Animals were divided into 4 groups: 2 treated with the LPS (T1/T2) and 2 controls (C1/C2). T1/C1 were slaughtered at 30 days post-infection (dpi) and T2/C2 at 85 dpi. Body weight and body condition were found higher in T1 and T2 than in controls, although differences were not significant. Treated sheep showed lower cumulative fecal egg count than controls (p<0.01). Levels of red blood cells (RBC), white blood cells, hemoglobin, and hematocrit (HCT) were higher in T1 and T2. Significant differences (p<0.05) in RBC and HCT were found between groups at 84 dpi. More severe macrocytic and hypochromic anemia was observed in C1 and C2 than in treated groups. Total protein and albumin values were higher in T1 and T2 (p<0.01) until 29 dpi. At the end of the trial, no significant differences were observed in hepatic enzymes, although gamma-glutamyl transferase and aspartate-amine transferase values were higher in C2, and alanine-amine transferase was higher in T2. At necropsy, the mean weight of liver, fibrosis in portal triads, and ganglion size were similar in all groups. The number and size of flukes was greater in C2 than in T2 (p<0.05). The histological examinations revealed a higher degree of parenchymatous fibrosis in T2 compared to C2 (p<0.05). The administration of LPS from O. intermedium increased the nonspecific resistance against F. hepatica in experimentally infected sheep.
3.- Immunological features of LPS from Ochrobactrum intermedium on sheep experimentally infected with Fasciola hepatica:
The effects of the lipopolysaccharide (LPS) from Ochrobactrum intermedium in sheep with fasciolosis was reported previously, resulting in lower faecal egg counts and fluke burden. In the current study, we analyzed its immunological effects in two groups of sheep, treated (T) and controls (C). Fasciolosis induces a T helper (Th) type-2 response, characterized by IL-4 and IL-10 production; however, at the beginning of the infection, the IFN-¿ production predominates (Th type-1 response). Although we did not find differences in IL-4 production or in the expression level of this gene in the hepatic lymph nodes, the expression level of IL-10 was higher (P<0.05) in the T group at 4 wpi. The IFN-¿ production was higher (P<0.01) at 12 wpi as well as its level of expression at 4 wpi (P<0.05) in the T group. We found higher expression level of TGF-ß at 4 wpi in the T group (P<0.05), associated with the previous report of thicker fibrous tracks in a treated group. Immunoglobulin G1, related with a Th type-2 response, was higher (P<0.01) in the T group at 4 and 12 wpi. In conclusion, the effects of LPS from O. intermedium could have resulted from a predominant Th type-2 immune response.
4.- Effect of dietary supplementation with flaxseed oil or vitamin E on sheep experimentally infected with Fasciola hepatica:
The effects of two diets supplemented with flaxseed oil (FO) or vitamin E (VE) were studied in sheep infected with Fasciola hepatica. The control group (CO), not supplemented, had higher weight gain and faecal egg count (P<0.05) at the end of the study. At necropsy, the number (71.2 ±26.5) and size of flukes were lower in the FO group (P<0.01), probably due to the higher levels of white blood cells and lymphocytes (P<0.01). This group also had higher red blood cell and haematocrit values (P<0.01). The CO group had more severe hypoproteinaemia and hypoalbuminaemia, due to having the highest fluke burden (123.0 ±35.2), and also the highest IgG1 titre (P<0.01). The diet did not affect production or gene expression levels of IFN-¿ and IL-4. On the other hand, the supplementation with VE led to a reduction in adult fluke burden (97.7 ±39.9) and lower lipid oxidation in the liver (P<0.001).
