Evaluation of a Cooperia oncophora double-domain ASP-based vaccine against Cooperia spp. infections in cattle and sheep.

A double-domain activation-associated secreted protein (dd-Co-ASP) isolated from the bovine small intestinal parasite Cooperia oncophora was previously shown to be an effective vaccine candidate to protect calves against a homologous challenge infection. The aim of this study was to investigate whether the dd-Co-ASP protein, purified from a Belgian C. oncophora isolate, would offer protection against a C. oncophora isolate from the southern hemisphere as well as other Cooperia species such as C. punctata in cattle and C. curticei in sheep. Two vaccination studies were performed, i.e. one in cattle and one in sheep, in which the protective effects of dd-Co-ASP, supplemented with Quil A as an adjuvant, were compared with an adjuvant control. Whereas our results showed a 75 % reduction in Cooperia spp. cumulative faecal egg counts, the results obtained in sheep demonstrated that dd-Co-ASP was ineffective in raising a protective immune response against a C. curticei challenge infection. Even though sequence analysis of the dd-Co-ASP gene revealed restricted sequence heterogeneity in the double domain ASP within and between bovine Cooperia species, the results of the vaccine study suggest that there is sufficient conservation at the protein level to yield cross-protection, holding promise for the development of a general Cooperia vaccine for use in cattle.

Identification of reference genes for real-time PCR cytokine gene expression studies in sheep experimentally infected with Fasciola hepatica.

The aim of this study was to validate reference genes for gene normalisation using qRT-PCR in hepatic lymph nodes (HLN) and livers from sheep infected with Fasciola hepatica during early and late stages of infection. To this end, a comprehensive statistical approach (RefFinder) encompassing four different methods of analysis (geNorm, BestKeeper, ΔCt method and NormFinder) was used to validate ten candidate reference genes. Stability analysis of gene expression followed by pairwise variation (Vn/Vn + 1) analysis revealed that PGK1, HSP90AA1 and GYPC were the most stable reference genes and suitable for qRT-PCR normalisation in both HLN and liver tissues. These three genes were validated against FoxP3, IL-10, TGF-ß, TNF-α and IL-1ß genes in the HLN tissue of sheep vaccinated with Cathepsin L1 from F. hepatica and unvaccinated infected and uninfected controls during early stages of infection. In the liver, the three reference genes were validated against TNF-α and IL-1ß during chronic stages of infection with F. hepatica and in uninfected controls. Our study is the first to evaluate and validate sheep reference genes in order to provide tools for monitoring cytokines in Fasciola hepatica infected sheep target organs. Our results present an approach to elucidate the role of different cytokines in F. hepatica vaccinated and infected sheep.

Pathological, immunological and parasitological study of sheep vaccinated with the recombinant protein 14-3-3z and experimentally infected with Fasciola hepatica.

In this study, the immunogenicity and protective capacity of a new recombinant vaccine candidate, the rFh14-3-3z protein was analysed in sheep experimentally challenged with Fasciola hepatica, in terms of fluke burden, faecal egg counts, hepatic damage and humoral immune response. Three groups of 8 animals each were used for study, group 1 was immunised with the rFh14-3-3z in Montanide adjuvant, whereas group 2 and 3 remained as adjuvant control and infection control groups, respectively. The parasitological analysis showed that no significant reduction in fluke burden, fluke size and faecal egg counts was detected. The extent of hepatic damage was very similar between groups. Nonetheless, animals immunised with the rFh14-3-3z protein induced the development of specific IgG1 and IgG2, being the IgG1 the predominant antibody; which confirms the immunogenicity of this protein in sheep. This is the first report of the 14-3-3z proteins as vaccine against the infection with F. hepatica.

Apoptosis of peritoneal leucocytes during early stages of Fasciola hepatica infections in sheep.

Several immunomodulatory properties have been described in Fasciola hepatica infections. Apoptosis has been shown to be an effective mechanism to avoid the immune response in helminth infections. The aim of the present work was to study apoptosis in peritoneal leucocytes of sheep experimentally infected with F. hepatica during the early stages of infection. Five groups (n=5) of sheep were used. Groups 2-5 were orally infected with 200 metacercariae (mc) and sacrificed at 1, 3, 9 and 18days post-infection (dpi), respectively. Group 1 was used as the uninfected control (UC). Apoptosis was detected using three different methods 1) immunocytochemistry (ICC) with a polyclonal antibody anti-active caspase-3; 2) an annexin V flow cytometry assay using the Annexin V-FITC/propidium iodide (PI); and 3) transmission electron microscopy (TEM). The differential leucocyte count revealed that the majority of peritoneal granulocytes were eosinophils, which increased significantly at 9 and 18 dpi with respect to the uninfected controls. The ICC study revealed that the percentage of caspase-3+ apoptotic peritoneal leucocytes increased significantly from 3 dpi onwards with respect to the uninfected controls. The flow cytometry annexin V assay detected a very significant (P<0.001) increase of apoptotic peritoneal macrophages, lymphocytes and granulocytes, which remained higher than in the UC until 18 dpi. Transmission electron microscopy studies also confirmed the presence of apoptosis in peritoneal eosinophils at 18 dpi. This is the first report of apoptosis induced by F. hepatica in the peritoneal leucocytes of sheep in vivo. The results of this work suggest the importance of apoptosis induction for the survival of the juvenile parasites in the peritoneal migratory stages of infection.

Severe hypoxaemia with a left ventricular assist device in a minipig model with an undiagnosed congenital cardiac disease.

We describe the placement of a left ventricular assist device (LVAD) in a pig with spontaneously occurring atrial septal defect (ASD) (incidental finding) that created a right-left cardiac shunt, with subsequent severe hypoxaemia. Early diagnosis was critical in order to prevent end-organ damage due to hypoxaemia. Adequate monitoring alerted us to the deterioration in oxygenation, haemodynamics and cerebral oxygen metabolism. This forced us to change the level of assistance provided by the pump, and thus dramatically correct this impairment. Necropsy revealed an ostium secundum ASD. In conclusion, if hypoxaemia presents after implementation of an LVAD, the presence of a right-left shunt must be ruled out. The first step must be a judicious reduction in assist device flow to minimize intracardiac shunting. Subsequently, atrial septal closure of the defect should be considered. We report an experimental model of severe hypoxaemia after placement of an LVAD as part of a larger research project.