SDS-PAGE and Western blot of urinary proteins in dogs with leishmaniasis

Canine leishmaniasis is an endemic disease in the Mediterranean area caused by the protozoan Leishmania infantum, which usually produces renal failure. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot using antibodies to IgG and IgA from dogs were carried out in the urine of 22 dogs with leishmaniasis diagnosed by ELISA and confirmed by PCR, and 20 healthy dogs. The results were compared to renal function laboratory tests and to those from a histopathological study of the kidneys from sick animals that died naturally or were euthanized. Five different bands with molecular weights ranging from 10 to 110 kDa were obtained from the electrophoresis of the urine of healthy dogs. 33.5% of total proteins corresponded to low molecular weight proteins and the other proteins had middle and high molecular weights. However, in the group with leishmaniasis, a maximum of 11 different bands with molecular weights ranging from 10 kDa to 150 kDa were displayed in the electrophoresis of the urine. The urine electrophoretic pattern in the sick dogs was classified as mixed (proteins with high and low molecular weights) because low molecular weight proteins made up 57.9% and the rest of the proteins had middle and high molecular weights. In Western blot, none of the healthy dogs showed excretion of IgG and/or IgA, whereas IgG and IgA were detected in the Western blot of urine of 68% and 55% respectively of dogs with leishmaniasis. The results obtained in the leishmaniasis group agreed with glomerular and tubular damage, which were confirmed by the histopathological findings.     

Differential serological testing by simultaneous indirect immunofluorescent antibody test in canine leishmaniosis and ehrlichiosis

A mixed indirect fluorescence antibody test (IFAT), based on cultured promastigotes Leishmania infantum and formol-inactivated suspension of cells infected with the bacteria Ehrlichia canis, was applied to make a differential diagnosis between canine ehrlichiosis and leishmaniosis. A titre greater than 80 was considered positive for antibodies to E. canis and suggestive of antibodies to L. infantum. Positive sera were titrated subsequently by serial dilutions to confirm antibodies positive to Leishmania and establishing the antibody titre of both pathogens. Fluorescence was absent with negative control sera and background staining was minimal. No serological cross-reactions between positive sera for L. infantum or E. canis were detected. Results obtained by mixed IFAT did not differ when the same serum IFAT standard was compared. The test showed equivalent sensitivity (100%). The specifities were 100% for L. infantum and 98.5% for E. canis. The equivalence in sensitivity was confirmed by calculating the correlation coefficient between IFAT standards and mixed IFAT (r>or=0.99 for both pathogens). The results of our investigations demonstrated that mixed IFAT is a specific means of establishing serological differential diagnosis of canine leishmaniosis and ehrlichiosis.     

Assessment of relevant factors and relationships concerning human dermal exposure to pesticides in greenhouse applications

Principal component analysis (PCA) was applied to the gas chromatographic data obtained from 23 different greenhouse trials. This was used to establish which factors, including application technique (very small, small, medium and large drop-size), crop characteristics (short/tall, thin/dense) and pattern application of the operator (walking towards or away from the treated area) are relevant to the dermal exposure levels of greenhouse applicators. The results showed that the highest exposure by pesticides during field applications in greenhouses, in the climatic conditions and in the crop conditions typical of a southern European country, occurs on the lower legs and front thighs of the applicators. Similar results were obtained by hierarchical cluster analysis (HCA). Drop-size seems to be very important in determining total exposure, while height and density of crops have little influence on total exposure under the conditions of the present study. No pesticide type is a major factor in total exposure. The application of multiple regression analysis (MRA) allowed assessment of the relationships between the pesticide exposure of the less affected parts of the body with the most affected parts.     

Effect of luteinising-hormone in vivo on ovarian cell subpopulations of newly-hatched chicks

1. We analysed the number and size of different ovarian cell subpopulations of newly-hatched chicks by ovarian cell suspension count and morphometric/stereological methods as well as delta5-3beta-hydroxysteroid dehydrogenase (delta5-3beta HSD) activity in these cells treated in vivo with LH during embryonic development. 2. Fertile White Leghorn eggs received 1 microg LH applied to the chorioallantoic membrane on days 13, 15, and 17 of incubation. All animals were killed within 24 h after hatching, the left ovary was dissected and processed. 3. The results indicate that the number of germ, pregranulosa, interstitial and undifferentiated cells was not affected by LH treatment. However, we observed an increase in the size of individual interstitial cells of the ovarian medulla. In these cells, delta5-3beta HSD activity was increased in response to LH. 4. These findings suggest that LH does not exert a proliferative effect on the cells of the prefollicular ovary of the chick and that interstitial cells can be target cells for LH, increasing their steroidogenic activity due to LH treatment.     

True hermaphroditism in a horned goat with 60XX/60XY chimerism

Anatomical, behavioral, histological, endocrinological, and cytogenetic characteristics were determined in a horned intersex goat. Histology of the gonads confirmed that the goat was a true hermaphrodite. Cytogenetic analysis showed that it was a chimera (60XX/60XY). Use of laparoscopy allowed determination of characteristics of the internal gonads.     

Antibacterial susceptibility patterns of Pseudomonas strains isolated from chronic canine otitis externa

Development of antibiotic resistance in bacteria is a problem of great concern. It is important to establish the convenience of antimicrobial susceptibility tests in animal infections. The aim of this study was to test the susceptibility to antibiotics of Pseudomonas strains isolated from chronic canine otitis externa. We tested 23 strains of Pseudomonas: 19 Ps. aeruginosa, three Ps. fluorescens and one Pseudomonas spp. The most effective antibiotics were tobramycin (100% susceptible), marbofloxacin (91.3%) and ceftazidime (91.3%). Ticarcillin and gentamicin, commonly used for the treatment of otitis externa also showed good results (susceptibility of strains was 86 and 65.2% respectively). Lower susceptibility was found using enrofloxacin (52.1%) probably due to its indiscriminate use. We emphasize the need for a rational policy of antibiotic prescribing in order to prevent the selection of resistant strains.     

Experimental infection model for Taenia solium cysticercosis in swine. Cysticercosis Working Group in Peru

A novel method for infecting pigs with Taenia solium using an intramuscular innoculum of oncospheres was investigated in a series of five experiments in 18 animals. The model is simple to perform, requires a minimal number of oncospheres, permits multiple infections per animal, and decreases the variation inherent in oral infection models. This intramuscular oncosphere assay (IMOA) may provide a valuable tool to evaluate therapeutic agents or potential vaccines for cysticercosis.     

Selection for ovulation rate in rabbits

An experiment of selection for ovulation rate was carried out. Animals were derived from a synthetic line first selected 12 generations for litter size, then 10 generations for uterine capacity. Selection was relaxed for 6 generations. Selection was based on the phenotypic value of ovulation rate with a selection pressure on does of 30%. Males were selected from litters of does with the highest ovulation rate. Males were selected within sire families in order to reduce inbreeding. Ovulation rate was measured in the second gestation by a laparoscopy, 12 days after mating. Each generation had about 80 females and 20 males. Results of three generations of selection were analyzed using Bayesian methods. Marginal posterior distributions of all unknowns were estimated by Gibbs sampling. Heritabilities of ovulation rate (OR), number of implanted embryos (IE), litter size (LS), embryo survival (ES), fetal survival (FS), and prenatal survival (PS) were 0.44, 0.32, 0.11, 0.26, 0.35, and 0.14, respectively. Genetic correlation between OR and LS was 0.56, indicating that selection for ovulation rate can augment litter size. Response to selection for OR was 1.80 ova. Correlated responses in IE and LS were 1.44 and 0.49, respectively. Selection for ovulation rate may be an alternative to improve litter size.     

Detecting<Emphasis Type="Italic">Orobanche</Emphasis> species by using cpDNA diagnostic markers

Some species of the genusOrobanche are among the most devastating parasitic weeds, causing extensive damage in agricultural fields. Considering the difficult control due to seed longevity in the soil, small seed size, high fecundity and a subterranean phase that allows them to parasitize the host before they emerge and become evident, the development of diagnostic markers is highly recommended. In our study we identified potential molecular diagnostic markers from the plastid genome in order to distinguish among the most importantOrobanche species attacking crops in Andalusia, the southern region of the Iberian Peninsula. The study has consideredO. crenata, O ramosa andO. cumana causing serious losses in legumes, solanaceous crops and sunflower fields, respectively, andO. minor that, although abundant in Andalusia, has to our knowledge not yet been found parasitizing agricultural hosts. We amplified a non-coding region from the plastid genome, studied sequence differences among the amplified fragments and digested those of the same length with selected restriction enzymes. Here, we propose a molecular protocol to distinguish the main parasitic plants in crop fields of southern Spain. Different applications such as identification ofOrobanche seeds in soil or crop seed lots are discussed in order to offer right crop recommendations or to prevent new infestation of parasite-free fields. Recommendations for further development of these diagnostic markers are also considered. http://www.phytoparasitica.org posting Jan. 15, 2007.     

Development of Pleospora allii on Garlic Debris Infected by Stemphylium vesicarium

Pseudothecia of Pleospora allii developed best on garlic leaf debris infected by Stemphylium vesicarium incubated at low temperature (5–10°C) and relative humidity (RH) close to saturation. RH of less than 96% prevented the formation of pseudothecia, while an incubation temperature of 15–20°C led to the early degeneration of pseudothecia. Under natural conditions, colonization by pseudothecia of unburied garlic leaf debris varied between seasons from 6.0 to 15.5 pseudothecia/mm2, whereas lower colonization levels were recorded when samples were buried. Pseudothecial maturity was reached 1–4mo after the deposition of garlic debris on the soil surface and 15 days after the burial of residues. In the later case, pseudothecia degenerated with degradation of the plant debris. Ascospore release, which required rainfall or dew periods, occurred between late January and late April depending upon the year. A high correlation was found between pseudothecia maturation and four meteorological variables. Two of which, i.e. the number of hours with RH98% and with a mean temperature of 4.5–10.5°C, and the accumulated rainfall, explained most variability (adjusted R2=0.82–0.98 depending upon the year). A multiple regression equation relating the pseudothecia maturity index with these two variables could be used to forecast the epidemic onset of Stemphylium leaf spots in Southern Spain. Temporal progress of pseudothecia maturation was best fitted by a monomolecular model.