Une technique ELISA pour la detection des anticorps anti-virus maedi-visna. Etude comparative avec l'immunodiffusion en gelose et la fixation du complement

An indirect microELISA test was performed for detection of maedi-visna antibodies in ovine and caprine species. The antigen consisted in viral particles, highly purified by successive ultracentrifugations.By comparative testing of 934 sera in ELISA and gel immunodiffusion, we found a good correlation between these two teṡts, and moreover, ELISA revealed another 11.3% of positive samples. The precocity of this ELISA was shown by experimental infection of sheep with different strains of maedi-visna: positive sera were detected 7 weeks post-infection, instead 4–5 months with gel immunodiffusion. The complement fixation test was compared with gel immunodiffusion and was found the less sensitive.This ELISA test appeared to be satisfactory, and may be used for early diagnosis of maedi-visna infection.     

Clostridium perfringens toxin-types in lambs and kids affected with gastroenteric pathologies in Italy

A study was carried out in the South of Italy to assess the role of clostridia in neonatal diseases of lambs and kids. Eighty-seven lambs and 15 kids belonging to 25 flocks were examined and Clostridium perfringens was the microorganism most commonly identified. C. perfringens isolates were analysed by polymerase chain reaction (PCR), in order to determine the prevalence of the genes cpa, cpb, cpb2, etx, iap and cpe. The most prevalent toxin-type of C. perfringens was found to be type A found in 84% of the cases with clostridial enterotoxaemia. No C. perfringens type B, C or E were found. C. perfringens type D was isolated in 16% of the cases. About 24% of the isolates were cpb2 positive. The prevalence of cpb2 across the different C. perfringens types varied. The beta(2)-toxin gene cpb2 was detected in 4/21 (19%) type A isolates, in 1/2 type D isolates, and in 1/2 type DE (cpe-carrying type D) isolates. The high rate of positivity to cpb2 among the isolates suggests that a vaccine based on the beta(2)-toxin, should be included in the vaccination schedule of the animals to confer adequate protection and to prevent the disease.     

Species composition of Gasterophilus spp. (Diptera, Oestridae) causing equine gastric myiasis in southern Italy: parasite biodiversity and risks for extinction

Horse gastrointestinal myiasis caused by larvae of Gasterophilus spp. (Diptera, Oestridae) flies has a worldwide distribution and, where present, it is primarily caused by larvae of Gasterophilus intestinalis and Gasterophilus nasalis. Other species, i.e., Gasterophilus inermis, Gasterophilus pecorum and Gasterophilus haemorrhoidalis, present in different or in the same regions of the gastrointestinal tract, were only occasionally reported in very limited areas of eastern European Countries and in central Italy. With the aim to contribute data on the species composition of Gasterophilus and on the seasonal variation of the infection pattern in southern Italy, 152 native horses were necropsied from January to December 2003 and Gasterophilus larvae were collected from the stomach, the small intestine and the rectum of each of them. On the whole, 125 (82.2%) horses were infected by larvae of Gasterophilus spp. and 214 second stage larvae (L2) and 13,342 third stage larvae (L3) collected. Five species of Gasterophilus were identified with the following prevalence: G. intestinalis=95.2%, G. nasalis=44.8%, G. inermis=15.2%, G. pecorum=2.6% and G. haemorrhoidalis=0.8%. L3 were retrieved throughout the observation period with the highest mean burdens from January to August 2003 while the lowest mean was registered from September to November 2003. L2 were collected in February-March 2003 and from September to December 2003. The majority of the animals (n=66, 43.4%) were infected with a single Gasterophilus species, however, 45 animals (29.6%) harboured 2 species, 12 animals (7.9%) 3 species and 2 animals (1.3%) 4 species. The trend of abundance in the L3 of G. intestinalis and G. nasalis, the most represented species, was highly concordant (r=0.5, p<0.001). A retrospective comparison of our results and of other data from four seasons of observation (1983-1986) in central Italy showed that the number of G. inermis, G. pecorum and G. haemorrhoidalis have been decreasing relative to G. intestinalis and G. nasalis which may suggest tendency toward the extinction of the three former species of Gasterophilus.     

The pathophysiology of epilepsy

Recent developments in the understanding of the cellular events in focal seizures are described. This is followed by a discussion of the pathogenesis of neuronal hyperexcitability, the mechanism of spread of the seizure discharge and the mechanisms involved in seizure termination. The pathophysiology of generalized seizures is described in terms of theories of the site of origin of the seizure discharge and possible mechanisms of the clinical phenomena associated with this discharge.     

Note: A comparison of molecular diagnostic procedures for the detection of aster yellows phytoplasmas (16Sr-I) in leafhopper vectors

Different molecular procedures were compared for the detection of aster yellows phytoplasmas (AYP) in the leafhopper vectorsMacrosteles quadripunctulatus (Kirschbaum),Euscelidius variegatus (Kirschbaum) andEuscelis incisus (Kirschbaum). Polymerase chain reaction (PCR) with universal and group-specific primers designed on the 16S-rDNA sequence was most sensitive in nested assays. A dot-blot procedure with an oligoprobe designed on the 16S-rDNA was less sensitive and consistent to detect phytoplasmas in total insect DNA, but consistently detected amplicons from direct PCR. The dot-blot assay with a probe based on a phytoplasma plasmid sequence detected AYP in most vector specimens and did not react with DNAs from leafhoppers infected by flavescence dorée and psyllids infected by apple proliferation phytoplasmas. This last assay is almost devoid of contamination risks, faster and cheaper compared to PCR, therefore it has to be preferred for field-scale analysis of leafhopper populations. http://www.phytoparasitica.org posting Feb. 24, 2004.     

Osteosarcoma with multiple metastases and subcutaneous involvement in a rabbit (Oryctolagus cuniculus)

BACKGROUND: Osteogenic sarcomas are rare in rabbits, but cases involving the mandible, ribs, frontal bones, and tibia have been reported. OBJECTIVE: The purpose of this study was to describe the clinical and pathologic findings in a rabbit with osteosarcoma and multiple metastases with subcutaneous involvement. METHODS: A 1-year-old female Polish rabbit was evaluated for dysorexia, lameness, gait difficulties with the hind limbs, and the presence of 2 subcutaneous nodules, on the left thigh and in the dorsal region. A hard swelling was also present at the base of the tail. Radiographic findings revealed multiple abnormalities in several bones, which were more severe in the sacrococcygeal region and femur. Several areas of calcification involving the thorax and abdomen as well as skeletal muscles, also were noted. The thigh nodule was surgically removed, and cytologic and histologic evaluations were performed. Following the rabbit's death, postmortem radiographs were taken and necropsy was performed. Samples of tissues and organs, involved bone segments, and other subcutaneous nodules were collected and examined by cytology and histology. RESULTS: Both the subcutaneous nodules and the majority of tissues and organs contained neoplastic cells whose appearance was consistent with osteogenic sarcoma. CONCLUSION: Clinical, radiologic, and pathologic data supported a diagnosis of osteosarcoma with multiple metastases and subcutaneous involvement.     

Colocalization of DNA fragmentation and caspase-3 activation during atresia in pig antral follicles

Apoptosis is the cellular mechanism of ovarian follicular atresia. The major downstream effector of this phenomenon in many tissues is caspase-3 but little is known about its role in pig ovarian apoptosis. In the present study, we detected the localization of caspase-3 in parallel with nuclear fragmentation (TUNEL) on healthy and early atretic antral follicles. In healthy antral follicles caspase-3 and TUNEL positivity were occasionally recorded within theca layer. The incidence of DNA fragmentation, as indicated also by the biochemical detection, increased mainly in the granulosa layer of early atretic follicles. Quantitative analysis revealed, besides, that atresia was accompanied by a higher incidence of caspase-3 (57.20 +/- 20.05 versus 3.64 +/- 0.61 positive cells in atretic versus healthy follicles, respectively; P < 0.05), of TUNEL positivity (20.13 +/- 9.33 versus 0.42 +/- 0.12; P < 0.05) and simultaneous immunostaining for caspase-3 and TUNEL (15.02 +/- 6.95 versus 0.31 +/- 0.05; P < 0.05) in the granulosa layer. In detached granulosa cells isolated from the follicular fluid of early atretic follicles a further significantly increase was recorded in the percentage of TUNEL positivity and in the incidence of cells that showed colocalization of caspase-3 activity and DNA fragmentation. Granulosa cells of early atretic follicles exhibited a higher positivity for caspase-3 localized in the cytoplasm and occasionally in the nucleus area of granulosa cells. These results indicate that capsase-3 was involved and precociously activated during the process of atresia. Finally, the progressively higher incidence of TUNEL positivity and of double immunostaining in atretic cells collected within the follicular fluid seems to indicate that proteases activity leads only tardily in a detectable DNA fragmentation.     

Cross-transmission studies with Hypoderma lineatum de Vill. (Diptera: Oestridae): attempted infestation of goats (Capra hircus)

The potential for cross-transmission of Hypoderma lineatum from cattle to domestic goats (Capra hircus) was examined using artificial infestation techniques. Two routes of infestation, subcutaneous injection and dermal penetration, were used to expose goats to newly hatched first instars. Presence of antibodies and appearance of circulating antigen (hypodermin C) were evaluated at selected intervals for up to 40 weeks post-infestation. In addition, immunoblots against H. lineatum first-instar proteins were conducted using sera taken at 10 weeks post-infestation. Goats were palpated for the presence of developing larvae at sub-dermal sites beginning at week 30 pi. No developing larvae were palpated at any time, regardless of the route of infestation nor was circulating antigen detected in any infested goats. Antibodies were present at weeks 6 and 10 and week 27 pi in both infested groups. Immunoblots indicated all infested goats produced antibodies to first instar H. lineatum antigens. H. lineatum appears to be incapable of completing development in domestic goats although the transient appearance of ELISA detectable antibodies and the presence of bands on immunoblots suggests that at least some larvae survive long-enough to engender a humoural response. The host specificity of H. lineatum is discussed in light of the general concepts of host-parasite relationships of oestrids.     

Iron chelation by the powerful antioxidant flavonoid quercetin

Chelation of the bare and hydrated iron(II) cation by quercetin has been investigated at the DF/B3LYP level in the gas phase. Several complexed species arising from neutral and anionic forms of the ligand have been taken into account. Both 1:1 and 1:2 metal/flavonoid stoichiometries have been considered. Results indicate that among the potential sites of chelation present on quercetin, the oxygen atoms belonging to the 3-hydroxy and 4-oxo, and to the 5-hydroxy and 4-oxo groups, are the preferred ones. Time-dependent density functional theory (TDDFT) calculations, used to reproduce the electronic UV-vis spectra of isolated quercetin and its complexes with Fe2+, were also performed in methanol and dimethylsulfoxide.